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Sample records for lysosomes rab gtpases

  1. Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

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    Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

    2010-09-01

    The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

  2. Rab2 promotes autophagic and endocytic lysosomal degradation.

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    Lőrincz, Péter; Tóth, Sarolta; Benkő, Péter; Lakatos, Zsolt; Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Takáts, Szabolcs; Scita, Giorgio; Juhász, Gábor

    2017-07-03

    Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. © 2017 Lőrincz et al.

  3. Are There Rab GTPases in Archaea?

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    Surkont, Jaroslaw; Pereira-Leal, Jose B

    2016-07-01

    A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Distinct actions of Rab3 and Rab27 GTPases on late stages of exocytosis of insulin.

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    Cazares, Victor A; Subramani, Arasakumar; Saldate, Johnny J; Hoerauf, Widmann; Stuenkel, Edward L

    2014-09-01

    Rab GTPases associated with insulin-containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase-activating protein overexpression in β-cells from wild-type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP-bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release-ready SGs in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway.

  5. Integrin-mediated function of Rab GTPases in cancer progression

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    Alahari Suresh K

    2010-12-01

    Full Text Available Abstract The RAS (rat sarcoma superfamily of small GTPases is broadly subdivided into five groups: Ras, Rho, Rab, Ran, and Arf. Rab family proteins are important in regulating signal transduction and cellular processes such as differentiation, proliferation, vesicle transport, nuclear assembly, and cytoskeleton formation. However, some Rab proteins have been reported to be necessary for the adhesion and migration of cancer cells. Although Ras and Rho family members have been strongly implicated in cancer progression, knowledge of Rabs action in this regard is limited. Some reports have also linked Rab GTPases with cancer cell migration and invasiveness. This review discusses the implications of the involvement of Rabs in malignant transformation and cancer therapy through integrin-mediated signaling events, with particular emphasis on breast cancer.

  6. Thousands of rab GTPases for the cell biologist.

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    Yoan Diekmann

    2011-10-01

    Full Text Available Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale

  7. Thousands of rab GTPases for the cell biologist.

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    Diekmann, Yoan; Seixas, Elsa; Gouw, Marc; Tavares-Cadete, Filipe; Seabra, Miguel C; Pereira-Leal, José B

    2011-10-01

    Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform

  8. The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

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    Emilie Fugier

    2009-06-01

    Full Text Available The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER-derived replicative organelle named the "Brucella-containing vacuole" (BCV. Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

  9. Rabifier2: an improved bioinformatic classifier of Rab GTPases.

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    Surkont, Jaroslaw; Diekmann, Yoan; Pereira-Leal, José B

    2017-02-15

    The Rab family of small GTPases regulates and provides specificity to the endomembrane trafficking system; each Rab subfamily is associated with specific pathways. Thus, characterization of Rab repertoires provides functional information about organisms and evolution of the eukaryotic cell. Yet, the complex structure of the Rab family limits the application of existing methods for protein classification. Here, we present a major redesign of the Rabifier, a bioinformatic pipeline for detection and classification of Rab GTPases. It is more accurate, significantly faster than the original version and is now open source, both the code and the data, allowing for community participation. Rabifier and RabDB are freely available through the web at http://rabdb.org . The Rabifier package can be downloaded from the Python Package Index at https://pypi.python.org/pypi/rabifier , the source code is available at Github https://github.com/evocell/rabifier . jsurkont@igc.gulbenkian.pt or jleal@igc.gulbenkian.pt. Supplementary data are available at Bioinformatics online.

  10. Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP)*

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    Lall, Patrick; Lindsay, Andrew J.; Hanscom, Sara; Kecman, Tea; Taglauer, Elizabeth S.; McVeigh, Una M.; Franklin, Edward; McCaffrey, Mary W.; Khan, Amir R.

    2015-01-01

    Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis. PMID:26032412

  11. Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP).

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    Lall, Patrick; Lindsay, Andrew J; Hanscom, Sara; Kecman, Tea; Taglauer, Elizabeth S; McVeigh, Una M; Franklin, Edward; McCaffrey, Mary W; Khan, Amir R

    2015-07-24

    Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.

  12. The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

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    Verma, Kuldeep; Datta, Sunando

    2017-03-24

    One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Role of Rab family GTPases and their effectors in melanosomal logistics.

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    Ohbayashi, Norihiko; Fukuda, Mitsunori

    2012-04-01

    Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.

  14. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

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    Hu, Dong, E-mail: austhudong@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wu, Jing, E-mail: wujing8008@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Hu, Fengyu; Yang, Yabo [Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Rongbo, E-mail: lory456@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China)

    2015-05-29

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.

  15. The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

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    Sanda Ljubicic

    Full Text Available Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.

  16. Identification and function of 11 Rab GTPases in giant freshwater prawn Macrobrachium rosenbergii.

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    Huang, Ying; Ren, Qian

    2015-03-01

    Rab GTPases, members of the Ras-like GTPase superfamily, are central elements in endocytic membrane trafficking. However, little is known of the Rab genes in the giant freshwater prawn Macrobrachium rosenbergii. In this study, 11 Rab genes were identified from M. rosenbergii. All MrRabs have a RAB domain. Phylogenetic analysis showed that these 11 MrRabs were divided into different groups. The MrRab genes were ubiquitously expressed in heart, hemocytes, hepatopancreas, gills, stomach, and intestines. Real-time polymerase chain reaction revealed that the MrRab genes were significantly upregulated by white spot syndrome virus (WSSV) in the prawns, indicating that MrRabs might play an important role in innate immune response against WSSV. Moreover, after challenge with Vibrio parahaemolyticus, the expression levels of all MrRabs in the hepatopancreas were also upregulated, which might indicated the involvement of MrRabs in prawns antibacterial immunity. In all, these preliminary results showed that MrRabs were involved in innate immunity of M. rosenbergii.

  17. A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.

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    Agola, Jacob O; Hong, Lin; Surviladze, Zurab; Ursu, Oleg; Waller, Anna; Strouse, J Jacob; Simpson, Denise S; Schroeder, Chad E; Oprea, Tudor I; Golden, Jennifer E; Aubé, Jeffrey; Buranda, Tione; Sklar, Larry A; Wandinger-Ness, Angela

    2012-06-15

    Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.

  18. Rab27 GTPases Distribute Extracellular Nanomaps for Invasive Growth and Metastasis: Implications for Prognosis and Treatment

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    Olivier De Wever

    2013-05-01

    Full Text Available The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.

  19. Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating proteins

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    Gabernet-Castello, Carme; O'Reilly, Amanda J.; Dacks, Joel B.; Field, Mark C.

    2013-01-01

    Rab GTPases serve as major control elements in the coordination and definition of specific trafficking steps and intracellular compartments. Rab activity is modulated in part by GTPase-activating proteins (GAPs), and many RabGAPs share a Tre-2/Bub2/Cdc16 (TBC)–domain architecture, although the majority of TBC proteins are poorly characterized. We reconstruct the evolutionary history of the TBC family using ScrollSaw, a method for the phylogenetic analysis of pan-eukaryotic data sets, and find...

  20. Rab27 GTPases distribute extracellular nanomaps for invasive growth and metastasis: implications for prognosis and treatment.

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    Hendrix, An; De Wever, Olivier

    2013-05-10

    The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.

  1. Endosomal maturation, Rab7 GTPase and phosphoinositides in African swine fever virus entry.

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    Miguel A Cuesta-Geijo

    Full Text Available Here we analyzed the dependence of African swine fever virus (ASFV infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs, late endosomes (LEs or lysosomes (LY. Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P which is involved in EE maturation and multivesicular body (MVB biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5P(2. Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection.

  2. The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia.

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    Xie, Bingxian; Chen, Qiaoli; Chen, Liang; Sheng, Yang; Wang, Hong Yu; Chen, Shuai

    2016-11-01

    The AS160 (Akt substrate of 160 kDa) is a Rab-GTPase activating protein (RabGAP) with several other functional domains, and its deficiency in mice or human patients lowers GLUT4 protein levels and causes severe insulin resistance. How its deficiency causes diminished GLUT4 proteins remains unknown. We found that the deletion of AS160 decreased GLUT4 levels in a cell/tissue-autonomous manner. Consequently, skeletal muscle-specific deletion of AS160 caused postprandial hyperglycemia and hyperinsulinemia. The pathogenic effects of AS160 deletion are mainly, if not exclusively, due to the loss of its RabGAP function since the RabGAP-inactive AS160(R917K) mutant mice phenocopied the AS160 knockout mice. The inactivation of RabGAP of AS160 promotes lysosomal degradation of GLUT4, and the inhibition of lysosome function could restore GLUT4 protein levels. Collectively, these findings demonstrate that the RabGAP activity of AS160 maintains GLUT4 protein levels in a cell/tissue-autonomous manner and its inactivation causes lysosomal degradation of GLUT4 and postprandial hyperglycemia and hyperinsulinemia.

  3. Rab And Arl GTPase Family Members Cooperate in the Localization of the Golgin GCC185

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    Burguete, A.Schweizer; Fenn, T.D.; Brunger, A.T.; Pfeffer, S.R.

    2009-05-27

    GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.

  4. Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating proteins.

    Science.gov (United States)

    Gabernet-Castello, Carme; O'Reilly, Amanda J; Dacks, Joel B; Field, Mark C

    2013-05-01

    Rab GTPases serve as major control elements in the coordination and definition of specific trafficking steps and intracellular compartments. Rab activity is modulated in part by GTPase-activating proteins (GAPs), and many RabGAPs share a Tre-2/Bub2/Cdc16 (TBC)-domain architecture, although the majority of TBC proteins are poorly characterized. We reconstruct the evolutionary history of the TBC family using ScrollSaw, a method for the phylogenetic analysis of pan-eukaryotic data sets, and find a sophisticated, ancient TBC complement of at least 10 members. Significantly, the TBC complement is nearly always smaller than the Rab cohort in any individual genome but also suggests Rab/TBC coevolution. Further, TBC-domain architecture has been well conserved in modern eukaryotes. The reconstruction also shows conservation of ancestral TBC subfamilies, continuing evolution of new TBCs, and frequent secondary losses. These patterns give additional insights into the sculpting of the endomembrane system.

  5. The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis

    Science.gov (United States)

    Onnis, A; Finetti, F; Patrussi, L; Gottardo, M; Cassioli, C; Spanò, S; Baldari, C T

    2015-01-01

    Accumulating evidence underscores the T-cell immune synapse (IS) as a site of intense vesicular trafficking, on which productive signaling and cell activation crucially depend. Although the T-cell antigen receptor (TCR) is known to exploit recycling to accumulate to the IS, the specific pathway that controls this process remains to be elucidated. Here we demonstrate that the small GTPase Rab29 is centrally implicated in TCR trafficking and IS assembly. Rab29 colocalized and interacted with Rab8, Rab11 and IFT20, a component of the intraflagellar transport system that regulates ciliogenesis and participates in TCR recycling in the non-ciliated T cell, as assessed by co-immunoprecipitation and immunofluorescence analysis. Rab29 depletion resulted in the inability of TCRs to undergo recycling to the IS, thereby compromizing IS assembly. Under these conditions, recycling TCRs accumulated in Rab11+ endosomes that failed to polarize to the IS due to defective Rab29-dependent recruitment of the dynein microtubule motor. Remarkably, Rab29 participates in a similar pathway in ciliated cells to promote primary cilium growth and ciliary localization of Smoothened. These results provide a function for Rab29 as a regulator of receptor recycling and identify this GTPase as a shared participant in IS and primary cilium assembly. PMID:26021297

  6. The RAB family GTPase Rab1A from Plasmodium falciparum defines a unique paralog shared by chromalveolates and rhizaria.

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    Elias, Marek; Patron, Nicola J; Keeling, Patrick J

    2009-01-01

    The RAB GTPases, which are involved in regulation of endomembrane trafficking, exhibit a complex but incompletely understood evolutionary history. We elucidated the evolution of the RAB1 subfamily ancestrally implicated in the endoplasmic reticulum-to-Golgi traffic. We found that RAB1 paralogs have been generated over the course of eukaryotic evolution, with some duplications coinciding with the advent of major eukaryotic lineages (e.g. Metazoa, haptophytes). We also identified a unique, derived RAB1 paralog, orthologous to the Plasmodium Rab1A, that occurs in stramenopiles, alveolates, and Rhizaria, represented by the chlorarachniophyte Gymnochlora stellata. This finding is consistent with the recently documented existence of a major eukaryotic clade ("SAR") comprising these three lineages. We further found a Rab1A-like protein in the cryptophyte Guillardia theta, but it exhibits unusual features among RAB proteins: absence of a C-terminal prenylation motif and an N-terminal extension with two MSP domains; and its phylogenetic relationships could not be established convincingly due to its divergent nature. Our results nevertheless point to a unique membrane trafficking pathway shared by at least some lineages of chromalveolates and Rhizaria, an insight that has implications towards interpreting the early evolution of eukaryotes and the endomembrane system.

  7. Assembly of the Biogenesis of Lysosome-related Organelles Complex-3 (BLOC-3) and Its Interaction with Rab9*

    Science.gov (United States)

    Kloer, Daniel P.; Rojas, Raul; Ivan, Viorica; Moriyama, Kengo; van Vlijmen, Thijs; Murthy, Namita; Ghirlando, Rodolfo; van der Sluijs, Peter; Hurley, James H.; Bonifacino, Juan S.

    2010-01-01

    The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs. PMID:20048159

  8. Interactions between Rab and Arf GTPases regulate endosomal phosphatidylinositol-4,5-bisphosphate during endocytic recycling.

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    Shi, Anbing; Grant, Barth D

    2013-01-01

    After endocytosis, a selective endocytic recycling process returns many endocytosed molecules back to the plasma membrane. The RAB-10/Rab10 GTPase is known to be a key recycling regulator for specific cargo molecules. New evidence, focused on C. elegans RAB-10 in polarized epithelia, points to a key role of RAB-10 in the regulation of endosomal phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) levels. In turn, PI(4,5)P2 levels strongly influence the recruitment of many peripheral membrane proteins, including those important for vesicle budding through their membrane bending activities. Part of the effect of RAB-10 on endosomal PI(4,5)P2 is through its newly identified effector CNT-1, a predicted GTPase activating protein (GAP) of the small GTPase ARF-6/Arf6. In mammals PI(4,5)P2 generating enzymes are known Arf6 effectors. In C. elegans we found that RAB-10, CNT-1 and ARF-6 are present on the same endosomes, that RAB-10 recruits CNT-1 to endosomes, and that loss of CNT-1 or RAB-10 leads to overaccumulation of endosomal PI(4,5)P2, presumably via hyperactivation of endosomal ARF-6. In turn this leads to over-recruitment of PI(4,5)P2-dependent membrane-bending proteins RME-1/Ehd and SDPN-1/Syndapin/PACSIN. Conversely, in arf-6 mutants, endosomal PI(4,5)P2 levels were reduced and endosomal recruitment of RME-1 and SDPN-1 failed. This work makes an unexpected link between distinct classes of small GTPases that control endocytic recycling, and provides insight into how this interaction affects endosome function at the level of lipid phosphorylation.

  9. Rab GTPases Regulate Vesicle Traffic%Rab蛋白调控胞内囊泡运输

    Institute of Scientific and Technical Information of China (English)

    冯婉娟; 徐子静; 孟令锋; 张蓉颖

    2012-01-01

    细胞内各个细胞器之间通过囊泡的膜转运是真核细胞存在的基本.Rab蛋白确保了转运蛋白被运输至正确的目的地.Rab蛋白是小GTP酶中的一大家族,它通过募集其效应物蛋白,其中包括接头蛋白,栓系因子,激酶,磷酸酶以及动力蛋白等,调控了细胞膜的选取,囊泡出芽,去包被,转运以及膜融合等过程.本文主要从Rab蛋白循环着手,依次论述了Rab蛋白在囊泡出芽,去包被,转运和膜融合等过程中起到的作用,从而使读者对Rab蛋白能有一个更加系统的了解.%Rab GTPases is a targe family of small GTPases. Rab GTPases regulate vesicle traffic which is fundamental to the existence of eukaryotic cells. Rab GTPases recruit their effect proteins including sorting adaptors, tethering factors, kinases, phosphatases and motors, control membrane identity and vesicle budding, uncoating, motility and fusion. This paper introduced the circuitry of Rab GTPases and their function in vesicle budding, uncoating, motility and fusion.

  10. The small GTPase Rab33A participates in regulation of amylase release from parotid acinar cells.

    Science.gov (United States)

    Imai, Akane; Tsujimura, Maiko; Yoshie, Sumio; Fukuda, Mitsunori

    2015-06-05

    Amylase is released from exocrine parotid acinar cells via typical exocytosis. Exocytosis of amylase-containing granules occurs through several steps, including formation, maturation, and transport of granules. These steps are thought to be regulated by members of the small GTPase Rab family. We previously demonstrated that Rab27 and its effectors mediate amylase release from parotid acinar cells, but the functional involvement of other Rab proteins in exocrine granule exocytosis remains largely unknown. Here, we studied isoproterenol (IPR)-induced amylase release from parotid acinar cells to investigate the possible involvement of Rab33A, which was recently suggested to regulate exocytosis in hippocampal neurons and PC12 cells. Rab33A was endogenously expressed in parotid acinar cells and present in secretory granules and the Golgi body. Functional ablation of Rab33A with anti-Rab33A antibody or a dominant-negative Rab33A-T50N mutant significantly reduced IPR-induced amylase release. Our results indicated that Rab33A is a novel component of IPR-stimulated amylase secretion from parotid acinar cells.

  11. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

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    María Milagros López de Armentia

    2016-03-01

    Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

  12. Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis

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    Amrutraj eZade

    2015-09-01

    Full Text Available Rab GTPases are the key regulators of intracellular membrane trafficking in eukaryotes. Many viruses and intracellular bacterial pathogens have evolved to hijack the host Rab GTPase functions, mainly through activators and effector proteins, for their benefit. Acanthamoeba polyphaga mimivirus (APMV is one of the largest viruses and belongs to the monophyletic clade of nucleo-cytoplasmic large DNA viruses (NCLDV. The inner membrane lining is integral to the APMV virion structure. APMV assembly involves extensive host membrane modifications, like vesicle budding and fusion, leading to the formation of a membrane sheet that is incorporated into the virion. Intriguingly, APMV and all group I members of the Mimiviridae family code for a putative Rab GTPase protein. APMV is the first reported virus to code for a Rab GTPase (encoded by R214 gene. Our thorough in silico analysis of the subfamily specific (SF region of Mimiviridae Rab GTPase sequences suggests that they are related to Rab5, a member of the group II Rab GTPases, of lower eukaryotes. Because of their high divergence from the existing three isoforms, A, B and C of the Rab5-family, we suggest that Mimiviridae Rabs constitute a new isoform, Rab5D. Phylogenetic analysis indicated probable horizontal acquisition from a lower eukaryotic ancestor followed by selection and divergence. Furthermore, interaction network analysis suggests that vps34 (a Class III P13K homolog, coded by APMV L615, Atg-8 and dynamin (host proteins are recruited by APMV Rab GTPase during capsid assembly. Based on these observations, we hypothesize that APMV Rab plays a role in the acquisition of inner membrane during virion assembly.

  13. The Rab2A GTPase Promotes Breast Cancer Stem Cells and Tumorigenesis via Erk Signaling Activation

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    Man-Li Luo

    2015-04-01

    Full Text Available Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs. Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

  14. Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

    Science.gov (United States)

    Temesvari, L; Rodriguez-Paris, J; Bush, J; Steck, T L; Cardelli, J

    1994-10-14

    Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

  15. Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases.

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    Elias, Marek; Brighouse, Andrew; Gabernet-Castello, Carme; Field, Mark C; Dacks, Joel B

    2012-05-15

    The presence of a nucleus and other membrane-bounded intracellular compartments is the defining feature of eukaryotic cells. Endosymbiosis accounts for the origins of mitochondria and plastids, but the evolutionary ancestry of the remaining cellular compartments is incompletely documented. Resolving the evolutionary history of organelle-identity encoding proteins within the endomembrane system is a necessity for unravelling the origins and diversification of the endogenously derived organelles. Comparative genomics reveals events after the last eukaryotic common ancestor (LECA), but resolution of events prior to LECA, and a full account of the intracellular compartments present in LECA, has proved elusive. We have devised and exploited a new phylogenetic strategy to reconstruct the history of the Rab GTPases, a key family of endomembrane-specificity proteins. Strikingly, we infer a remarkably sophisticated organellar composition for LECA, which we predict possessed as many as 23 Rab GTPases. This repertoire is significantly greater than that present in many modern organisms and unexpectedly indicates a major role for secondary loss in the evolutionary diversification of the endomembrane system. We have identified two Rab paralogues of unknown function but wide distribution, and thus presumably ancient nature; RabTitan and RTW. Furthermore, we show that many Rab paralogues emerged relatively suddenly during early metazoan evolution, which is in stark contrast to the lack of significant Rab family expansions at the onset of most other major eukaryotic groups. Finally, we reconstruct higher-order ancestral clades of Rabs primarily linked with endocytic and exocytic process, suggesting the presence of primordial Rabs associated with the establishment of those pathways and giving the deepest glimpse to date into pre-LECA history of the endomembrane system.

  16. The large conductance calcium-activated K(+) channel interacts with the small GTPase Rab11b.

    Science.gov (United States)

    Sokolowski, Sophia; Harvey, Margaret; Sakai, Yoshihisa; Jordan, Amy; Sokolowski, Bernd

    2012-09-21

    The transduction of sound by the receptor or hair cells of the cochlea leads to the activation of ion channels found in the basal and lateral regions of these cells. Thus, the processing of these transduced signals to the central nervous system is tied to the regulation of baso-lateral ion channels. The large conductance calcium-activated potassium or BK channel was revealed to interact with the small GTPase, Rab11b, which is one of many Rabs found in various endosomal pathways. Immunoelectron microscopy showed the colocalization of these two proteins in receptor cells and auditory neurons. Using Chinese hamster ovary cells as a heterologous expression system, Rab11b increased or decreased BK expression, depending on the overexpression or RNAi knockdown of Rab, respectively. Additional mutation analyses, using a yeast two-hybrid assay, suggested that this GTPase moderately interacts within a region of BK exclusive of the N- or C-terminal tails. These data suggest that this small GTPase regulates BK in a slow recycling process through the endocytic compartment and to the plasmalemma.

  17. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

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    Katherine Maringer

    2014-08-01

    Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  18. Conservation and function of Rab small GTPases in Entamoeba: annotation of E. invadens Rab and its use for the understanding of Entamoeba biology.

    Science.gov (United States)

    Nakada-Tsukui, Kumiko; Saito-Nakano, Yumiko; Husain, Afzal; Nozaki, Tomoyoshi

    2010-11-01

    Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.

  19. Prenylated Rab acceptor protein is a receptor for prenylated small GTPases.

    Science.gov (United States)

    Figueroa, C; Taylor, J; Vojtek, A B

    2001-07-27

    Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.

  20. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes.

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    Katrin Kremer

    2013-03-01

    Full Text Available The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

  1. Revealing localization and regulation of GTPase PmRab7 in lymphoid cells ofPenaeus monodon after WSSV infection

    Institute of Scientific and Technical Information of China (English)

    Amrendra Kumar; Vaishnavi Ramasubbu; Kiran D Rasal; Saravanankumar Ayyappan

    2016-01-01

    Objective:To identify white spot syndrome virus (WSSV) entry into the host-cells of the cultured shrimpPenaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain) which is playing a vital role in theWSSV internalization. Methods:In this study, we have cloned PmRab7 and expressed inEscherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number ofWSSV has been studied by using real-timePCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colonyPCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B) 651 bp gene transformed successfully inRosetta and after 6 h of induction expressed rPmRab7 was onSDS page, furthermore soluble fraction of rPmRab7 (26 kDa) was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number ofWSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other handWSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.

  2. Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

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    Amrendra Kumar

    2016-10-01

    Full Text Available Objective: To identify white spot syndrome virus (WSSV entry into the host-cells of the cultured shrimp Penaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain which is playing a vital role in the WSSV internalization. Methods: In this study, we have cloned PmRab7 and expressed in Escherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B 651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page, furthermore soluble fraction of rPmRab7 (26 kDa was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other hand WSSV may follow the same route of entry. WSSV internalization has directly linked with regulation of PmRab7.

  3. Phylogeny and evolution of Rab7 and Rab9 proteins

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    Wyroba Elżbieta

    2009-05-01

    Full Text Available Abstract Background An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network. Results Although Rab7 and Rab9 proteins are quite small and show heterogeneous rates of substitution in different lineages, we found a phylogenetic signal and inferred evolutionary relationships between them. Rab7 proteins evolved before radiation of main eukaryotic supergroups while Rab9 GTPases diverged from Rab7 before split of choanoflagellates and metazoans. Additional duplication of Rab9 and Rab7 proteins resulting in several isoforms occurred in the early evolution of vertebrates and next in teleost fishes and tetrapods. Three Rab7 lineages emerged before divergence of monocots and eudicots and subsequent duplications of Rab7 genes occurred in particular angiosperm clades. Interestingly, several Rab7 copies were identified in some representatives of excavates, ciliates and amoebozoans. The presence of many Rab copies is correlated with significant differences in their expression level. The diversification of analysed Rab subfamilies is also manifested by non-conserved sequences and structural features, many of which are involved in the interaction with regulators and effectors. Individual sites discriminating different subgroups of Rab7 and Rab9 GTPases have been identified. Conclusion Phylogenetic reconstructions of Rab7 and Rab9 proteins were performed by a variety of methods. These Rab GTPases show diversification

  4. Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase from Entamoeba histolytica.

    Science.gov (United States)

    Kumar Srivastava, Vijay; Chandra, Mintu; Datta, Sunando

    2014-07-01

    Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasite Entamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes. Entamoeba histolytica RabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8-16% sequence identity with other GTPases. Recent studies suggested that EhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action of EhRabX3. To provide structural insights into the inclusive action of this unique GTPase, EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single native EhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space group C2, with unit-cell parameters a=198.6, b=119.3, c=89.2 Å, β=103.1°. Preliminary analysis of the data using the Matthews Probability Calculator suggested the presence of four to six molecules in the asymmetric unit.

  5. The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.

    Directory of Open Access Journals (Sweden)

    Matthias Stein

    Full Text Available BACKGROUND: Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics. CONCLUSIONS/SIGNIFICANCE: We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

  6. Role of Rab GTPases and their interacting proteins in mediating metabolic signalling and regulation.

    Science.gov (United States)

    Chua, Christelle En Lin; Tang, Bor Luen

    2015-06-01

    The vesicular transport pathways, which shuttle materials to and from the cell surface and within the cell, and the metabolic (growth factor and nutrient) signalling pathways, which integrate a variety of extracellular and intracellular signals to mediate growth, proliferation or survival, are both important for cellular physiology. There is evidence to suggest that the transport and metabolic signalling pathways intersect-vesicular transport can affect the regulation of metabolic signals and vice versa. The Rab family GTPases regulate the specificity of vesicular transport steps in the cell. Together with their interacting proteins, Rabs would likely constitute the points of intersection between vesicular transport and metabolic signalling pathways. Examples of these points would include growth factor signalling, glucose and lipid metabolism, as well as autophagy. Many of these processes involve mechanistic/mammalian target of rapamycin (mTOR) complex 1 (mTORC1) in downstream cascades, or are regulated by TORC signalling. A general functionality of the vesicular transport processes controlled by the Rabs is also important for spatial and temporal regulation of the transmission of metabolic signals between the cell surface and the nucleus. In other cases, specific Rabs and their interacting proteins are known to function in recruiting metabolism-related proteins to target membranes, or may compete with other factors in the TORC signalling pathway as a means of metabolic regulation. We review and discuss herein examples of how Rabs and their interacting proteins can mediate metabolic signalling and regulation in cells.

  7. Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

    Science.gov (United States)

    Inada, Noriko; Betsuyaku, Shigeyuki; Shimada, Takashi L; Ebine, Kazuo; Ito, Emi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Takano, Yoshitaka; Fukuda, Hiroo; Nakano, Akihiko; Ueda, Takashi

    2016-09-01

    RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

  8. Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection.

    Science.gov (United States)

    Hilbi, Hubert; Rothmeier, Eva; Hoffmann, Christine; Harrison, Christopher F

    2014-01-01

    Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires' disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells.

  9. The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures

    Science.gov (United States)

    Patrussi, Laura; Baldari, Cosima T.

    2016-01-01

    ABSTRACT Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Among them, Rab8 is an essential participant in primary cilium formation. In a report recently published in the Journal of Cell Science, Finetti and colleagues identify Rab8 as a novel player in vesicular traffic in the non-ciliated T lymphocytes, which contributes to the assembly of the specialized signaling platform known as the immune synapse. By interacting with the v-SNARE VAMP-3, Rab8 is indeed responsible for the final docking/fusion step in T cell receptor (TCR) recycling to the immune synapse. A second important take-home message which comes to light from this work is that VAMP-3 also interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened at the plasma membrane. Hence the data presented in this report, in addition to identifying Rab8 as a novel player in vesicular traffic to the immune synapse, reveal how both ciliated and non-ciliated cells take advantage of a conserved pathway to build highly specific cellular structures. PMID:26587735

  10. Dynamic Changes in the Intracellular Association of Selected Rab Small GTPases with MHC Class II and DM during Dendritic Cell Maturation.

    Science.gov (United States)

    Pérez-Montesinos, Gibrán; López-Ortega, Orestes; Piedra-Reyes, Jessica; Bonifaz, Laura C; Moreno, José

    2017-01-01

    Antigen processing for presentation by major histocompatibility complex class II (MHCII) molecules requires the latter to travel through the endocytic pathway together with its chaperons: the invariant chain (Ii) and DM. Nevertheless, the nature of the compartments where MHCII molecules travel to acquire peptides lacks definition regarding molecules involved in intracellular vesicular trafficking, such as Rab small GTPases. We aimed to define which Rab proteins are present during the intracellular transport of MHCII, DM, and Ii through the endocytic pathway on their route to the cell surface during dendritic cell (DC) maturation. We examined, by means of three-color confocal microscopy, the association of MHCII, DM, and Ii with Rab5, Rab7, Rab9, and Rab11 during the maturation of bone marrow-derived or spleen DC in response to LPS as an inflammatory stimulus. Prior to the stage of immature DC, MHCII migrated from diffuse small cytoplasmic vesicles, predominantly Rab5+Rab7- and Rab5+Rab7+ into a pericentriolar Rab5+Rab7+Rab9+ cluster, with Rab11+ areas. As DC reached the mature phenotype, MHCII left the pericentriolar endocytic compartments toward the cell surface in Rab11+ and Rab9+Rab11+ vesicles. The invariant chain and MHCII transport pathways were not identical. DM and MHCII appeared to arrive to pericentriolar endocytic compartments of immature DC through partially different routes. The association of MHCII molecules with distinct Rab GTPases during DC maturation suggests that after leaving the biosynthetic pathway, MHCII sequentially traffic from typical early endosomes to multivesicular late endosomes to finally arrive at the cell surface in Rab11+ recycling-type endosomes. In immature DCs, DM encounters transiently MHCII in the Rab5+Rab7+Rab9+ compartments, to remain there in mature DC.

  11. Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis.

    Science.gov (United States)

    Su, Hua; Liu, Bingchen; Fröhlich, Otto; Ma, Heping; Sands, Jeff M; Chen, Guangping

    2013-10-01

    The UT-A1 urea transporter plays an important role in the urinary concentration mechanism. However, the molecular mechanisms regarding UT-A1 trafficking, endocytosis, and degradation are still unclear. In this study, we identified the small GTPase Rab14 as a binding partner to the C terminus of UT-A1 in a yeast 2-hybrid assay. Interestingly, UT-A1 binding is preferential for the GDP-bound inactive form of Rab14. Coinjection of Rab14 in Xenopus oocytes results in a decrease of UT-A1 urea transport activity, suggesting that Rab14 acts as a negative regulator of UT-A1. We subsequently found that Rab14 reduces the cell membrane expression of UT-A1, as evidenced by cell surface biotinylation. This effect is blocked by chlorpromazine, an inhibitor of the clathrin-mediated endocytic pathway, but not by filipin, an inhibitor of the caveolin-mediated endocytic pathway. In kidney, Rab14 is mainly expressed in IMCD epithelial cells with a pattern identical to UT-A1 expression. Consistent with its role in participating in clathrin-mediated endocytosis, Rab14 localizes in nonlipid raft microdomains and codistributes with Rab5, a marker of the clathrin-mediated endocytic pathway. Taken together, our study suggests that Rab14, as a novel UT-A1 partner, may have an important regulatory function for UT-A1 urea transport activity in the kidney inner medulla.

  12. [Role of the adaptins, dynamin like GTP-ases and Rab proteins in metabolic disorders and various infections].

    Science.gov (United States)

    Kierczak, Marcin; Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2003-01-01

    Numerous metabolic and genetic diseases are due to mutations in adaptins, dynamin-like GTP-ases or disorders in trafficking machinery mediated by Rab proteins. A great number of pathogenes including viruses (HIV, SIV), bacteria and protozoa use various elements of endocytic/trafficking machinery to get into the host cells and to make their infection successful. Their different strategies are discussed.

  13. Structurally Distinct Bacterial TBC-like GAPs Link Arf GTPase to Rab1 Inactivation to Counteract Host Defenses

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    Dong, Na; Zhu, Yongqun; Lu, Qiuhe; Hu, Liyan; Zheng, Yuqing; Shao, Feng (NIBS-China); (Zhejiang)

    2012-10-10

    Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.

  14. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  15. RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

    Science.gov (United States)

    Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

    2016-01-15

    Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.

  16. The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

    DEFF Research Database (Denmark)

    Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

    2012-01-01

    The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL...... be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice....... In conclusion, TBC1D1 plays a role in regulation of glucose metabolism in skeletal muscle. Moreover, functional TBC1D1 is required for AICAR- or contraction-induced metabolic responses, implicating a role in energy-sensing signals....

  17. Nutritional, hormonal, and depot-dependent regulation of the expression of the small GTPase Rab18 in rodent adipose tissue.

    Science.gov (United States)

    Pulido, Marina R; Rabanal-Ruiz, Yoana; Almabouada, Farid; Díaz-Ruiz, Alberto; Burrell, María A; Vázquez, María J; Castaño, Justo P; Kineman, Rhonda D; Luque, Raúl M; Diéguez, Carlos; Vázquez-Martínez, Rafael; Malagón, María M

    2013-02-01

    There is increasing evidence that proteins associated with lipid droplets (LDs) play a key role in the coordination of lipid storage and mobilization in adipocytes. The small GTPase, RAB18, has been recently identified as a novel component of the protein coat of LDs and proposed to play a role in both β-adrenergic stimulation of lipolysis and insulin-induced lipogenesis in 3T3-L1 adipocytes. In order to better understand the role of Rab18 in the regulation of lipid metabolism in adipocytes, we evaluated the effects of age, fat location, metabolic status, and hormonal milieu on Rab18 expression in rodent white adipose tissue (WAT). Rab18 mRNA was undetectable at postnatal day 15 (P15), but reached adult levels by P45, in both male and female rats. In adult rats, Rab18 immunolocalized around LDs, as well as within the cytoplasm of mature adipocytes. A weak Rab18 signal was also detected in the stromal-vascular fraction of WAT. In mice, fasting significantly increased, though with a distinct time-course pattern, Rab18 mRNA and protein levels in visceral and subcutaneous WAT. The expression of Rab18 was also increased in visceral and subcutaneous WAT of obese mice (diet-induced, ob/ob, and New Zealand obese mice) compared with lean controls. Rab18 expression in rats was unaltered by castration, adrenalectomy, or GH deficiency but was increased by hypophysectomy, as well as hypothyroidism. When viewed together, our results suggest the participation of Rab18 in the regulation of lipid processing in adipose tissue under both normal and pathological conditions.

  18. Endoplasmic reticulum‐resident Rab8A GTPase is involved in phagocytosis in the protozoan parasite Entamoeba histolytica

    Science.gov (United States)

    Hanadate, Yuki; Saito‐Nakano, Yumiko; Nakada‐Tsukui, Kumiko

    2016-01-01

    Summary Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans‐Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down‐regulation of EhRab8A by small antisense RNA‐mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS‐PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene‐silenced strain. Further, overexpression of wild‐type EhRab8A augmented phagocytosis, whereas expression of the dominant‐negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER‐resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis. PMID:26807810

  19. Small GTPase Rab21 mediates fibronectin induced actin reorganization in Entamoeba histolytica: implications in pathogen invasion.

    Directory of Open Access Journals (Sweden)

    Merlyn Emmanuel

    2015-03-01

    Full Text Available The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as 'invadosomes', promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots.

  20. Small GTPase Rab21 Mediates Fibronectin Induced Actin Reorganization in Entamoeba histolytica: Implications in Pathogen Invasion

    Science.gov (United States)

    Emmanuel, Merlyn; Nakano, Yumiko Saito; Nozaki, Tomoyoshi; Datta, Sunando

    2015-01-01

    The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as ‘invadosomes’, promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots. PMID:25730114

  1. Exogenous gene transfer of Rab(3)8 small GTPase ameliorates aberrant lung surfactant homeostasis in Ruby rats.

    Science.gov (United States)

    Osanai, Kazuhiro; Nakase, Keisuke; Sakuma, Takashi; Nishiki, Kazuaki; Nojiri, Masafumi; Kato, Ryo; Saito, Masatoshi; Fujimoto, Yuki; Mizuno, Shiro; Toga, Hirohisa

    2017-04-24

    Rab(3)8 small GTPase regulates intracellular transport in melanocytes and alveolar type II epithelial cells. Ruby rats carrying Rab(3)8 and other gene mutations exhibit oculocutaneous albinism, bleeding diathesis, and hence, are a rat model of human Hermansky-Pudlak syndrome (HPS). We previously showed that Long Evans Cinnamon (LEC) rats, one strain of the Ruby rats, developed aberrant lung surfactant homeostasis with remarkably enlarged lamellar bodies in alveolar type II cells. A replication-deficient recombinant adenovirus expressing rat Rab(3)8 (Ad-Rab(3)8) was constructed. Alveolar type II cells were isolated from the LEC rats and tested for lung surfactant phosphatidylcholine secretion. The rats were also examined whether exogenous expression of Ad- Rab(3)8 could rescue the altered lung surfactant homeostasis in the lungs. Isolated type II cells infected with Ad-Rab(3)8 exhibited improved secretion patterns of [(3)H]phosphatidylcholine, i.e. increased basal hyposecretion and decreased agonist-induced hypersecretion. Endobronchial administration of Ad-Rab(3)8 improved the morphology of type II cells and lamellar bodies, reducing their sizes close to those of wild-type rats. The increased amounts of phosphatidylcholine and surfactant protein B in the lamellar body fractions were decreased in the Ad-Rab(3)8 infected lungs. These results provide strong evidence that the aberrant lung surfactant homeostasis in the LEC rats is caused by Rab(3)8 deficit, and suggest that endobronchial delivery of the responsive transgene could be an effective method to ameliorate the abnormal lung phenotype in the animal model of HPS.

  2. Interaction between small GTPase Rab7 and PI3KC3 links autophagy and endocytosis: A new Rab7 effector protein sheds light on membrane trafficking pathways.

    Science.gov (United States)

    Lin, Mary Grace; Zhong, Qing

    2011-03-01

    Endocytosis and autophagy are both membrane trafficking pathways vital for cell survival. Endocytosis, the primary means by which cells internalize material such as cell-surface receptors and their protein ligands, is essential for proper cell growth and communication. Autophagy is a catabolic process that degrades cargo ranging from organelles to protein aggregates to bacteria, and it is important for maintaining cellular homeostasis. Defects in both endosome and autophagosome maturation lead to an array of human diseases, including cancer; however, the molecular mechanisms underlying endosome and autophagosome maturation are not well characterized. In the case of endocytosis, small GTPases, key players in membrane organization, are required for endosome maturation. Specifically, activation of the small GTPase Rab7 is required for the initiation of the early-to-late endosome transition, although how this is regulated is largely unknown. Now recent findings from our laboratory show that Rubicon, a component of the PI3KC3 complex, inhibits endosome maturation by preventing activation of Rab7. Not only do our results clarify the molecular link between PI3KC3 and Rab7 function in endosome maturation, they lead us to propose new models for PI3KC3 involvement in membrane trafficking, particularly at the convergence between the endosome and autophagosome pathways.

  3. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily.

    Science.gov (United States)

    Pereira-Leal, J B; Seabra, M C

    2000-08-25

    The Rab/Ypt/Sec4 family forms the largest branch of the Ras superfamily of GTPases, acting as essential regulators of vesicular transport pathways. We used the large amount of information in the databases to analyse the mammalian Rab family. We defined Rab-conserved sequences that we designate Rab family (RabF) motifs using the conserved PM and G motifs as "landmarks". The Rab-specific regions were used to identify new Rab proteins in the databases and suggest rules for nomenclature. Surprisingly, we find that RabF regions cluster in and around switch I and switch II regions, i.e. the regions that change conformation upon GDP or GTP binding. This finding suggests that specificity of Rab-effector interaction cannot be conferred solely through the switch regions as is usually inferred. Instead, we propose a model whereby an effector binds to RabF (switch) regions to discriminate between nucleotide-bound states and simultaneously to other regions that confer specificity to the interaction, possibly Rab subfamily (RabSF) specific regions that we also define here. We discuss structural and functional data that support this model and its general applicability to the Ras superfamily of proteins.

  4. Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco.

    Science.gov (United States)

    George, Suja; Parida, Ajay

    2011-03-01

    Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.

  5. Insights into the GTP/GDP cycle of RabX3, a novel GTPase from Entamoeba histolytica with tandem G-domains.

    Science.gov (United States)

    Chandra, Mintu; Mukherjee, Madhumita; Srivastava, Vijay Kumar; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2014-02-25

    Members of the small GTPase Ras superfamily regulate a host of systems through their ability to catalyze the GTP/GDP cycle. All family members reported thus far possess a single GTPase domain with a P-loop containing a nucleoside triphosphate hydrolase fold. Here for the first time we report a novel member from Entamoeba histolytica, EhRabX3, which harbors two GTPase domains in tandem and exhibits unique biochemical properties. A combination of biochemical and microcalorimetric studies revealed that EhRabX3 binds to a single guanine nucleotide through its N-terminal domain. Unlike most of the members of the Ras superfamily, the dissociation of the nucleotide from EhRabX3 is independent of Mg(2+), perhaps indicating a novel mechanism of nucleotide exchange by this protein. We found that EhRabX3 is extremely sluggish in hydrolyzing GTP, and that could be attributed to its atypical nucleotide binding pocket. It harbors substitutions at two positions that confer oncogenicity to Ras because of impaired GTP hydrolysis. Engineering these residues into the conserved counterparts enhanced their GTPase activity by at least 20-fold. In contrast to most of the members of the Ras superfamily, EhRabX3 lacks the prenylation motif. Using indirect immunofluorescence and biochemical fractionation, we demonstrated that the protein is distributed all over the cytosol in amoebic trophozoites. Collectively, this unique ancient GTPase exhibits a striking evolutionary divergence from the other members of the superfamily.

  6. Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells.

    Science.gov (United States)

    Wilson, A L; Sheridan, K M; Erdman, R A; Maltese, W A

    1996-09-15

    Previous studies have reached differing conclusions as to whether or not guanine-nucleotide-dependent conformational changes affect the ability of Rab proteins to undergo post-translational modification by Rab:geranylgeranyltransferase (Rab-GGTase). We now show that the ability of a Rab1B mutant [Q67L (Gln-67-->Leu)] with reduced intrinsic GTPase activity to undergo geranylgeranylation in cell-free assays depends on the guanine nucleotide composition of the system. When GTP is the predominant nucleotide in the assay, Rab1BQ67L is a poor substrate. However, when GDP is present and GTP is omitted, prenylation of the Q67L mutant is comparable with that of the wild-type (WT) protein. These studies, coupled with the poor prenylation of Rab1BWT in the presence of the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, support the notion that Rab-GGTase prefers substrates in the GDP conformation. When the abilities of Rab1BQ67L and Rab1BWT to undergo prenylation were compared by metabolic labelling of transiently expressed proteins in cultured human 293 cells, we did not observe a decline in prenylation of the mutant protein as predicted on the basis of the cell-free assays. Moreover, the Q67L mutant was comparable with the wild-type Rab1B in its ability to associate with co-expressed Rab GDP dissociation inhibitors in 293 cells. These findings raise the possibility that unidentified proteins present in intact cells may compensate for the reduced intrinsic GTPase activity of the Q67L mutant, allowing a significant proportion of the nascent Rab1BQ67L to assume a GDP conformation. The differential prenylation of Rab1BQ67L in cell-free systems versus intact cells underscores the importance of evaluating the post-translational modification of specific Rab mutants in vivo, where poorly characterized regulatory proteins may have a significant effect on GTPase activity or nucleotide exchange rates.

  7. Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic.

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    Poteryaev, Dmitry; Fares, Hanna; Bowerman, Bruce; Spang, Anne

    2007-01-24

    The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND-1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.

  8. The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse.

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    Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J; Baldari, Cosima T

    2015-07-15

    IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11(+) endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR(+) endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis.

  9. Rab蛋白在自噬形成过程中的调节作用及机制%Role of Rab GTPases in the Regulation of Autophagy and Its Mecha-nism

    Institute of Scientific and Technical Information of China (English)

    黄敏; 李承新

    2014-01-01

    自噬是指双层膜包裹细胞内细胞器、蛋白质等物质形成自噬体,并与内体形成自噬内体,最终与溶酶体融合形成自噬溶酶体,降解其所包裹内容物,以实现细胞稳态的过程。多种激酶、磷酸酶、鸟苷三磷酸酶( GTP酶)参与了自噬的形成过程。近年研究发现Rab GTPases家族中的许多分子在自噬形成过程中发挥中重要作用。%Autophagy is an evolutionarily conserved process in which cytoplasmic contents ( in-cluding organelles and proteins ) are engulfed by a double-membrane autophagosome , which eventu-ally has its content degraded at lysosome.The main role of autophagy is to maintain intracellular ho-meostasis.Various kinases , phosphatases , and guanosine triphosphatases ( GTPases ) take part in the autophagy.Recently , it was found that many molecules of Rab GTPase family play a pivotal role in the process of autophagy.

  10. Crystal Structure Analysis of Wild Type and Fast Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba histolytica.

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    Srivastava, Vijay Kumar; Chandra, Mintu; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2016-01-16

    The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess "phosphate binding loop containing nucleoside triphosphate hydrolases fold", only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis.

  11. Recessive Inactivating Mutations in TBCK, Encoding a Rab GTPase-Activating Protein, Cause Severe Infantile Syndromic Encephalopathy

    Science.gov (United States)

    Chong, Jessica X.; Caputo, Viviana; Phelps, Ian G.; Stella, Lorenzo; Worgan, Lisa; Dempsey, Jennifer C.; Nguyen, Alina; Leuzzi, Vincenzo; Webster, Richard; Pizzuti, Antonio; Marvin, Colby T.; Ishak, Gisele E.; Ardern-Holmes, Simone; Richmond, Zara; Bamshad, Michael J.; Ortiz-Gonzalez, Xilma R.; Tartaglia, Marco; Chopra, Maya; Doherty, Dan

    2016-01-01

    Infantile encephalopathies are a group of clinically and biologically heterogeneous disorders for which the genetic basis remains largely unknown. Here, we report a syndromic neonatal encephalopathy characterized by profound developmental disability, severe hypotonia, seizures, diminished respiratory drive requiring mechanical ventilation, brain atrophy, dysgenesis of the corpus callosum, cerebellar vermis hypoplasia, and facial dysmorphism. Biallelic inactivating mutations in TBCK (TBC1-domain-containing kinase) were independently identified by whole-exome sequencing as the cause of this condition in four unrelated families. Matching these families was facilitated by the sharing of phenotypic profiles and WES data in a recently released web-based tool (Geno2MP) that links phenotypic information to rare variants in families with Mendelian traits. TBCK is a putative GTPase-activating protein (GAP) for small GTPases of the Rab family and has been shown to control cell growth and proliferation, actin-cytoskeleton dynamics, and mTOR signaling. Two of the three mutations (c.376C>T [p.Arg126∗] and c.1363A>T [p.Lys455∗]) are predicted to truncate the protein, and loss of the major TBCK isoform was confirmed in primary fibroblasts from one affected individual. The third mutation, c.1532G>A (p.Arg511His), alters a conserved residue within the TBC1 domain. Structural analysis implicated Arg511 as a required residue for Rab-GAP function, and in silico homology modeling predicted impaired GAP function in the corresponding mutant. These results suggest that loss of Rab-GAP activity is the underlying mechanism of disease. In contrast to other disorders caused by dysregulated mTOR signaling associated with focal or global brain overgrowth, impaired TBCK function results in progressive loss of brain volume. PMID:27040692

  12. Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum lindemuthianum.

    Science.gov (United States)

    Siriputthaiwan, Piyawan; Jauneau, Alain; Herbert, Corentin; Garcin, Daphné; Dumas, Bernard

    2005-01-15

    In eukaryotic cells, Rab/GTPases are major regulators of vesicular trafficking and are involved in essential processes including exocytosis, endocytosis and cellular differentiation. To investigate the role of these proteins in fungal pathogenicity, a dominant-negative mutant allele of CLPT1, a Rab/GTPase of the bean pathogen Colletotrichum lindemuthianum, was expressed in transgenic strains. This mutated gene encodes the amino-acid substitution N123I analogous to the N133I substitution in a known trans-dominant inhibitor of the Sec4 Rab/GTPase from Saccharomyces cerevisiae. A pectinase gene promoter was used to drive the CLPT1(N123I) allele in C. lindemuthianum, allowing the expression of the foreign gene on pectin medium and during pathogenesis, but not on glucose. The same strategy was used to overexpress the wild-type CLPT1 allele. During growth on pectin medium, production of extracellular pectinases was strongly impaired only in CLPT1(N123I)-expressing strains. Cytological analysis revealed that CLPT1(N123I) strains accumulated intracellular aggregates only on pectin, resulting from the fusion of vesicles containing polysaccharides or glycoproteins. Moreover, these strains showed a severe reduction of pathogenesis and were unable to penetrate the host cells. These results indicated that the Rab/GTPase CLPT1 is essential for fungal pathogenesis by regulating the intracellular transport of secretory vesicles involved in the delivery of proteins to the extracellular medium and differentiation of infectious structures.

  13. Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene

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    Huxley Clare

    2001-02-01

    Full Text Available Abstract Background Rab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b. Results The human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb. The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a. Conclusions Our results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18.

  14. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus

    Science.gov (United States)

    Xu, Kai; Nagy, Peter D.

    2016-01-01

    Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment. PMID:27760128

  15. Tropheryma whipplei, the agent of Whipple's disease, affects the early to late phagosome transition and survives in a Rab5- and Rab7-positive compartment.

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    Giovanna Mottola

    Full Text Available Tropheryma whipplei, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where Tropheryma whipplei resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the Tropheryma whipplei phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that Tropheryma whipplei blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the Tropheryma whipplei genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH homologous with the GAPDH of Listeria monocytogenes, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, Tropheryma whipplei is the first pathogen described to induce a "chimeric" phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.

  16. Small GTPase Rab4b participates in the gene transcription of 20-hydroxyecdysone and insulin pathways to regulate glycogen level and metamorphosis.

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    Hou, Li; Cai, Mei-Juan; Liu, Wen; Song, Qian; Zhao, Xiao-Fan

    2012-11-01

    The insulin and 20-hydroxyecdysone (20E) pathways coordinately regulate insect growth and metamorphosis. However, the molecular mechanism of the interaction of these two pathways in regulating insect development is not well understood. In the present study, we found that a small GTPase Rab4b from a lepidopteran insect Helicoverpa armigera participates in gene transcription in the two pathways. The results show that RNA interference of Rab4b in larvae results in a decrease in glycogen levels, small pupae, abnormal metamorphic transition, or larval death. The molecular mechanisms are demonstrated that knockdown of Rab4b in the larvae suppresses the transcription of glycogen synthase (GS), as well as the metamorphic-initiating factor (Br) and hormone receptor 3 (HR3), but increases the transcription of Forkhead box class O (FOXO). Further studies in the cell line confirm that Rab4b is necessary for gene transcription in the insulin and 20E pathways. Rab4b locates in the cytoplasm and takes part in regulation on FOXO cytoplasmic location by insulin induction, but travels toward the cell membrane upon 20E induction without affecting the FOXO location. The transcription of Rab4b could be upregulated by insulin injection or glucose feeding to the larvae, but not by 20E or juvenile hormone analogy methoprene. Our data suggest that Rab4b takes part in metamorphosis by regulating gene transcription and glycogen level in the insulin and 20E pathways.

  17. C9orf72’s interaction with Rab GTPases - modulation of membrane traffic and autophagy

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    Bor Luen Tang

    2016-10-01

    Full Text Available Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 (C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS and Frontotemporal Dementia (FTD. While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1 autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings

  18. Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

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    Youhei Egami

    Full Text Available Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

  19. GTPase of the Immune-Associated Nucleotide Protein 5 Regulates the Lysosomal Calcium Compartment in T Lymphocytes

    Science.gov (United States)

    Serrano, Daniel; Ghobadi, Farnaz; Boulay, Guylain; Ilangumaran, Subburaj; Lavoie, Christine; Ramanathan, Sheela

    2017-01-01

    T lymphocytes from Gimap5lyp/lyp rats carrying a recessive mutation in the GTPase of immune-associated protein 5 (Gimap5) gene undergo spontaneous apoptosis. Molecular mechanisms underlying this survival defect are not yet clear. We have shown that Gimap5lyp/lyp T lymphocytes display reduced calcium influx following T cell antigen receptor (TCR) stimulation that was associated with impaired buffering of calcium by mitochondria. Here, we investigated the subcellular localization of GIMAP5 and its influence on Ca2+ response in HEK293T cells and T lymphocytes. The more abundantly expressed GIMAP5v2 localizes to the lysosome and certain endosomal vesicles. Gimap5lyp/lyp T lymphocytes showed increased accumulation of calcium in the lysosomes as evidenced by Gly-Phe β-naphthylamide (GPN) triggered Ca2+ release. As a corollary, GPN-induced Ca2+ flux was decreased in HEK293T cells expressing GIMAP5v2. Strikingly, TCR stimulation of rat, mouse, and human T lymphocytes increased lysosomal calcium content. Overall, our findings show that lysosomes modulate cellular Ca2+ response during T cell activation and that GIMAP5 regulates the lysosomal Ca2+ compartment in T lymphocytes. PMID:28223986

  20. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  1. Cloning of two genes encoding Rab7 in Paramecium.

    Science.gov (United States)

    Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2006-01-01

    Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.

  2. Structural determinants allowing endolysosomal sorting and degradation of endosomal GTPases.

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    Valero, Ruth A; Oeste, Clara L; Stamatakis, Konstantinos; Ramos, Irene; Herrera, Mónica; Boya, Patricia; Pérez-Sala, Dolores

    2010-09-01

    Rapid control of protein degradation is usually achieved through the ubiquitin-proteasome pathway. We recently found that the short-lived GTPase RhoB is degraded in lysosomes. Moreover, the fusion of the RhoB C-terminal sequence CINCCKVL, containing the isoprenylation and palmitoylation sites, to other proteins directs their sorting into multivesicular bodies (MVBs) and rapid lysosomal degradation. Here, we show that this process is highly specific for RhoB. Alteration of late endosome lipid dynamics produced the accumulation of RhoB, but not of other endosomal GTPases, including Rab5, Rab7, Rab9 or Rab11, into enlarged MVB. Other isoprenylated and bipalmitoylated GTPases, such as H-Ras, Rap2A, Rap2B and TC10, were not accumulated into MVB and were stable. Remarkably, although TC10, which is highly homologous to RhoB, was stable, a sequence derived from its C-terminus (CINCCLIT) elicited MVB sorting and degradation of a green fluorescent protein (GFP)-chimeric protein. This led us to identify a cluster of basic amino acids (KKH) in the TC10 hypervariable region, constituting a secondary signal potentially involved in electrostatic interactions with membrane lipids. Mutation of this cluster allowed TC10 MVB sorting and degradation, whereas inserting it into RhoB hypervariable region rescued this protein from its lysosomal degradation pathway. These findings define a highly specific structural module for entering the MVB pathway and rapid lysosomal degradation.

  3. The glycine brace: a component of Rab, Rho, and Ran GTPases associated with hinge regions of guanine- and phosphate-binding loops

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    Neuwald Andrew F

    2009-03-01

    Full Text Available Abstract Background Ras-like GTPases function as on-off switches in intracellular signalling pathways and include the Rab, Rho/Rac, Ran, Ras, Arf, Sar and Gα families. How these families have evolutionarily diverged from each other at the sequence level provides clues to underlying mechanisms associated with their functional specialization. Results Bayesian analysis of divergent patterns within a multiple alignment of Ras-like GTPase sequences identifies a structural component, termed here the glycine brace, as the feature that most distinguishes Rab, Rho/Rac, Ran and (to some degree Ras family GTPases from other Ras-like GTPases. The glycine brace consists of four residues: An aromatic residue that forms a stabilizing CH-π interaction with a conserved glycine at the start of the guanine-binding loop; a second aromatic residue, which is nearly always a tryptophan, that likewise forms stabilizing CH-π and NH-π interactions with a glycine at the start of the phosphate-binding P-loop; and two other residues (typically an aspartate and a serine or threonine that, together with a conserved buried water molecule, form a network of interactions connecting the two aromatic residues. Conclusion It is proposed that the two glycine residues function as hinges and that the glycine brace influences guanine nucleotide binding and release by interacting with these hinges.

  4. Identification and characterization of Rab7 from orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Fu, Jing; Huang, Youhua; Cai, Jia; Wei, Shina; Ouyang, Zhengliang; Ye, Fuzhou; Huang, Xiaohong; Qin, Qiwei

    2014-01-01

    Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. During the virus-host co-evolution, host Rab7 was also exploited by virus to complete their life cycle. To date, however, the roles of fish Rab7 in virus infection remained largely unknown. Here, we cloned and characterized a Rab7 gene from grouper, Epinephelus coioides (Ec-Rab7). The full-length Ec-Rab7 cDNA was composed of 1182 bp and encoded a polypeptide of 207 amino acids which shared 99% identity with that from Anoplopoma fimbria or Oreochromis niloticus. Ec-Rab7 contained five conserved domains of Rab GTPase family including GTP-binding or GTPase regions as well as an effector site. RT-PCR analysis revealed that Ec-Rab7 ubiquitously expressed in all detected tissues and its transcript in spleen was up-regulated after challenge with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-Rab7 was distributed in the cytoplasm as spots and mostly colocalized with lysosomes. Notably, the ectopic expressed Ec-Rab7 partly aggregated into the viral factories in cells infected by SGIV. Furthermore, overexpression of Ec-Rab7 accelerated the occurrence of cytopathic effect (CPE) induced by SGIV infection and promoted viral gene transcription. In addition, far western blotting assay revealed that Ec-Rab7 might interact with viral proteins, including SGIV VP69 and VP101. Taken together, our data suggested that Ec-Rab7 might be potentially involved in SGIV replication.

  5. Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

    Directory of Open Access Journals (Sweden)

    Victor L Jensen

    2016-12-01

    Full Text Available Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS, frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT, which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue, CCDC-104 (CCDC104, and RPI-1 (RP1/RP1L1, which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new

  6. Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

    Science.gov (United States)

    Jensen, Victor L; Carter, Stephen; Sanders, Anna A W M; Li, Chunmei; Kennedy, Julie; Timbers, Tiffany A; Cai, Jerry; Scheidel, Noemie; Kennedy, Breandán N; Morin, Ryan D; Leroux, Michel R; Blacque, Oliver E

    2016-12-01

    Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for

  7. 阴道毛滴虫Rab11鸟苷三磷酸酶cDNA克隆和序列分析%Molecular Cloning and Sequence Analysis of Rab11 GTPase in Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    张仁利; 许铭炎; 许锦阶; 高世同; 黄达娜; 耿艺介; 傅玉才

    2006-01-01

    Objective Rab11 GTPases play an essential role in regulating membrane trafficking pathways in eukaryotic cells. Nonetheless, there has been little work done on characterizing the transport machinery of Trichomonas. The aim of this study is to clone and characterize a Rab11 gene of Trichomonas vaginalis.Methods A cDNA expression library was constructed with T. vaginalis total RNA. A cDNA clone, which showed a high degree of homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs. The genomic DNA corresponding to the cDNA sequence was amplified using PCR techniques and following by sequencing. Results cDNA with a length of 710 base pairs and an open reading frame of 636 bp was obtained. The deduced amino acid sequence from the open reading frame was found to possess 211 residuals. Sequence analysis demonstrated that this cDNA clone was homologous to the Rab11 subfamily of different species (60% identity and 79% similarity with Arabidopsis thaliana Rab11c, 58% identity and 78% similarity with human Rab11b), and that the amino acid sequence contains all the well known conserved sequence elements of Rab family. Specific Rab motifs were also detected in the deduced amino acid sequence. Phylogenetic analysis showed that its closest homologues are Rab11 proteins from other species. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5'-ATG and 3'-stop codon is identical to the cDNA sequence.Conclusion A cDNA clone corresponding to the T. vaginalis Rab11 gene was obtained.The function of this gene in regulating membrane trafficking pathways of the parasitic protist is still under investigation.%目的近年研究表明Rab11鸟苷三磷酸酶在调节各种真核细胞的膜转运通道中发挥着重要作用.但是,对于原生动物毛滴虫膜转运系统的研究仍甚少.本研究旨在克隆和分析阴道毛滴虫Rab

  8. Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.

    Science.gov (United States)

    Osińska, Magdalena; Wiejak, Jolanta; Wypych, Emilia; Bilski, Henryk; Bartosiewicz, Rafał; Wyroba, Elżbieta

    2011-01-01

    Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala(140) in Rab7a for Ser(140) in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.

  9. Lysosome

    Directory of Open Access Journals (Sweden)

    Ursula Matte BSc, PhD

    2016-12-01

    Full Text Available Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules without being digested by autolysis. For those working on the field of lysosomal storage disorders, the lack of one such hydrolase would lead to undegraded or partially degraded substrate storage inside engorged organelles disturbing cellular function by yet poorly explored mechanisms. However, in recent years, a much more complex scenario of lysosomal function has emerged, beyond and above the cellular “digestive” system. Knowledge on how the impairment of this organelle affects cell functioning may shed light on signs and symptoms of lysosomal disorders and open new roads for therapy.

  10. Lysosome

    National Research Council Canada - National Science Library

    Ursula Matte BSc, PhD; Gabriela Pasqualim BSc, MSc

    2016-01-01

    Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules...

  11. Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation.

    Science.gov (United States)

    Brewer, Paul Duffield; Habtemichael, Estifanos N; Romenskaia, Irina; Mastick, Cynthia Corley; Coster, Adelle C F

    2016-01-08

    The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.

  12. E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy.

    Directory of Open Access Journals (Sweden)

    Madhavi Latha Somaraju Chalasani

    Full Text Available The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.

  13. Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice

    OpenAIRE

    Lippuner, Christoph; Paape, Daniel; Paterou, Athina; Brand, Janko; Richardson, Melville; Smith, Andrew; Hoffmann, Kirstin; Brinkmann, Volker; Blackburn, Clare; Aebischer, Toni

    2009-01-01

    The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5(+) stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to a...

  14. Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice

    OpenAIRE

    Lippuner, Christoph; Paape, Daniel; Paterou, Athina; Brand, Janko; Richardson, Melville; Smith, Andrew; Hoffmann, Kirstin; Brinkmann, Volker; Blackburn, Clare; Aebischer, Toni

    2009-01-01

    The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5(+) stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to a...

  15. Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

    Directory of Open Access Journals (Sweden)

    Michael G Connor

    2015-10-01

    Full Text Available Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

  16. Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis.

    Science.gov (United States)

    Lindsay, Andrew J; McCaffrey, Mary W

    2004-07-30

    Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.

  17. Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line.

    Science.gov (United States)

    Mascia, Anna; Gentile, Flaviana; Izzo, Antonella; Mollo, Nunzia; De Luca, Maria; Bucci, Cecilia; Nitsch, Lucio; Calì, Gaetano

    2016-08-01

    Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.

  18. PmTBC1D20, a Rab GTPase-activating protein from the black tiger shrimp, Penaeus monodon, is involved in white spot syndrome virus infection.

    Science.gov (United States)

    Yingvilasprasert, Wanchart; Supungul, Premruethai; Tassanakajon, Anchalee

    2014-02-01

    TBC (TRE2/BUB2/CDC16) domain proteins contain an ≈ 200-amino-acid motif and function as Rab GTPase-activating proteins that are required for regulating the activity of Rab proteins, and so, in turn, endocytic membrane trafficking in cells. TBC domain family member 20 (TBC1D20) has recently been reported to mediate Hepatitis C virus replication. Herein, PmTBC1D20 identified from the black tiger shrimp, Penaeus monodon, was characterized and evaluated for its role in white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of PmTBC1D20 contains 2003 bp with a predicted 1443 bp open reading frame encoding a deduced 480 amino acid protein. Its transcript levels were significantly up-regulated at 24 and 48 h by ≈ 2.3- and 2.1-fold, respectively, after systemic infection with WSSV. In addition, depletion of PmTBC1D20 transcript in shrimps by double stranded RNA interference led to a decrease in the level of transcripts of three WSSV genes (VP28, ie1 and wsv477). This suggests the importance of PmTBC1D20 in WSSV infection. This is the first report of TBC1D20 in a crustacean and reveals the possible mechanism used by WSSV to modulate the activity of the host protein, PmTBC1D20, for its benefit in viral trafficking and replication.

  19. A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

    Science.gov (United States)

    Karim, Sazzad; Alezzawi, Mohamed; Garcia-Petit, Christel; Solymosi, Katalin; Khan, Nadir Zaman; Lindquist, Emelie; Dahl, Peter; Hohmann, Stefan; Aronsson, Henrik

    2014-04-01

    A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

  20. MERTK signaling in the retinal pigment epithelium regulates the tyrosine phosphorylation of GDP dissociation inhibitor alpha from the GDI/CHM family of RAB GTPase effectors.

    Science.gov (United States)

    Shelby, Shameka J; Feathers, Kecia L; Ganios, Anna M; Jia, Lin; Miller, Jason M; Thompson, Debra A

    2015-11-01

    Photoreceptor outer segments (OS) in the vertebrate retina undergo a process of continual renewal involving shedding of disc membranes that are cleared by phagocytic uptake into the retinal pigment epithelium (RPE). In dystrophic Royal College of Surgeons (RCS) rats, OS phagocytosis is blocked by a mutation in the gene encoding the receptor tyrosine kinase MERTK. To identify proteins tyrosine-phosphorylated downstream of MERTK in the RPE, MALDI-mass spectrometry with peptide-mass fingerprinting was used in comparative studies of RCS congenic and dystrophic rats. At times corresponding to peak phagocytic activity, the RAB GTPase effector GDP dissociation inhibitor alpha (GDI1) was found to undergo tyrosine phosphorylation only in congenic rats. In cryosections of native RPE/choroid, GDI1 colocalized with MERTK and the intracellular tyrosine-kinase SRC. In cultured RPE-J cells, and in transfected heterologous cells, MERTK stimulated SRC-mediated tyrosine phosphorylation of GDI1. In OS-fed RPE-J cells, GDI1 colocalized with MERTK and SRC on apparent phagosomes located near the apical membrane. In addition, both GDI1 and RAB5, a regulator of vesicular transport, colocalized with ingested OS. Taken together, these findings identify a novel role of MERTK signaling in membrane trafficking in the RPE that is likely to subserve mechanisms of phagosome formation.

  1. A functional interplay between the small GTPase Rab11a and mitochondria-shaping proteins regulates mitochondrial positioning and polarization of the actin cytoskeleton downstream of Src family kinases.

    Science.gov (United States)

    Landry, Marie-Claude; Champagne, Claudia; Boulanger, Marie-Chloé; Jetté, Alexandra; Fuchs, Margit; Dziengelewski, Claire; Lavoie, Josée N

    2014-01-24

    It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

  2. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7

    Directory of Open Access Journals (Sweden)

    Vanessa M. D’Costa

    2015-09-01

    Full Text Available Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

  3. In vitro reconstitution of Rab GTPase-dependent vesicle clustering by the yeast lethal giant larvae/tomosyn homolog, Sro7.

    Science.gov (United States)

    Rossi, Guendalina; Watson, Kelly; Demonch, Mallory; Temple, Brenda; Brennwald, Patrick

    2015-01-01

    Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.

  4. Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion

    NARCIS (Netherlands)

    Singh, M.K.; Krüger, F.; Beckmann, H.; Brumm, S.; Vermeer, J.E.M.; Munnik, T.; Mayer, U.; Stierhof, Y.D.; Grefen, C.; Schumacher, K.; Jürgens, G.

    2014-01-01

    Plasma-membrane proteins such as ligand-binding receptor kinases, ion channels, or nutrient transporters are turned over by targeting to a lytic compartment--lysosome or vacuole--for degradation. After their internalization, these proteins arrive at an early endosome, which then matures into a late

  5. Crystal Structures of Human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating Protein (RabGAP) Domains Reveal Critical Elements for GLUT4 Translocation

    Energy Technology Data Exchange (ETDEWEB)

    S Park; W Jin; S Shoelson

    2011-12-31

    We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 {angstrom} resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 {alpha}-helices and no {beta}-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met{sup 930}, corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met{sup 930} and Leu{sup 1019} predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.

  6. Molecular characterization of CLPT1, a SEC4-like Rab/GTPase of the phytopathogenic fungus Colletotrichum lindemuthianum which is regulated by the carbon source.

    Science.gov (United States)

    Dumas, B; Borel, C; Herbert, C; Maury, J; Jacquet, C; Balsse, R; Esquerré-Tugayé, M T

    2001-07-11

    The gene CLPT1 (Colletotrichum lindemuthianum Protein Transport 1) encoding a Rab/GTPase was isolated from the filamentous fungus Colletotrichum lindemuthianum, the causal agent of bean anthracnose. At the amino acid level, CLPT1 shows between 54 and 80% identity to SEC4-like proteins, a class of molecules required for intracellular vesicular transport in yeasts. In particular, typical SEC4 domains involved in nucleotide binding and membrane attachment are present in the CLPT1 sequence. Functional identity of CLPT1 with SEC4 was confirmed by complementation of the Saccharomyces cerevisiae sec4-8 mutation. This is the first report of a gene involved in the control of intracellular vesicular trafficking in a phytopathogenic fungus. RNA blot analyses of CLPT1 expression were performed during in vitro growth of the fungus on synthetic media containing glucose or pectin, as single carbon source. The accumulation of CLPT1 mRNA was strongly increased on pectin, a plant cell wall polysaccharide that induces the production of extracellular pectinases, whereas the level of CLPT1 mRNA was below the detection threshold on glucose. These results suggest that CLPT1 is mainly involved in protein secretion and that the production of extracellular enzymes potentially involved in pathogenesis in filamentous fungi is sustained by induction of the genes involved in the secretory machinery.

  7. Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

    Directory of Open Access Journals (Sweden)

    Coreen Johnson

    Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

  8. Bioinformatic approaches to identifying and classifying Rab proteins.

    Science.gov (United States)

    Diekmann, Yoan; Pereira-Leal, José B

    2015-01-01

    The bioinformatic annotation of Rab GTPases is important, for example, to understand the evolution of the endomembrane system. However, Rabs are particularly challenging for standard annotation pipelines because they are similar to other small GTPases and form a large family with many paralogous subfamilies. Here, we describe a bioinformatic annotation pipeline specifically tailored to Rab GTPases. It proceeds in two steps: first, Rabs are distinguished from other proteins based on GTPase-specific motifs, overall sequence similarity to other Rabs, and the occurrence of Rab-specific motifs. Second, Rabs are classified taking either a more accurate but slower phylogenetic approach or a slightly less accurate but much faster bioinformatic approach. All necessary steps can either be performed locally or using the referenced online tools. An implementation of a slightly more involved version of the pipeline presented here is available at RabDB.org.

  9. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  10. Rab, Arf, and Arl-Regulated Membrane Traffic in Cortical Neuron Migration.

    Science.gov (United States)

    Tang, Bor Luen

    2016-07-01

    The migration of projection neurons from its birthplace in the subventricular zone to their final destination in the cortical plate is a complex process that requires a series of highly coordinated cellular events. Amongst the key factors involved in the processes are modulators of cytoskeletal dynamics, as well as cellular membrane traffic. Members of the small GTPases family responsible for the latter process, the Rabs and Arfs, have been recently implicated in cortical neuron migration. Rab5 and Rab11, which are key modulators of endocytosis and endocytic recycling respectively, ensure proper surface expression and distribution of N-cadherin, a key adhesion protein that tethers migrating neurons to the radial glia fiber tracts during pia-directed migration. Rab7, which is associated with lysosomal biogenesis and function, is important for the final step of terminal translocation when N-cadherin is downregulated by lysosomal degradation. Arf6 activity, which is known to be important in neuronal processes outgrowth, may negatively impact the multipolar-bipolar transition of cortical neurons undergoing radial migration, but the downstream effector of Arf6 in this regard is not yet known. In addition to the above, members of the Arl family which have been recently shown to be important in radial glia scaffold formation, would also be important for cortical neuron migration. In this short review, we discuss recent advances in our understanding of the importance of membrane traffic regulated by the Rab, Arf, and Arl family members in cortical neuron migration.

  11. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    Directory of Open Access Journals (Sweden)

    Margrét H Ögmundsdóttir

    Full Text Available Mammalian Target of Rapamycin Complex 1 (mTORC1 is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K/Akt/Rheb signalling and extracellular amino acids (AAs to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs, subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293 cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  12. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    Science.gov (United States)

    Ögmundsdóttir, Margrét H; Heublein, Sabine; Kazi, Shubana; Reynolds, Bruno; Visvalingam, Shivanthy M; Shaw, Michael K; Goberdhan, Deborah C I

    2012-01-01

    Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+)-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  13. Dmon1 controls recruitment of Rab7 to maturing endosomes in Drosophila.

    Science.gov (United States)

    Yousefian, Jahan; Troost, Tobias; Grawe, Ferdi; Sasamura, Takeshi; Fortini, Mark; Klein, Thomas

    2013-04-01

    The small GTPases Rab5 and Rab7 are important organisers of endosome formation and maturation. In addition, they orchestrate the trafficking of cargo through the endosomal pathway. A crucial event during maturation of endosomes is the replacement of the early organiser Rab5 with the late organiser Rab7 in a process called Rab conversion. Rab conversion is a prerequisite for late events, chief among them the fusion of matured endosomes with the lysosome. Recent work identifies members of the Sand1/Mon1 protein family as crucial factors during this process. Here, we present an analysis of the function of the Drosophila ortholog of mon1/sand1, Dmon1. We found that loss of function of Dmon1 results in an enlargement of maturing endosomes and loss of their association with Rab7. The enlarged endosomes contain Notch and other trans-membrane proteins as cargo. We report the first electron microscopy analysis of Dmon1 cells in a metazoan and extend the analysis of the endosomes in mutant cells. Our results suggest that the phenotype can be explained by the loss of function of Rab7. Moreover, the endosomes of Dmon1 cells mature normally in many aspects, despite the loss of association with Rab7. Surprisingly, we did not observe overactive or ectopic signalling through receptors such as Notch and RTKs in Dmon1 mutant cells, as would have been expected because of the accumulation of receptors in the maturing endosomes of these cells. This was the case even when receptor uptake into intraluminal vesicles was suppressed.

  14. Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

    Directory of Open Access Journals (Sweden)

    Neha Dikshit

    2015-08-01

    Full Text Available Recurrent urinary tract infections (UTIs caused by uropathogenic E. coli (UPEC are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR, the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

  15. Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

    Science.gov (United States)

    Dikshit, Neha; Bist, Pradeep; Fenlon, Shannon N; Pulloor, Niyas Kudukkil; Chua, Christelle En Lin; Scidmore, Marci A; Carlyon, Jason A; Tang, Bor Luen; Chen, Swaine L; Sukumaran, Bindu

    2015-08-01

    Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

  16. Important relationships between Rab and MICAL proteins in endocytic trafficking

    Institute of Scientific and Technical Information of China (English)

    Juliati; Rahajeng; Sai; Srinivas; Panapakkam; Giridharan; Naava; Naslavsky; Steve; Caplan

    2010-01-01

    The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.

  17. Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis.

    Science.gov (United States)

    Dutta, Dipannita; Donaldson, Julie G

    2015-09-01

    Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.

  18. Characterization of a Rab11 homologue, EoRab11a, in Euplotes octocarinatus.

    Science.gov (United States)

    Li, Jiangjiao; Nie, Yu; Dang, Xuhong; Liang, Aihua; Chai, Baofeng; Wang, Wei

    2009-03-01

    Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. EF061065), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53-61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min(-1) detected by HPLC at 30 degrees C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro. Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus. These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus.

  19. Large nucleotide-dependent conformational change in Rab28

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sung Haeng; Baek, Kyuwon; Dominguez, Roberto (UPENN-MED)

    2008-12-01

    Rab GTPases are essential regulators of membrane trafficking. We report crystal structures of Rab28 in the active (GppNHp-bound) and inactive (GDP-3'P-bound) forms at 1.5 and 1.1 {angstrom} resolution. Rab28 is a distant member of the Rab family. While the overall fold of Rab28 resembles that of other Rab GTPases, it undergoes a larger nucleotide-dependent conformational change than other members of this family. Added flexibility resulting from a double-glycine motif at the beginning of switch 2 might partially account for this observation. The double-glycine motif, which is conserved in the Arf family, only occurs in Rab28 and Rab7B of the Rab family, and may have a profound effect on their catalytic activities.

  20. A complete Rab screening reveals novel insights in Weibel-Palade body exocytosis.

    Science.gov (United States)

    Zografou, Sofia; Basagiannis, Dimitris; Papafotika, Alexandra; Shirakawa, Ryutaro; Horiuchi, Hisanori; Auerbach, Daniel; Fukuda, Mitsunori; Christoforidis, Savvas

    2012-10-15

    Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

  1. Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein.

    Science.gov (United States)

    Lindsay, Andrew J; Hendrick, Alan G; Cantalupo, Giuseppina; Senic-Matuglia, Francesca; Goud, Bruno; Bucci, Cecilia; McCaffrey, Mary W

    2002-04-05

    Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

  2. Rabs set the stage for polarity.

    Science.gov (United States)

    Parker, Sara S; Cox, Christopher; Wilson, Jean M

    2017-01-26

    Cell polarity refers to the asymmetric localization of cellular components that allows cells to carry out their specialized functions, be they epithelial barrier function, transmission of action potentials in nerve cells, or modulation of the immune response. The establishment and maintenance of cell polarity requires the directed trafficking of membrane proteins and lipids - essential processes that are mediated by Rab GTPases. Interestingly, several of the Rabs that impact polarity are present in the earliest eukaryotes, and the Rab polarity repertoire has expanded as cells have become more complex. There is a substantial conservation of Rab function across diverse cell types. Rabs act through an assortment of effector proteins that include scaffolding proteins, cytoskeletal motors, and other small GTPases. In this review we highlight the similarities and differences in Rab function for the instruction of polarity in diverse cell types.

  3. Loss of Rab27 function results in abnormal lung epithelium structure in mice.

    Science.gov (United States)

    Bolasco, Giulia; Tracey-White, Dhani C; Tolmachova, Tanya; Thorley, Andrew J; Tetley, Teresa D; Seabra, Miguel C; Hume, Alistair N

    2011-03-01

    Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.

  4. Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation

    Indian Academy of Sciences (India)

    Katherine Maringer; Azure Yarbrough; Sunder Sims-Lucas; Entsar Saheb; Sanaa Jawed; John Bush

    2016-06-01

    Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.

  5. Small GTPases and cilia.

    Science.gov (United States)

    Li, Yujie; Hu, Jinghua

    2011-01-01

    Small GTPases are key molecular switches that bind and hydrolyze GTP in diverse membrane- and cytoskeleton-related cellular processes. Recently, mounting evidences have highlighted the role of various small GTPases, including the members in Arf/Arl, Rab, and Ran subfamilies, in cilia formation and function. Once overlooked as an evolutionary vestige, the primary cilium has attracted more and more attention in last decade because of its role in sensing various extracellular signals and the association between cilia dysfunction and a wide spectrum of human diseases, now called ciliopathies. Here we review recent advances about the function of small GTPases in the context of cilia, and the correlation between the functional impairment of small GTPases and ciliopathies. Understanding of these cellular processes is of fundamental importance for broadening our view of cilia development and function in normal and pathological states and for providing valuable insights into the role of various small GTPases in disease processes, and their potential as therapeutic targets.

  6. Rab proteins specify motorized vesicle transport

    NARCIS (Netherlands)

    Wanschers, B.F.J.

    2008-01-01

    Small GTPases of the Rab-family are key regulators of intracellular membrane traffic. These proteins constantly cycle between an 'active' GTP-bound and 'inactive' GDP-bound state. In their GTP-bound conformation Rab proteins can engage in complex formation with so called effector proteins. It is at

  7. Rab6 and Rab11 regulate Chlamydia trachomatis development and golgin-84-dependent Golgi fragmentation.

    Directory of Open Access Journals (Sweden)

    Anette Rejman Lipinski

    2009-10-01

    Full Text Available Many intracellular pathogens that replicate in special membrane bound compartments exploit cellular trafficking pathways by targeting small GTPases, including Rab proteins. Members of the Chlamydiaceae recruit a subset of Rab proteins to their inclusions, but the significance of these interactions is uncertain. Using RNA interference, we identified Rab6 and Rab11 as important regulators of Chlamydia infections. Depletion of either Rab6 or Rab11, but not the other Rab proteins tested, decreased the formation of infectious particles. We further examined the interplay between these Rab proteins and the Golgi matrix components golgin-84 and p115 with regard to Chlamydia-induced Golgi fragmentation. Silencing of the Rab proteins blocked Chlamydia-induced and golgin-84 knockdown-stimulated Golgi disruption, whereas Golgi fragmentation was unaffected in p115 depleted cells. Interestingly, p115-induced Golgi fragmentation could rescue Chlamydia propagation in Rab6 and Rab11 knockdown cells. Furthermore, transport of nutrients to Chlamydia, as monitored by BODIPY-Ceramide, was inhibited by Rab6 and Rab11 knockdown. Taken together, our results demonstrate that Rab6 and Rab11 are key regulators of Golgi stability and further support the notion that Chlamydia subverts Golgi structure to enhance its intracellular development.

  8. The I-BAR protein Ivy1 is an effector of the Rab7 GTPase Ypt7 involved in vacuole membrane homeostasis

    NARCIS (Netherlands)

    Numrich, Johannes; Péli-Gulli, Marie-Pierre; Arlt, Henning; Sardu, Alessandro; Griffith, Janice; Levine, Tim; Engelbrecht-Vandré, Siegfried; Reggiori, Fulvio; De Virgilio, Claudio; Ungermann, Christian

    2015-01-01

    Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show

  9. The RCP-Rab11 complex regulates endocytic protein sorting.

    Science.gov (United States)

    Peden, Andrew A; Schonteich, Eric; Chun, John; Junutula, Jagath R; Scheller, Richard H; Prekeris, Rytis

    2004-08-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.

  10. Structural Insights into Rab27 Recruitment by its Effectors

    Science.gov (United States)

    M. G. Chavas, Leonard; Ihara, Kentaro; Kawasaki, Masato; Wakatsuki, Soichi

    An increasing number of Rab GTPases associated with partial dysfunction has been linked to several human diseases characterized by a diminution in vesicle transport. Due to its direct implication in human disorders, the Rab27 subfamily is considered as a standard for vesicle docking studies. By which mechanism Rab27 effectors distinguish among the pool of Rab GTPases? What is the underneath machinery rendering the interaction of eleven distinct effectors specific of Rab27 when compared to other Rabs of the secretory pathway? By solving the X-ray structures of Rab27, both in its inactive form and active form bound to the effector protein Slp2-a, attempts have been given to unravel the molecular basis of regulation of the delivering process of vesicles to fusion by the Rab27 subfamily.

  11. Crystal structure of inactive form of Rab3B

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Shen, Yang [Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, Ontario, Canada M5G 1L7 (Canada); Jiao, Ronghong [Department of Function Inspection, Hebei Provincial People' s Hospital, Shijiazhuang 050051 (China); Liu, Yanli; Deng, Lingfu [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Qi, Chao, E-mail: qichao@mail.ccnu.edu.cn [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer This is the first structural information of human Rab3B. Black-Right-Pointing-Pointer To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. Black-Right-Pointing-Pointer The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 A resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  12. The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

    Directory of Open Access Journals (Sweden)

    Senthil Kumar A Natesan

    Full Text Available BACKGROUND: Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes. METHODOLOGY/PRINCIPAL FINDINGS: Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies. CONCLUSIONS/SIGNIFICANCE: RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non

  13. Crystal structure of inactive form of Rab3B

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei; Shen, Yang; Jiao, Ronghong; Liu, Yanli; Deng, Lingfu; Qi, Chao (Hebei); (Toronto); (Huazhong)

    2012-06-28

    Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 {angstrom} resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  14. 31 Flavors of Drosophila Rab proteins

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jun; Schulze, Karen L.; Hiesinger, P. Robin; Suyama, Kaye; Wang, Stream; Fish, Matthew; Acar, Melih; Hoskins, Roger A.; Bellen, HugoJ.; Scott, Matthew P.

    2007-04-03

    Rab proteins are small GTPases that play important roles intransport of vesicle cargo and recruitment, association of motor andother proteins with vesicles, and docking and fusion of vesicles atdefined locations. In vertebrates, more than 75 Rab genes have beenidentified, some of which have been intensively studied for their rolesin endosome and synaptic vesicle trafficking. Recent studies of thefunctions of certain Rab proteins have revealed specific roles inmediating developmental signal transduction. We have begun a systematicgenetic study of the 33 Rab genes in Drosophila. Most of the fly proteinsare clearly related to specific vertebrate proteins. We report here thecreation of a set of transgenic fly lines that allow spatially andtemporally regulated expression of Drosophila Rab proteins. We generatedfluorescent protein-tagged wild-type, dominant-negative, andconstitutively active forms of 31 Drosophila Rab proteins. We describeDrosophila Rab expression patterns during embryogenesis, the subcellularlocalization of some Rab proteins, and comparisons of the localization ofwild-type, dominant-negative, and constitutively active forms of selectedRab proteins. The high evolutionary conservation and low redundancy ofDrosophila Rab proteins make these transgenic lines a useful toolkit forinvestigating Rab functions in vivo.

  15. GTPases in intracellular trafficking: an overview.

    Science.gov (United States)

    Segev, Nava

    2011-02-01

    Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.

  16. Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.

    Science.gov (United States)

    Mullock, B M; Smith, C W; Ihrke, G; Bright, N A; Lindsay, M; Parkinson, E J; Brooks, D A; Parton, R G; James, D E; Luzio, J P; Piper, R C

    2000-09-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

  17. Syntaxin 7 Is Localized to Late Endosome Compartments, Associates with Vamp 8, and Is Required for Late Endosome–Lysosome Fusion

    Science.gov (United States)

    Mullock, Barbara M.; Smith, Chez W.; Ihrke, Gudrun; Bright, Nicholas A.; Lindsay, Margaret; Parkinson, Emma J.; Brooks, Doug A.; Parton, Robert G.; James, David E.; Luzio, J. Paul; Piper, Robert C.

    2000-01-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, α and γ SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells. PMID:10982406

  18. Role of rab proteins in epithelial membrane traffic

    NARCIS (Netherlands)

    van Ijzendoorn, SCD; Mostov, KE; Hoekstra, D

    2003-01-01

    Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such m

  19. Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo.

    Science.gov (United States)

    Sorvina, Alexandra; Shandala, Tetyana; Brooks, Douglas A

    2016-01-01

    The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

  20. Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo

    Directory of Open Access Journals (Sweden)

    Alexandra Sorvina

    2016-06-01

    Full Text Available The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10, appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

  1. RAB5A — EDRN Public Portal

    Science.gov (United States)

    From UniProtKB/Swiss-Prot: The small GTPases Rab are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes. Rabs cycle between an inactive GDP-bound form and an active GTP-bound form that is able to recruit to membranes different sets of downstream effectors directly responsible for vesicle formation, movement, tethering and fusion. RAB5A is required for the fusion of plasma membranes and early endosomes. Contributes to the regulation of filopodia extension.

  2. Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.

    Science.gov (United States)

    Magalhaes, Joana; Gegg, Matthew E; Migdalska-Richards, Anna; Doherty, Mary K; Whitfield, Phillip D; Schapira, Anthony H V

    2016-08-15

    Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD. © The Author 2016. Published by Oxford University Press.

  3. Staphylococcus aureus Alpha-Toxin Induces the Formation of Dynamic Tubules Labeled with LC3 within Host Cells in a Rab7 and Rab1b-Dependent Manner

    Directory of Open Access Journals (Sweden)

    María M. López de Armentia

    2017-10-01

    Full Text Available Staphylococcus aureus is a pathogen that causes severe infectious diseases that eventually lead to septic and toxic shock. S. aureus infection is characterized by the production of virulence factors, including enzymes and toxins. After internalization S. aureus resides in a phagosome labeled with Rab7 protein. Here, we show that S. aureus generates tubular structures marked with the small GTPases Rab1b and Rab7 and by the autophagic protein LC3 at early times post-infection. As shown by live cell imaging these tubular structures are highly dynamic, extend, branch and grow in length. We have named them S. aureus induced filaments (Saf. Furthermore, we demonstrate that the formation of these filaments depends on the integrity of microtubules and the activity of the motor protein Kinesin-1 (Kif5B and the Rab-interacting lysosomal protein (RILP. Our group has previously reported that α-hemolysin, a secreted toxin of S. aureus, is responsible of the activation of the autophagic pathway induced by the bacteria. In the present report, we demonstrate that the autophagic protein LC3 is recruited to the membrane of S. aureus induced filaments and that α-hemolysin is the toxin that induces Saf formation. Interestingly, increasing the levels of intracellular cAMP significantly inhibited Saf biogenesis. Remarkably in this report we show the formation of tubular structures that emerge from the S. aureus-containing phagosome and that these tubules generation seems to be required for efficient bacteria replication.

  4. Diversification of the RAB Guanosine Triphosphatase Family in Dicots and Monocots

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    RAB guanosine triphosphatases (GTPases) are key regulators of vesicle trafficking and are essential to the growth and development of all eukaryotic cells. During evolution, the RAB family has expanded in different patterns to facilitate distinct cellular, developmental and physiological adaptations. Yeast has only 11 family members, whereas mammalian RABs have expanded to 18 RAB subfamilies. Plant RABs have diversified primarily by duplicating members within a single subfamily. Plant RABs are divided into eight subfamilies, corresponding to mammalian RAB1, RAB2, RAB5, RAB6,RAB7, RAB8, RAB11 and RAB18. Functional diversification of these is exemplified by the RAB11s, orthologs of which are partitioned into unique cell compartments in plants where they function to transport vesicles during localized tip growth.Similarly, the RAB2 family in grasses is likely involved in vesicle secretion associated with wall expansion, as determined by analysis of over-expression mutants. We propose that dicots and monocots have also diverged in their RAB profiles to accommodate unique cellular functions between the two groups. Here we present a bioinformatics analysis comparing the RAB sub-families of rice, maize and Arabidopsis. These results will guide future functional studies to test for the role of diversification of subfamilies unique to monocots compared to dicots.

  5. The RCP–Rab11 Complex Regulates Endocytic Protein SortingD⃞

    Science.gov (United States)

    Peden, Andrew A.; Schonteich, Eric; Chun, John; Junutula, Jagath R.; Scheller, Richard H.; Prekeris, Rytis

    2004-01-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP–Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes. PMID:15181150

  6. The Rab4 effector Rabip4 plays a role in the endocytotic trafficking of Glut 4 in 3T3-L1 adipocytes

    NARCIS (Netherlands)

    Mari, Muriel; Monzo, Pascale; Kaddai, Vincent; Keslair, Frédérique; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Cormont, Mireille

    2006-01-01

    Insulin regulates glucose uptake in the adipocytes by modulating Glut 4 localization, a traffic pathway involving the endocytic small GTPases Rab4, Rab5, and RabThe expression of the Rab4 effector Rabip4 leads to a 30% increase in glucose uptake and Glut 4 translocation in the presence of insulin, w

  7. Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis.

    Science.gov (United States)

    Caviglia, Sara; Brankatschk, Marko; Fischer, Elisabeth J; Eaton, Suzanne; Luschnig, Stefan

    2016-07-01

    A crucial yet ill-defined step during the development of tubular networks, such as the vasculature, is the formation of connections (anastomoses) between pre-existing lumenized tubes. By studying tracheal tube anastomosis in Drosophila melanogaster, we uncovered a key role of secretory lysosome-related organelle (LRO) trafficking in lumen fusion. We identified the conserved calcium-binding protein Unc-13-4/Staccato (Stac) and the GTPase Rab39 as critical regulators of this process. Stac and Rab39 accumulate on dynamic vesicles, which form exclusively in fusion tip cells, move in a dynein-dependent manner, and contain late-endosomal, lysosomal, and SNARE components characteristic of LROs. The GTPase Arl3 is necessary and sufficient for Stac LRO formation and promotes Stac-dependent intracellular fusion of juxtaposed apical plasma membranes, thereby forming a transcellular lumen. Concomitantly, calcium is released locally from ER exit sites and apical membrane-associated calcium increases. We propose that calcium-dependent focused activation of LRO exocytosis restricts lumen fusion to appropriate domains within tip cells.

  8. The Autophagic Process Occurs in Human Bone Metastasis and Implicates Molecular Mechanisms Differently Affected by Rab5a in the Early and Late Stages

    Directory of Open Access Journals (Sweden)

    Paola Maroni

    2016-03-01

    Full Text Available Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination.

  9. The Autophagic Process Occurs in Human Bone Metastasis and Implicates Molecular Mechanisms Differently Affected by Rab5a in the Early and Late Stages

    Science.gov (United States)

    Maroni, Paola; Bendinelli, Paola; Resnati, Massimo; Matteucci, Emanuela; Milan, Enrico; Desiderio, Maria Alfonsina

    2016-01-01

    Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination. PMID:27023526

  10. Arabidopsis RabF1 (ARA6) Is Involved in Salt Stress and Dark-Induced Senescence (DIS)

    Science.gov (United States)

    Yin, Congfei; Karim, Sazzad; Zhang, Hongsheng; Aronsson, Henrik

    2017-01-01

    Arabidopsis small GTPase RabF1 (ARA6) functions in endosomal vesicle transport and may play a crucial role in recycling and degradation of molecules, thus involved in stress responses. Here we have reported that complementary overexpression lines RabF1OE (overexpression), GTPase mutants RabF1Q93L (constitutively active) and RabF1S47N (dominant negative) lines show longer root growth than wild-type, rabF1 knockout and N-myristoylation deletion (Δ1−29, N-terminus) complementary overexpression mutant plants under salt induced stress, which indicates that N-myristoylation of RabF1 is indispensable for salt tolerance. Moreover, RabF1 is highly expressed during senescence and RabF1OE lines were more tolerant of dark-induced senescence (DIS) than wild-type and rabF1. PMID:28157156

  11. Rab Proteins and the Secretory Pathway: The Case of Rab18 in Neuroendocrine Cells

    Directory of Open Access Journals (Sweden)

    RAFAEL eVAZQUEZ-MARTINEZ

    2011-01-01

    Full Text Available The secretory pathway is a process characteristic of cells specialized in secretion such as endocrine cells and neurons. It consists of different stages that are dependent on specific transport of proteins in vesicular-tubular carriers. Biochemical analyses have unveiled a number of protein families that confer identity to carrier vesicles and specificity to their transport. Among them is the family of Rab proteins, Ras-like small GTPases that anchor to the surface of transport vesicles and participate in vesicle formation from the donor compartment, transport along cytoskeletal tracks, and docking and fusion with the acceptor compartment. All of these functions are accomplished through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors. The numerous Rab proteins have distinct subcellular distributions throughout the endomembrane system, which ensures efficient cargo transfer. Rab proteins act as molecular switches that alternate between a cytosolic GDP-bound, inactive form and a membrane-associated GTP-bound, active conformation. Cycling between inactive and active states is a highly regulated process that enables Rabs to confer spatio-temporal precision to the different stages through which a vesicle passes during its lifespan. This review focuses on our current knowledge on Rab functioning, from their structural features to the multiple regulatory proteins and effectors that control Rab activity and translate Rab function. Furthermore, we also summarize the information available on a particular Rab protein, Rab18, which has been linked to the control of secretory granule traffic in neuroendocrine cells.

  12. Locking GTPases covalently in their functional states

    Science.gov (United States)

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-07-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase-acryl-nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.

  13. The C. elegans rab family: identification, classification and toolkit construction.

    Science.gov (United States)

    Gallegos, Maria E; Balakrishnan, Sanjeev; Chandramouli, Priya; Arora, Shaily; Azameera, Aruna; Babushekar, Anitha; Bargoma, Emilee; Bokhari, Abdulmalik; Chava, Siva Kumari; Das, Pranti; Desai, Meetali; Decena, Darlene; Saramma, Sonia Dev Devadas; Dey, Bodhidipra; Doss, Anna-Louise; Gor, Nilang; Gudiputi, Lakshmi; Guo, Chunyuan; Hande, Sonali; Jensen, Megan; Jones, Samantha; Jones, Norman; Jorgens, Danielle; Karamchedu, Padma; Kamrani, Kambiz; Kolora, Lakshmi Divya; Kristensen, Line; Kwan, Kelly; Lau, Henry; Maharaj, Pranesh; Mander, Navneet; Mangipudi, Kalyani; Menakuru, Himabindu; Mody, Vaishali; Mohanty, Sandeepa; Mukkamala, Sridevi; Mundra, Sheena A; Nagaraju, Sudharani; Narayanaswamy, Rajhalutshimi; Ndungu-Case, Catherine; Noorbakhsh, Mersedeh; Patel, Jigna; Patel, Puja; Pendem, Swetha Vandana; Ponakala, Anusha; Rath, Madhusikta; Robles, Michael C; Rokkam, Deepti; Roth, Caroline; Sasidharan, Preeti; Shah, Sapana; Tandon, Shweta; Suprai, Jagdip; Truong, Tina Quynh Nhu; Uthayaruban, Rubatharshini; Varma, Ajitha; Ved, Urvi; Wang, Zeran; Yu, Zhe

    2012-01-01

    Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).

  14. Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

    Science.gov (United States)

    Rastogi, Ruchir; Verma, Jitender Kumar; Kapoor, Anjali; Langsley, Gordon; Mukhopadhyay, Amitabha

    2016-07-08

    Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

  15. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    Science.gov (United States)

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

  16. Bacteria and protozoa differentially modulate the expression of Rab proteins.

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    Elsa Seixas

    Full Text Available Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.

  17. Cdk5 Regulation of the GRAB-Mediated Rab8-Rab11 Cascade in Axon Outgrowth.

    Science.gov (United States)

    Furusawa, Kotaro; Asada, Akiko; Urrutia, Pamela; Gonzalez-Billault, Christian; Fukuda, Mitsunori; Hisanaga, Shin-Ichi

    2017-01-25

    Neurons communicate with each other through their axons and dendrites. However, a full characterization of the molecular mechanisms involved in axon and dendrite formation is still incomplete. Neurite outgrowth requires the supply of membrane components for surface expansion. Two membrane sources for axon outgrowth are suggested: Golgi secretary vesicles and endocytic recycling endosomes. In non-neuronal cells, trafficking of secretary vesicles from Golgi is regulated by Rab8, a member of Rab small GTPases, and that of recycling endosomes is by Rab11, another member of Rabs. However, whether these vesicles are coordinately or independently transported in growing axons is unknown. Herein, we find that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. Knockdown of GRAB suppressed axon outgrowth of cultured mouse brain cortical neurons. GRAB mediates the interaction between Rab11A and Rab8A, and this activity is regulated by phosphorylation at Ser169 and Ser180 by Cdk5-p35. The nonphosphorylatable GRAB mutant S169/180A promoted axonal outgrowth to a greater extent than did the phosphomimetic GRAB mutant S169/180D. Phosphorylation of GRAB suppressed its guanine nucleotide exchange factor activity and its ability to recruit Rab8A- to Rab11A-positive endosomes. In vivo function of GRAB and its Cdk5-phophorylation were shown in migration and process formation of developing neurons in embryonic mouse brains. These results indicate that GRAB regulates axonal outgrowth via activation and recruitment of Rab8A- to Rab11A-positive endosomes in a Cdk5-dependent manner. While axon outgrowth requires membrane supply for surface expansion, the molecular mechanisms regulating the membrane transport in growing axons remain unclear. Here, we demonstrate that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. GRAB promotes the axonal membrane transport by mediating the interaction between Rab11 and Rab

  18. Complementation and Expression Analysis of SoRab1A and SoRab2A in Sugarcane Demonstrates Their Functional Diversification

    Institute of Scientific and Technical Information of China (English)

    Jia-Ming Zhang; Anne W Sylvester; Ding-Qin Li; Xue-Piao Sun

    2006-01-01

    Mammalian and plant Rab1 and Rab2 are small GTPases that regulate vesicle trafficking in the endoplasmic reticulum (ER) to Golgi compartments. Little is known about their functional diversification or potential interaction. We cloned sugarcane (Saccharum officinarum L.) Rab1A and Rab2A genes and studied their functional differences by expression and complementation experiments. We found differential expression of the two genes during sugarcane leaf development: SoRab2A expression declined from the dividing base to the maturing tip of the growing leaves, whereas SoRab1A was constitutively expressed, suggesting that SoRab2A is required for cell division and expansion and SoRab1A is required for cells at all developmental stages. We used a yeast temperature sensitive ypt1-A136D mutant strain to further investigate these shared and unique functions. Ypt1 is a small GTPase that regulates vesicle transport in the same cellular location as Rab1 and Rab2. Neither SoRab1A nor SoRab2A alone could restore the growth of the mutant at restrictive temperatures when SoRab1A and SoRab2A were transformed separately. However, SoRab1A transformants maintained normal morphology and viability at non-permissive temperature, and resumed growth when returned to permissive temperature, whereas SoRab2A transformants died at non-permissive temperature, suggesting that SoRab1A function is required for a cell's viability. Mutant growth was fully restored when SoRab1A and SoRab2A were co-transformed, indicating that SoRab1A and SoRab2A complement each other and they both are needed to restore the function of ypt1-A136D. These results demonstrate that SoRab1A and SoRab2A serve distinct but overlapping functions, mostly by regulating the transportation of different sets of proteins.

  19. Rab23 is a flagellar protein in Trypanosoma brucei

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    Field Mark C

    2011-06-01

    Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

  20. Two Rab5 Homologs Are Essential for the Development and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae

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    Cheng D. Yang

    2017-05-01

    Full Text Available The rice blast fungus, Magnaporthe oryzae, infects many economically important cereal crops, particularly rice. It has emerged as an important model organism for studying the growth, development, and pathogenesis of filamentous fungi. RabGTPases are important molecular switches in regulation of intracellular membrane trafficking in all eukaryotes. MoRab5A and MoRab5B are Rab5 homologs in M. oryzae, but their functions in the fungal development and pathogenicity are unknown. In this study, we have employed a genetic approach and demonstrated that both MoRab5A and MoRab5B are crucial for vegetative growth and development, conidiogenesis, melanin synthesis, vacuole fusion, endocytosis, sexual reproduction, and plant pathogenesis in M. oryzae. Moreover, both MoRab5A and MoRab5B show similar localization in hyphae and conidia. To further investigate possible functional redundancy between MoRab5A and MoRab5B, we overexpressed MoRAB5A and MoRAB5B, respectively, in MoRab5B:RNAi and MoRab5A:RNAi strains, but neither could rescue each other’s defects caused by the RNAi. Taken together, we conclude that both MoRab5A and MoRab5B are necessary for the development and pathogenesis of the rice blast fungus, while they may function independently.

  1. A bioinformatic analysis of the RAB genes of Trypanosoma brucei.

    Science.gov (United States)

    Ackers, John P; Dhir, Vivek; Field, Mark C

    2005-05-01

    RAB proteins are small GTPases with vital roles in eukaryotic intracellular transport; orthologous RABs appear to fulfil similar functions in diverse organisms. Trypanosoma brucei spp., the causative organisms of Old World trypanosomiasis of humans and domestic animals, have extremely effective endocytic and exocytic mechanisms that are likely to be involved in maintenance of infection, making study of these systems of importance. Taking advantage of the essential completion of the T. brucei genome, we have re-examined the T. brucei RABs (TbRABs) so far described and identified a total of 16. BLAST searches and phylogenetic analysis show that nine of the TbRABs can confidently be assigned as orthologues or homologues of known RAB proteins from higher eukaryotes, and four more with reasonable probability. The core endocytic pathway is probably similar in complexity to yeast, whilst the early exocytic pathway appears to be more complex than in yeast. Two of the TbRAB family (RAB23 and 28) with clear mammalian orthologues appear to be unusual, and may be involved in nuclear processes and are described in more detail in an accompanying paper. Three TbRABs appear, however, to have no close homologues and may fulfil specialised functions in this organism. The availability of a complete set of TbRABs--which includes orthologues of the RABs responsible for control of the core of the endomembrane system (i.e. RAB1, 2, 4-7 and 11)--provides a first overview of the trafficking complexity that is present within a kinetoplastid parasite. Based on these homologies we suggest a systematic nomenclature for the TbRABs to reflect their functional homologies. This information is of importance both from the perspective of understanding the evolution and diversity of eukaryotic trafficking, but also in providing a framework by which to understand protein processing, trafficking, endocytosis and other related processes in these parasites.

  2. Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

    NARCIS (Netherlands)

    Aligianis, IA; Johnson, CA; Gissen, P; Chen, DR; Hampshire, D; Hoffmann, K; Maina, EN; Morgan, NV; Tee, L; Morton, J; Ainsworth, [No Value; Horn, D; Rosser, E; Cole, TRP; Stolte-Dijkstra, [No Value; Fieggen, K; Clayton-Smith, J; Megarbane, A; Shield, JP; Newbury-Ecob, R; Dobyns, WB; Graham, JM; Kjaer, KW; Warburg, M; Bond, J; Trembath, RC; Harris, LW; Takai, Y; Mundlos, S; Tannahill, D; Woods, CG; Maher, ER

    2005-01-01

    Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of t

  3. Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

    DEFF Research Database (Denmark)

    Aligianis, Irene A; Johnson, Colin A; Gissen, Paul;

    2005-01-01

    Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator...

  4. Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility.

    Science.gov (United States)

    Lindsay, Andrew J; McCaffrey, Mary W

    2016-07-09

    Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of α5β1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells.

  5. A Pan-GTPase Inhibitor as a Molecular Probe.

    Science.gov (United States)

    Hong, Lin; Guo, Yuna; BasuRay, Soumik; Agola, Jacob O; Romero, Elsa; Simpson, Denise S; Schroeder, Chad E; Simons, Peter; Waller, Anna; Garcia, Matthew; Carter, Mark; Ursu, Oleg; Gouveia, Kristine; Golden, Jennifer E; Aubé, Jeffrey; Wandinger-Ness, Angela; Sklar, Larry A

    2015-01-01

    Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

  6. A Pan-GTPase Inhibitor as a Molecular Probe.

    Directory of Open Access Journals (Sweden)

    Lin Hong

    Full Text Available Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

  7. Loss-of-function mutations in Rab escort protein 1 (REP-1 affect intracellular transport in fibroblasts and monocytes of choroideremia patients.

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    Natalia V Strunnikova

    Full Text Available BACKGROUND: Choroideremia (CHM is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1, an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo BioParticles conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1, pigment epithelial derived factor (PEDF, tumor necrosis factor (TNF alpha, fibroblast growth factor (FGF beta and interleukin (lL-8. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different

  8. Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

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    Marina R Pulido

    Full Text Available Lipid droplets (LDs are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K, the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

  9. Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

    Science.gov (United States)

    Pulido, Marina R; Diaz-Ruiz, Alberto; Jiménez-Gómez, Yolanda; Garcia-Navarro, Socorro; Gracia-Navarro, Francisco; Tinahones, Francisco; López-Miranda, José; Frühbeck, Gema; Vázquez-Martínez, Rafael; Malagón, Maria M

    2011-01-01

    Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

  10. LRRK2 transport is regulated by its novel interacting partner Rab32.

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    Dieter Waschbüsch

    Full Text Available Leucine-rich repeat kinase 2 (LRRK2 is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD. Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.

  11. The Rab11-FIP1/RCP gene codes for multiple protein transcripts related to the plasma membrane recycling system.

    Science.gov (United States)

    Jin, Min; Goldenring, James R

    2006-06-01

    Rab11a is a member of the Rab11 small GTPase family, and plays an important role in plasma membrane recycling. Rab11-Family Interacting Protein 1 (Rab11-FIP1) binds to Rab11 through a carboxyl-terminal amphipathic alpha helix. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from human chromosome 8. Among them, Rab11-FIP1A-D have carboxyl terminal Rab11 binding domains, while Rab11-FIP1E-H do not contain the Rab11 binding domain. While Rab11-FIP1B and F gene transcripts are ubiquitous, other Rab11-FIP1 transcripts demonstrate more limited patterns of expression in human tissue cDNAs. EGFP-Rab11-FIP1A-D proteins over-expressed in HeLa cells targeted to Rab11a-containing membranes, while EGFP-Rab11-FIP1E/F and H proteins did not localize with recycling system membranes. However, transferrin trafficking was not significantly altered in HeLa cells over-expressing expressing any of the EGFP-Rab11-FIP1 proteins. Rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP demonstrated that Rab11-FIP1B and Rab11-FIP1C/RCP are expressed endogenously. Strikingly, endogenous staining for Rab11-FIP1C/RCP only partially co-localized with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, and EGFP-Rab11a in the perinuclear region, indicating that Rab11-FIP1C/RCP resides in a differentiable subcellular compartment within the plasma membrane recycling system compared with Rab11-FIP1A and Rab11-FIP1B. These data suggest that Rab11-FIP1 proteins may play coordinated roles in regulating plasma membrane recycling with regional specificity within the Rab11a-containing recycling system.

  12. Interplay between Rab27a effectors in pancreatic β-cells

    Institute of Scientific and Technical Information of China (English)

    Mami Yamaoka; Toshimasa Ishizaki; Toshihide Kimura

    2015-01-01

    The small GTPase Rab27a is a member of the Rab familythat is involved in membrane trafficking in various kindsof cells. Rab27a has GTP- and GDP-bound forms, andtheir interconversion regulates intracellular signalingpathways. Typically, only a GTP-bound GTPase binds itsspecific effectors with the resulting downstream signalscontrolling specific cellular functions. We previouslyidentified novel Rab27a-interacting proteins. Surprisingly,some of these proteins interacted with GDP-boundRab27a. The present study reviews recent progressin our understanding of the roles of Rab27a and itseffectors in the secretory process. In pancreatic β-cells,GTP-bound Rab27a regulates insulin secretion at the preexocytoticstages via its GTP-specific effectors such asExophilin8/Slac2-c/MyRIP and Slp4/Granuphilin. Glucosestimulation causes insulin exocytosis. Glucose stimulationalso converts Rab27a from its GTP- to its GDP-boundform. GDP-bound Rab27a interacts with GDP-specificeffectors and controls endocytosis of the secretorymembrane. Thus, Rab27a cycling between GTP- andGDP-bound forms synchronizes with the recycling ofsecretory membrane to re-use the membrane and keepthe β-cell volume constant.

  13. Identification and characterization of Iporin as a novel interaction partner for rab1

    Directory of Open Access Journals (Sweden)

    Konczal Magdalena

    2005-03-01

    Full Text Available Abstract Background The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. Results Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD. Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. Conclusion Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus.

  14. Rab25 upregulation correlates with the proliferation, migration, and invasion of renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuanyuan; Jia, Qingzhu [Biomedical Analysis Center, Third Military Medical University, Chongqing (China); Chongqing Key Laboratory of Cytomics, Chongqing (China); Zhang, Qian [Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing (China); Wan, Ying, E-mail: wanying_cn@163.com [Biomedical Analysis Center, Third Military Medical University, Chongqing (China); Chongqing Key Laboratory of Cytomics, Chongqing (China)

    2015-03-20

    Renal cell carcinoma (RCC) is a common urological cancer with a poor prognosis. A recent cohort study revealed that the median survival of RCC patients was only 1.5 years and that <10% of the patients in the study survived up to 5 years. In tumor development, Rab GTPase are known to play potential roles such as regulation of cell proliferation, migration, invasion, communication, and drug resistance in multiple tumors. However, the correlation between Rabs expression and the occurrence, development, and metastasis of RCC remains unclear. In this study, we analyzed the transcriptional levels of 52 Rab GTPases in RCC patients. Our results showed that high levels of Rab25 expression were significantly correlated with RCC invasion classification (P < 0.01), lymph-node metastasis (P < 0.001), and pathological stage (P < 0.01). Conversely, in 786-O and A-498 cells, knocking down Rab25 protein expression inhibited cell proliferation, migration, and invasion. Our results also demonstrated that Rab25 is a target gene of let-7d, and further suggested that Rab25 upregulation in RCC is due to diminished expression of let-7d. These findings indicate that Rab25 might be a novel candidate molecule involved in RCC development, thus identifying a potential biological therapeutic target for RCC. - Highlights: • The transcriptional levels of 52 Rab GTPases were analyzed in renal cell carcinoma (RCC). • High levels of Rab25 expression were significantly correlated with clinicopathological factors of RCC. • Knockdown of Rab25 protein expression reduced RCC cells proliferation, migration, and invasion. • Rab25 is a target gene of let-7d in RCC.

  15. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  16. Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells.

    Science.gov (United States)

    Sun, Yi; Chiu, Tim T; Foley, Kevin P; Bilan, Philip J; Klip, Amira

    2014-04-01

    Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase-dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)-MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP-MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP-MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA-mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.

  17. The jaw of the worm: GTPase-activating protein EAT-17 regulates grinder formation in Caenorhabditis elegans.

    Science.gov (United States)

    Straud, Sarah; Lee, Inhwan; Song, Bomi; Avery, Leon; You, Young-Jai

    2013-09-01

    Constitutive transport of cellular materials is essential for cell survival. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that EAT-17, a novel GTPase-activating protein (GAP), regulates RAB-6.2 function in grinder formation in Caenorhabditis elegans. We identified EAT-17 as a novel RabGAP that interacts with RAB-6.2, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. EAT-17 has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in C. elegans shows that RNAi against rab-6.2 produces a phenotype identical to eat-17. A directed yeast two-hybrid screen using EAT-17 as bait and each of the 25 RAB proteins as prey identifies RAB-6.2 as the interacting partner of EAT-17, confirming that RAB-6.2 is a specific substrate of EAT-17. Additionally, deletion mutants of rab-6.2 show grinder defects identical to those of eat-17 loss-of-function mutants, and both RAB-6.2 and EAT-17 are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that EAT-17 is a specific GTPase-activating protein for RAB-6.2. Based on the conserved function of Rab6 in vesicular transport, we propose that EAT-17 regulates the turnover rate of RAB-6.2 activity in cargo trafficking for grinder formation.

  18. The CORVET subunit Vps8 cooperates with the Rab5 homolog Vps21 to induce clustering of late endosomal compartments

    NARCIS (Netherlands)

    Markgraf, Daniel F; Ahnert, Franziska; Arlt, Henning; Mari, Muriel; Peplowska, Karolina; Epp, Nadine; Griffith, Janice; Reggiori, Fulvio; Ungermann, Christian

    2009-01-01

    Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypi

  19. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    Energy Technology Data Exchange (ETDEWEB)

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  20. CSFV proliferation is associated with GBF1 and Rab2

    Indian Academy of Sciences (India)

    WULONG LIANG; MINPING ZHENG; CHANGLEI BAO; YANMING ZHANG

    2017-03-01

    The Golgi apparatus and its resident proteins are utilized and regulated by viruses to facilitate their proliferation. Inthis study, we investigated Classical swine fever virus (CSFV) proliferation when the function of the Golgi wasdisturbed. Golgi function was disturbed using chemical inhibitors, namely, brefeldin A (BFA) and golgicide A (GCA),and RNA interfering targets, such as the Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1)and Rab2 GTPases. CSFV proliferation was significantly inhibited during RNA replication and viral particlegeneration after BFA and GCA treatment. CSFV multiplication dynamics were retarded in cells transfected withGBF1 and Rab2 shRNA. Furthermore, CSFV proliferation was promoted by GBF1 and Rab2 overexpression using alentiviral system. Hence, Golgi function is important for CSFV multiplication, and GBF1 and Rab2 participate inCSFV proliferation. Further studies must investigate Golgi-resident proteins to elucidate the mechanism underlyingCSFV replication.

  1. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    Science.gov (United States)

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

  2. Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

    Directory of Open Access Journals (Sweden)

    Huxley Clare

    2002-10-01

    Full Text Available Abstract Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM, a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1 gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.

  3. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    Energy Technology Data Exchange (ETDEWEB)

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  4. Microglial migration mediated by ATP-induced ATP release from lysosomes

    Institute of Scientific and Technical Information of China (English)

    Ying Dou; Qing-ming Luo; Shumin Duan; Hang-jun Wu; Hui-quan Li; Song Qin; Yin-er Wang; Jing Li; Hui-fang Lou; Zhong Chen; Xiao-ming Li

    2012-01-01

    Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system.Attracted by factors released from damaged cells,microglia are recruited towards the damaged or infected site,where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris.ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury.However,the mechanisms of the long-range migration of microglia remain to be clarified.Here,we found that lysosomes in microglia contain abundant ATP and exhibit Ca2+-dependent exocytosis in response to various stimuli.By establishing an efficient in vitro chemotaxis assay,we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia,a response that was significantly inhibited in microglia treated with an agent inducing iysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice),a small GTPase required for the trafficking and exocytosis of secretory iysosomes.These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis,thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

  5. α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity*♦

    Science.gov (United States)

    Chen, Robert H. C.; Wislet-Gendebien, Sabine; Samuel, Filsy; Visanji, Naomi P.; Zhang, Gang; Marsilio, Diana; Langman, Tammy; Fraser, Paul E.; Tandon, Anurag

    2013-01-01

    α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity. PMID:23344955

  6. A Novel Aberrant Splice Site Mutation in RAB23 Leads to an Eight Nucleotide Deletion in the mRNA and Is Responsible for Carpenter Syndrome in a Consanguineous Emirati Family

    OpenAIRE

    2012-01-01

    Carpenter syndrome is caused by mutations in the RAB23 gene that encodes a small GTPase of the Rab subfamily of proteins. Rab proteins are known to be involved in the regulation of cellular trafficking and signal transduction. Currently, only few mutations in RAB23 have been reported in patients with Carpenter syndrome. In this paper, we report the clinical features, molecular and functional analysis of 2 children from an Emirati consanguineous family with this syndrome. The affected children...

  7. Expression profile of Rab5, Rab7, tryptophan aspartate-containing coat protein, leprae lipoarabinomannan, and phenolic glycolipid-1 on the failure of the phagolysosome process in macrophages of leprosy patients as a viability marker of Mycobacterium leprae

    Directory of Open Access Journals (Sweden)

    Cita Rosita Sigit Prakoeswa

    2016-01-01

    Conclusion: In M. leprae infection, Rab5, Rab7, and Lep-LAM play important roles in the failure of phagolysosome process via a membrane trafficking pathway, while PGL-1 plays a role via blocking lysosomal activities. These inventions might be used for the development of an early diagnostic device in the future.

  8. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

    Science.gov (United States)

    Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

    2012-04-10

    Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

  9. Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory granules.

    Science.gov (United States)

    Vazquez-Martinez, Rafael; Cruz-Garcia, David; Duran-Prado, Mario; Peinado, Juan R; Castaño, Justo P; Malagon, Maria M

    2007-07-01

    Rab proteins comprise a complex family of small GTPases involved in the regulation of intracellular membrane trafficking and reorganization. In this study, we identified Rab18 as a new inhibitory player of the secretory pathway in neuroendocrine cells. In adrenal chromaffin PC12 cells and pituitary AtT20 cells, Rab18 is located at the cytosol but associates with a subpopulation of secretory granules after stimulation of the regulated secretory pathway, strongly suggesting that induction of secretion provokes Rab18 activation and recruitment to these organelles. In support of this, a dominant-inactive Rab18 mutant was found to distribute diffusely in the cytosol, whereas a dominant-active Rab18 mutant was predominantly associated to secretory granules. Furthermore, interaction of Rab18 with secretory granules was associated to an inhibition in the secretory activity of PC12 and AtT20 cells in response to stimulatory challenges. Association of Rab18 with secretory granules was also observed by immunoelectron microscopy in normal, non-tumoral endocrine cells (pituitary melanotropes), wherein Rab18 protein content is inversely correlated to the level of secretory activity of cells. Taken together, these findings suggest that, in neuroendocrine cells, Rab18 acts as a negative regulator of secretory activity, likely by impairing secretory granule transport.

  10. Rab25 acts as an oncogene in luminal B breast cancer and is causally associated with Snail driven EMT.

    Science.gov (United States)

    Mitra, Shreya; Federico, Lorenzo; Zhao, Wei; Dennison, Jennifer; Sarkar, Tapasree Roy; Zhang, Fan; Takiar, Vinita; Cheng, Kwai W; Mani, Sendurai; Lee, Ju Seog; Mills, Gordon B

    2016-06-28

    The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions.

  11. RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

    Science.gov (United States)

    Singh, Chabungbam Orville; Xin, Hu-hu; Chen, Rui-ting; Wang, Mei-xian; Liang, Shuang; Lu, Yan; Cai, Zi-zheng; Zhang, Deng-pan; Miao, Yun-gen

    2015-06-01

    Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

  12. Rab proteins: the key regulators of intracellular vesicle transport.

    Science.gov (United States)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.

  13. The small GTP-binding protein rab4 is associated with early endosomes

    Energy Technology Data Exchange (ETDEWEB)

    van der Sluijs, P.; Hull, M.; Mellman, I. (Yale Univ. School of Medicine, New Haven, CT (United States)); Zahraoui, A.; Tavitian, A. (INSERM U 248, Paris (France)); Goud, B. (Unite de Genetique Somatique, Paris (France))

    1991-07-15

    Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. The authors demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of {approx} 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing {sup 125}-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. They also localized rab4p to tansferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4p cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular tansport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

  14. Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.

    Science.gov (United States)

    Díaz, Jorge; Mendoza, Pablo; Ortiz, Rina; Díaz, Natalia; Leyton, Lisette; Stupack, Dwayne; Quest, Andrew F G; Torres, Vicente A

    2014-06-01

    Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion.

  15. Critical importance of RAB proteins for synaptic function.

    Science.gov (United States)

    Mignogna, Maria Lidia; D'Adamo, Patrizia

    2017-02-01

    Neurons are highly polarized cells that exhibit one of the more complex morphology and function. Neuronal intracellular trafficking plays a key role in dictating the directionality and specificity of vesicle formation, transport and fusion, allowing the transmission of information in sophisticate cellular network. Thus, the integrity of protein trafficking and spatial organization is especially important in neuronal cells. RAB proteins, small monomeric GTPases belonging to the RAS superfamily, spatially and temporally orchestrate specific vesicular trafficking steps. In this review we summarise the known roles of RAB GTPases involved in the maintenance of neuronal vesicular trafficking in the central nervous system. In particular, we discriminate the axonal pre-synaptic trafficking and dendritic post-synaptic trafficking, to better underlie how a correct orchestration of vesicle movement is necessary to maintain neuronal polarity and then, to permit an accurate architecture and functionality of synaptic activity.

  16. Vps33B is required for delivery of endocytosed cargo to lysosomes

    NARCIS (Netherlands)

    Galmes, Romain; ten Brink, Corlinda; Oorschot, Viola; Veenendaal, Tineke; Jonker, Caspar; van der Sluijs, Peter; Klumperman, Judith

    2015-01-01

    In mammalian cells Vps33B forms a complex with VIPAS-39 that is recruited to recycling endosomes. Here we show that when Vps33B is expressed together with Rab7-interacting lysosomal protein (RILP) it is recruited to late endosomes-lysosomes and that depletion of Vps33B impairs late

  17. Spatial-Temporal Study of Rab1b Dynamics and Function at the ER-Golgi Interface.

    Science.gov (United States)

    Martinez, Hernán; García, Iris A; Sampieri, Luciana; Alvarez, Cecilia

    2016-01-01

    The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic.

  18. A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes.

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    Antonia E G Booth

    Full Text Available The mechanism(s by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1 as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP, is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.

  19. A Role for Na+,K+-ATPase α1 in Regulating Rab27a Localisation on Melanosomes

    Science.gov (United States)

    Booth, Antonia E. G.; Tarafder, Abul K.; Hume, Alistair N.; Recchi, Chiara; Seabra, Miguel C.

    2014-01-01

    The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation. PMID:25051489

  20. The novel Rab11-FIP/Rip/RCP family of proteins displays extensive homo- and hetero-interacting abilities.

    Science.gov (United States)

    Wallace, Deborah M; Lindsay, Andrew J; Hendrick, Alan G; McCaffrey, Mary W

    2002-04-12

    The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Here, we characterise several of these proteins with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. We now demonstrate that two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4 and show that several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end which includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. We identify a new (sixth) member of the protein family, which we propose to name Rab11-FIP4, and report the family evolutionary complexity and chromosomal distribution. Furthermore, we propose that the ability of these proteins to bind each other will be important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity.

  1. Spatial-Temporal Study of Rab1b Dynamics and Function at the ER-Golgi Interface

    Science.gov (United States)

    Martinez, Hernán; García, Iris A.; Sampieri, Luciana

    2016-01-01

    The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic. PMID:27500526

  2. RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport.

    Directory of Open Access Journals (Sweden)

    Caitlin A Taylor

    2015-12-01

    Full Text Available The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1.

  3. Rab from the white shrimp Litopenaeus vannamei: characterization and its regulation upon environmental stress.

    Science.gov (United States)

    Wang, Lei; Wang, Xiao-Rong; Liu, Jin; Chen, Chu-Xian; Liu, Yuan; Wang, Wei-Na

    2015-10-01

    With the destruction of the ecological environment, shrimp cultivation in China has been seriously affected by outbreaks of infectious diseases. Rab, which belong to small GTPase Ras superfamily, can regulate multiple steps in eukaryotic vesicle trafficking including vesicle budding, vesicle tethering, and membrane fusion. Knowledge of Rab in shrimp is essential to understanding regulation and detoxification mechanisms of environmental stress. In this study, we analyzed the functions of Rab from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNA of Rab was obtained, which was 751 bp long, with open reading frame encoding 206 amino acids. In this study, for the first time, the gene expression of Rab of L. vannamei was analyzed by quantitative real-time PCR after exposure to five kinds of environmental stresses (bacteria, pH, Cd, salinity and low temperature). The results demonstrate that Rab is sensitive and involved in bacteria, pH, and Cd stress responses and Rab is more sensitive to bacteria than other stresses. Therefore we infer that Rab may have relationship with the anti-stress mechanism induced by environment stress in shrimp and Rab could be used as critical biomarkers for environmental quality assessment.

  4. Endocytic turnover of Rab8 controls cell polarization

    Science.gov (United States)

    Vidal-Quadras, Maite; Holst, Mikkel R.; Larsson, Elin; Hachimi, Mariam; Yau, Wai-Lok; Peränen, Johan; Martín-Belmonte, Fernando

    2017-01-01

    ABSTRACT Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis. PMID:28137756

  5. Knockdown of Rab5a expression decreases cancer cell motility and invasion through integrin-mediated signaling pathway

    Directory of Open Access Journals (Sweden)

    Shi Shu-liang

    2011-08-01

    Full Text Available Abstract Background Rab GTPases function as modulators in intracellular transport. Rab5a, a member of the Rab subfamily of small GTPases, is an important regulator of vesicle traffic from the plasma membrane to early endosomes. Recent findings have reported that Rab5a gene was involved in the progression of cancer. In the present study, we investigated the effect of Rab5a on cervical cancer invasion and metastasis and the molecular mechanism underlying the involvement of Rab5a. Methods Rab5a expression was assessed by immunohistochemical analysis on a cervical cancer tissue microarray. RNA interference (RNAi was performed to knock down the endogenous expression of Rab5a gene in HeLa and SiHa cells. Cell motility was evaluated using invasion assay and wound migration assay in vitro. The expression levels of integrin-associated molecules were detected by Western blot and immunofluorescence. Results We found that Rab5a was expressed at a high level in cervical cancer tissues. Silencing of Rab5a expression significantly decreased cancer cell motility and invasiveness. The down-regulation of integrin-associated focal adhesion signaling molecules was further detected in Rab5a knockdown cells. Meanwhile, active GTP-bound Rac1, Cdc42, and RhoA were also down-regulated, accompanied with the reduction in the number and size of filopodia and lamellipodia. Conclusions Taken together, these data suggest that Rab5a functions in regulating the invasion phenotype, and we propose that this regulation may be via integrin-mediated signaling pathway in cervical cancer cells.

  6. Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs.

    Science.gov (United States)

    Sadacca, L Amanda; Bruno, Joanne; Wen, Jennifer; Xiong, Wenyong; McGraw, Timothy E

    2013-08-01

    Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

  7. The role of the small FTPase rab4 in endosome function

    NARCIS (Netherlands)

    Mohrmann, K.

    2001-01-01

    An important goal of studying membrane transport is to identify and characterize the regulatory proteins. RabGTPases are known to play an important role in the regulation of membrane transport (Mohrmann and van der Sluijs, 1999; Olkkonen and Stenmark, 1997; Takai et al., 2001). Initially it was thou

  8. Rab32 is important for autophagy and lipid storage in Drosophila.

    Directory of Open Access Journals (Sweden)

    Chao Wang

    Full Text Available Lipids are essential components of all organisms. Within cells, lipids are mainly stored in a specific type of organelle, called the lipid droplet. The molecular mechanisms governing the dynamics of lipid droplets have been little explored. The protein composition of lipid droplets has been analyzed in numerous proteomic studies, and a large number of lipid droplet-associated proteins have been identified, including Rab small GTPases. Rab proteins are known to participate in many intracellular membranous events; however, their exact role in lipid droplets is largely unexplored. Here we systematically investigate the roles of Drosophila Rab family proteins in lipid storage in the larval adipose tissue, fat body. Rab32 and several other Rabs were found to affect the size of lipid droplets as well as lipid levels. Further studies showed that Rab32 and Rab32 GEF/Claret may be involved in autophagy, consequently affecting lipid storage. Loss-of-function mutants of several components in the autophagy pathway result in similar effects on lipid storage. These results highlight the potential functions of Rabs in regulating lipid metabolism.

  9. Rab18 is reduced in pituitary tumors causing acromegaly and its overexpression reverts growth hormone hypersecretion.

    Science.gov (United States)

    Vazquez-Martinez, Rafael; Martinez-Fuentes, Antonio J; Pulido, Marina R; Jimenez-Reina, Luis; Quintero, Ana; Leal-Cerro, Alfonso; Soto, Alfonso; Webb, Susan M; Sucunza, Nuria; Bartumeus, Frederic; Benito-Lopez, Pedro; Galvez-Moreno, Maria A; Castaño, Justo P; Malagon, Maria M

    2008-06-01

    Rab proteins regulate the sequential steps of intracellular membrane transport. Alterations of these GTPases and their associated proteins are emerging as the underlying cause for several human diseases involving dysregulated secretory activities. Herein we investigated the role of Rab18, which negatively regulates hormone secretion by interacting with secretory granules, in relation to the altered functioning of tumoral pituitary somatotropes causing acromegaly. A total of 18 patients diagnosed with pituitary tumors causing acromegaly (nine patients) or nonfunctioning adenomas (nine patients) underwent endoscopic transsphenoidal surgery. Adenomas were subsequently processed to evaluate Rab18 production in relation to GH secretion. We found that somatotropinoma cells are characterized by a high secretory activity concomitantly with a remarkably reduced Rab18 expression (15%) and protein content levels (30%), as compared with cells from nonfunctioning pituitary adenomas derived from patients with normal or reduced GH plasma levels (100%). Furthermore, immunoelectron microscopy revealed that Rab18 association with the surface of GH-containing secretory granules was significantly lower in somatotropes from acromegalies than nonfunctioning pituitary adenomas. Finally, we provide evidence that modulation of Rab18 gene expression can revert substantially the hypersecretory activity of cells because Rab18 overexpression reduced by 40% the capacity of cells from acromegalies to respond to GHRH stimulation. These results suggest that molecular alterations affecting individual components of the secretory granule traffic machinery can contribute to maintain a high level of GH in plasma. Accordingly, Rab18 constitutes a valuable target as a diagnostic, prognostic, and/or therapeutic tool for human acromegaly.

  10. Four GTPases differentially regulate the Sec7 Arf-GEF to direct traffic at the trans-golgi network.

    Science.gov (United States)

    McDonold, Caitlin M; Fromme, J Christopher

    2014-09-29

    Traffic through the Golgi complex is controlled by small GTPases of the Arf and Rab families. Guanine nucleotide exchange factor (GEF) proteins activate these GTPases to control Golgi function, yet the full assortment of signals regulating these GEFs is unknown. The Golgi Arf-GEF Sec7 and the homologous BIG1/2 proteins are effectors of the Arf1 and Arl1 GTPases. We demonstrate that Sec7 is also an effector of two Rab GTPases, Ypt1 (Rab1) and Ypt31/32 (Rab11), signifying unprecedented signaling crosstalk between GTPase pathways. The molecular basis for the role of Ypt31/32 and Rab11 in vesicle formation has remained elusive. We find that Arf1, Arl1, and Ypt1 primarily affect the membrane localization of Sec7, whereas Ypt31/32 exerts a dramatic stimulatory effect on the nucleotide exchange activity of Sec7. The convergence of multiple signaling pathways on a master regulator reveals a mechanism for balancing incoming and outgoing traffic at the Golgi.

  11. Role of host GTPases in infection by Listeria monocytogenes.

    Science.gov (United States)

    Ireton, Keith; Rigano, Luciano A; Dowd, Georgina C

    2014-09-01

    The bacterial pathogen Listeria monocytogenes induces internalization into mammalian cells and uses actin-based motility to spread within tissues. Listeria accomplishes this intracellular life cycle by exploiting or antagonizing several host GTPases. Internalization into human cells is mediated by the bacterial surface proteins InlA or InlB. These two modes of uptake each require a host actin polymerization pathway comprised of the GTPase Rac1, nucleation promotion factors, and the Arp2/3 complex. In addition to Rac1, InlB-mediated internalization involves inhibition of the GTPase Arf6 and participation of Dynamin and septin family GTPases. After uptake, Listeria is encased in host phagosomes. The bacterial protein GAPDH inactivates the human GTPase Rab5, thereby delaying phagosomal acquisition of antimicrobial properties. After bacterial-induced destruction of the phagosome, cytosolic Listeria uses the surface protein ActA to stimulate actin-based motility. The GTPase Dynamin 2 reduces the density of microtubules that would otherwise limit bacterial movement. Cell-to-cell spread results when motile Listeria remodel the host plasma membrane into protrusions that are engulfed by neighbouring cells. The human GTPase Cdc42, its activator Tuba, and its effector N-WASP form a complex with the potential to restrict Listeria protrusions. Bacteria overcome this restriction through two microbial factors that inhibit Cdc42-GTP or Tuba/N-WASP interaction.

  12. Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

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    Yanan Hou

    Full Text Available The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1. Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

  13. Structure of Rab11-FIP3-Rabin8 reveals simultaneous binding of FIP3 and Rabin8 effectors to Rab11.

    Science.gov (United States)

    Vetter, Melanie; Stehle, Ralf; Basquin, Claire; Lorentzen, Esben

    2015-09-01

    The small GTPase Rab11 and its effectors FIP3 and Rabin8 are essential to membrane-trafficking pathways required for cytokinesis and ciliogenesis. Although effector binding is generally assumed to be sequential and mutually exclusive, we show that Rab11 can simultaneously bind FIP3 and Rabin8. We determined crystal structures of human Rab11-GMPPNP-Rabin8 and Rab11-GMPPNP-FIP3-Rabin8. The structures reveal that the C-terminal domain of Rabin8 adopts a previously undescribed fold that interacts with Rab11 at an unusual effector-binding site neighboring the canonical FIP3-binding site. We show that Rab11-GMPPNP-FIP3-Rabin8 is more stable than Rab11-GMPPNP-Rabin8, owing to direct interaction between Rabin8 and FIP3 within the dual effector-bound complex. The data allow us to propose a model for how membrane-targeting complexes assemble at the trans-Golgi network and recycling endosomes, through multiple weak interactions that create high-avidity complexes.

  14. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis.

    Science.gov (United States)

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F G; Torres, Vicente A

    2016-05-17

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis.

  15. Carpenter syndrome: extended RAB23 mutation spectrum and analysis of nonsense-mediated mRNA decay

    OpenAIRE

    2011-01-01

    Abstract Carpenter syndrome, a rare autosomal recessive disorder characterized by a combination of craniosynostosis, polysyndactyly, obesity, and other congenital malformations, is caused by mutations in RAB23, encoding a member of the Rab-family of small GTPases. In 15 out of 16 families previously reported, the disease was caused by homozygosity for truncating mutations, and currently only a single missense mutation has been identified in a compound heterozygote. Here, we describ...

  16. RAB23 Mutations in Carpenter Syndrome Imply an Unexpected Role for Hedgehog Signaling in Cranial-Suture Development and Obesity

    OpenAIRE

    2007-01-01

    Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative...

  17. RabGAP22 is required for defense to the vascular pathogen Verticillium longisporum and contributes to stomata immunity.

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    Jonas Roos

    Full Text Available Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro :GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL signaling gene BRI1-associated receptor kinase 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed serine:glyoxylate aminotransferase (AGT1 as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA and abscisic acid (ABA. An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity.

  18. Small GTPases and Brucella entry into the endoplasmic reticulum.

    Science.gov (United States)

    de Bolle, Xavier; Letesson, Jean-Jacques; Gorvel, Jean-Pierre

    2012-12-01

    A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.

  19. Rho GTPases and cancer

    DEFF Research Database (Denmark)

    Li, Hui; Peyrollier, Karine; Kilic, Gülcan;

    2014-01-01

    Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases...... are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho...

  20. Rab1b overexpression modifies Golgi size and gene expression in HeLa cells and modulates the thyrotrophin response in thyroid cells in culture.

    Science.gov (United States)

    Romero, Nahuel; Dumur, Catherine I; Martinez, Hernán; García, Iris A; Monetta, Pablo; Slavin, Ileana; Sampieri, Luciana; Koritschoner, Nicolas; Mironov, Alexander A; De Matteis, Maria Antonietta; Alvarez, Cecilia

    2013-03-01

    Rab1b belongs to the Rab-GTPase family that regulates membrane trafficking and signal transduction systems able to control diverse cellular activities, including gene expression. Rab1b is essential for endoplasmic reticulum-Golgi transport. Although it is ubiquitously expressed, its mRNA levels vary among different tissues. This work aims to characterize the role of the high Rab1b levels detected in some secretory tissues. We report that, in HeLa cells, an increase in Rab1b levels induces changes in Golgi size and gene expression. Significantly, analyses applied to selected genes, KDELR3, GM130 (involved in membrane transport), and the proto-oncogene JUN, indicate that the Rab1b increase acts as a molecular switch to control the expression of these genes at the transcriptional level, resulting in changes at the protein level. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element-binding protein consensus binding site in those target promoter regions. Moreover, our results reveal that, in a secretory thyroid cell line (FRTL5), Rab1b expression increases in response to thyroid-stimulating hormone (TSH). Additionally, changes in Rab1b expression in FRTL5 cells modify the specific TSH response. Our results show, for the first time, that changes in Rab1b levels modulate gene transcription and strongly suggest that a Rab1b increase is required to elicit a secretory response.

  1. The Role of Rab3a in Secretory Vesicle Docking Requires Association/Dissociation of Guanidine Phosphates and Munc18-1

    Science.gov (United States)

    van Weering, Jan R.T.; Toonen, Ruud F.; Verhage, Matthijs

    2007-01-01

    Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1. PMID:17637832

  2. The role of Rab3a in secretory vesicle docking requires association/dissociation of guanidine phosphates and Munc18-1.

    Directory of Open Access Journals (Sweden)

    Jan R T van Weering

    Full Text Available Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1.

  3. Rab14 limits the sorting of Glut4 from endosomes into insulin-sensitive regulated secretory compartments in adipocytes.

    Science.gov (United States)

    Brewer, Paul Duffield; Habtemichael, Estifanos N; Romenskaia, Irina; Coster, Adelle C F; Mastick, Cynthia Corley

    2016-05-15

    Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptor-related protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin.

  4. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  5. Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium.

    Science.gov (United States)

    Yang, Junjun; Yao, Wei; Qian, Guisheng; Wei, Zhenghua; Wu, Guangyu; Wang, Guansong

    2015-12-01

    The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.

  6. CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2.

    Directory of Open Access Journals (Sweden)

    Lin Sun

    Full Text Available Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.

  7. Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

    Science.gov (United States)

    Micaroni, Massimo; Stanley, Amanda C.; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X. F.; Lim, Jet P.; Marsh, Brad J.; Storrie, Brian; Gleeson, Paul A.; Stow, Jennifer L.

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6–GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages. PMID:23437303

  8. Rab6a/a' are important Golgi regulators of pro-inflammatory TNF secretion in macrophages.

    Science.gov (United States)

    Micaroni, Massimo; Stanley, Amanda C; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X F; Lim, Jet P; Marsh, Brad J; Storrie, Brian; Gleeson, Paul A; Stow, Jennifer L

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.

  9. A role for Rab14 in the endocytic trafficking of GLUT4 in 3T3-L1 adipocytes.

    Science.gov (United States)

    Reed, Sam E; Hodgson, Lorna R; Song, Shuang; May, Margaret T; Kelly, Eoin E; McCaffrey, Mary W; Mastick, Cynthia C; Verkade, Paul; Tavaré, Jeremy M

    2013-05-01

    Insulin enhances the uptake of glucose into adipocytes and muscle cells by promoting the redistribution of the glucose transporter isoform 4 (GLUT4) from intracellular compartments to the cell surface. Rab GTPases regulate the trafficking itinerary of GLUT4 and several have been found on immunopurified GLUT4 vesicles. Specifically, Rab14 has previously been implicated in GLUT4 trafficking in muscle although its role, if any, in adipocytes is poorly understood. Analysis of 3T3-L1 adipocytes using confocal microscopy demonstrated that endogenous GLUT4 and endogenous Rab14 exhibited a partial colocalisation. However, when wild-type Rab14 or a constitutively-active Rab14Q70L mutant were overexpressed in these cells, the colocalisation with both GLUT4 and IRAP became extensive. Interestingly, this colocalisation was restricted to enlarged 'ring-like' vesicular structures (mean diameter 1.3 µm), which were observed in the presence of overexpressed wild-type Rab14 and Rab14Q70L, but not an inactive Rab14S25N mutant. These enlarged vesicles contained markers of early endosomes and were rapidly filled by GLUT4 and transferrin undergoing endocytosis from the plasma membrane. The Rab14Q70L mutant reduced basal and insulin-stimulated cell surface GLUT4 levels, probably by retaining GLUT4 in an insulin-insensitive early endosomal compartment. Furthermore, shRNA-mediated depletion of Rab14 inhibited the transit of GLUT4 through early endosomal compartments towards vesicles and tubules in the perinuclear region. Given the previously reported role of Rab14 in trafficking between endosomes and the Golgi complex, we propose that the primary role of Rab14 in GLUT4 trafficking is to control the transit of internalised GLUT4 from early endosomes into the Golgi complex, rather than direct GLUT4 translocation to the plasma membrane.

  10. Crystallization of an engineered RUN domain of Rab6-interacting protein 1/DENND5

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, Humberto; Franklin, Edward; Khan, Amir R. (Trinity)

    2011-08-29

    Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains. Previously, successful purification of the RUN-PLAT tandem domains (residues 683-1061) of the 1263-residue Rab6-interacting protein 1 (R6IP1) required co-expression with Rab6, as attempts to solubly express the effector alone were unsuccessful. R6IP1 is also known as DENN domain-containing protein 5 (DENND5) and is expressed as two isoforms, R6IP1A/B (DENND5A/B), which differ by 24 amino acids at the N-terminus. Here, a deletion in R6IP1 was engineered to enable soluble expression and to improve the quality of the crystals grown in complex with Rab6. A large 23-residue loop linking two -helices in the RUN1 domain was removed and replaced with a short linker. This loop resides on the opposite face to the Rab6-binding site and is not conserved in the RUN-domain family. In contrast to wild-type R6IP1-Rab6 crystals, which took several weeks to grow to full size, the engineered R6IP1 (RPdel)-Rab6 crystals could be grown in a matter of days.

  11. GTPases in semaphorin signaling.

    Science.gov (United States)

    Püschel, Andreas W

    2007-01-01

    A hallmark of semaphorin receptors is their interaction with multiple GTPases. Plexins, the signal transducing component of semaphorin receptors, directly associate with several GTPases. In addition, they not only recruit guaninine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) but also are the only known integral membrane proteins that show a catalytic activity as GAPs for small GTPases. GTPases function upstream of semaphorin receptors and regulate the activity of plexins through an interaction with the cytoplasmic domain. The association of Plexin-Al (Sema3A receptor) or Plexin-B1 (Sema4D receptor) with the GTPase Rnd1 and ligand-dependent receptor clustering are required for their activity as R-Ras GAPs. The GTPases R-Ras and Rho function downstream of plexins and are required for the repulsive effects of semaphorins. In this review, I will focus on the role of GTPases in signaling by two plexins that have been analyzed in most detail, Plexin-A1 and Plexin-B1.

  12. RAB8B Is Required for Activity and Caveolar Endocytosis of LRP6

    Directory of Open Access Journals (Sweden)

    Kubilay Demir

    2013-09-01

    Full Text Available Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis.

  13. OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking.

    Science.gov (United States)

    Heo, Jae Bok; Rho, Hee Sun; Kim, Se Won; Hwang, Sung Min; Kwon, Hyun Jin; Nahm, Min Yeop; Bang, Woo Young; Bahk, Jeong Dong

    2005-12-01

    The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.

  14. Computational model explains high activity and rapid cycling of Rho GTPases within protein complexes.

    Directory of Open Access Journals (Sweden)

    Andrew B Goryachev

    2006-12-01

    Full Text Available Formation of multiprotein complexes on cellular membranes is critically dependent on the cyclic activation of small GTPases. FRAP-based analyses demonstrate that within protein complexes, some small GTPases cycle nearly three orders of magnitude faster than they would spontaneously cycle in vitro. At the same time, experiments report concomitant excess of the activated, GTP-bound form of GTPases over their inactive form. Intuitively, high activity and rapid turnover are contradictory requirements. How the cells manage to maximize both remains poorly understood. Here, using GTPases of the Rab and Rho families as a prototype, we introduce a computational model of the GTPase cycle. We quantitatively investigate several plausible layouts of the cycling control module that consist of GEFs, GAPs, and GTPase effectors. We explain the existing experimental data and predict how the cycling of GTPases is controlled by the regulatory proteins in vivo. Our model explains distinct and separable roles that the activating GEFs and deactivating GAPs play in the GTPase cycling control. While the activity of GTPase is mainly defined by GEF, the turnover rate is a sole function of GAP. Maximization of the GTPase activity and turnover rate places conflicting requirements on the concentration of GAP. Therefore, to achieve a high activity and turnover rate at once, cells must carefully maintain concentrations of GEFs and GAPs within the optimal range. The values of these optimal concentrations indicate that efficient cycling can be achieved only within dense protein complexes typically assembled on the membrane surfaces. We show that the concentration requirement for GEF can be dramatically reduced by a GEF-activating GTPase effector that can also significantly boost the cycling efficiency. Interestingly, we find that the cycling regimes are only weakly dependent on the concentration of GTPase itself.

  15. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

    Science.gov (United States)

    Wyroba, E; Surmacz, L; Osinska, M; Wiejak, J

    2007-01-01

    Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  16. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

    Directory of Open Access Journals (Sweden)

    E Wyroba

    2009-08-01

    Full Text Available Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein and LAMP-2 (lysosomal membrane protein 2 as well as a7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2 in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100 000 x g of equal load were quantified by immunoblotting. LAMP-2 crossreacting polypeptide of ~106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The a7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILPrelated polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  17. Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells.

    Science.gov (United States)

    Zou, Jianlong; Hu, Bo; Arpag, Sezgi; Yan, Qing; Hamilton, Audra; Zeng, Yuan-Shan; Vanoye, Carlos G; Li, Jun

    2015-04-29

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

  18. Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

    Science.gov (United States)

    Lu, Yingying; Hao, Bai-Xia; Graeff, Richard; Wong, Connie W M; Wu, Wu-Tian; Yue, Jianbo

    2013-08-16

    Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

  19. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

    Science.gov (United States)

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2016-06-01

    The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation.

  20. Rho GTPase function in development

    DEFF Research Database (Denmark)

    Pedersen, Esben; Brakebusch, Cord

    2012-01-01

    Rho GTPase functions have been carefully investigated for many years using cell biological models. In recent years, mouse models with targeted mutations in Rho GTPase genes enabled the study of Rho GTPase function in vivo, partially confirming and partially contradicting expectations based...... on earlier in vitro experiments. This review sums up recent findings on the role of Rho GTPases in development, underlining the importance of in vivo research for our understanding of Rho GTPases in living organisms, and describing challenges for the future....

  1. Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica.

    Science.gov (United States)

    Liu, Zhao-liang; Luo, Cong; Dong, Long; Van Toan, Can; Wei, Peng-xiao; He, Xin-hua

    2014-04-25

    The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984bp and contained an open reading frame of 600bp, which encoded a 200 amino acid protein with a molecular weight of 21.83kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.

  2. Expression profile of Rab5, Rab7, tryptophan aspartate-containing coat protein, leprae lipoarabinomannan, and phenolic glycolipid-1 on the failure of the phagolysosome process in macrophages of leprosy patients as a viability marker of Mycobacterium leprae.

    Science.gov (United States)

    Prakoeswa, Cita Rosita Sigit; Wahyuni, Ratna; Iswahyudi; Adriaty, Dinar; Yusuf, Irawan; Sutjipto; Agusni, Indropo; Izumi, Shinzo

    2016-06-01

    Phagolysosome process in macrophage of leprosy patients' is important in the early phase of eliminating Mycobacterium leprae invasion. This study was to clarify the involvement of Rab5, Rab7, and trytophan aspartate-containing coat protein (TACO) from host macrophage and leprae lipoarabinomannan (Lep-LAM) and phenolic glycolipid-1 (PGL-1) from M. leprae cell wall as the reflection of phagolysosome process in relation to 16 subunit ribosomal RNA (16S rRNA) M. leprae as a marker of viability of M. leprae. Using a cross sectional design study, skin biopsies were obtained from 47 newly diagnosed, untreated leprosy at Dr Soetomo Hospital, Surabaya, Indonesia. RNA isolation and complementary DNA synthesis were performed. Samples were divided into two groups: 16S rRNA M. leprae-positive and 16S rRNA M. leprae-negative. The expressions of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 were assessed with an immunohistochemistry technique. Using Mann-Whitney U analysis, a significant difference in the expression profile of Rab5, Rab7, Lep-LAM, and PGL-1 was found (p.05). Spearman analysis revealed that there was a significant correlation between the score of Rab5, Rab7, Lep-LAM, and PGL-1 and the score of 16S rRNA M. leprae (p<.05). In M. leprae infection, Rab5, Rab7, and Lep-LAM play important roles in the failure of phagolysosome process via a membrane trafficking pathway, while PGL-1 plays a role via blocking lysosomal activities. These inventions might be used for the development of an early diagnostic device in the future. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  3. The large GTPase Mx1 is involved in apical transport in MDCK cells.

    Science.gov (United States)

    Hoff, Florian; Greb, Christoph; Hollmann, Christina; Hönig, Ellena; Jacob, Ralf

    2014-09-01

    In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin-related large GTPase Mx1 is a component of post-Golgi vesicles carrying the neurotrophin receptor p75(NTR) . Moreover, siRNA-mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.

  4. A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor.

    Directory of Open Access Journals (Sweden)

    Shicong Wang

    Full Text Available Rab29 (also referred as Rab7L1 is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN, and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR, and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR or calcium independent M6PR (ciM6PR, but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G. Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs.

  5. A Role of Rab29 in the Integrity of the Trans-Golgi Network and Retrograde Trafficking of Mannose-6-Phosphate Receptor

    Science.gov (United States)

    Wang, Shicong; Ma, Zexu; Xu, Xiaohui; Wang, Zhen; Sun, Lixiang; Zhou, Yunhe; Lin, Xiaosi; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    Rab29 (also referred as Rab7L1) is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN), and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR), and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR) or calcium independent M6PR (ciM6PR), but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G). Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs. PMID:24788816

  6. The localization of the Golgin GCC185 is independent of Rab6A/A' and Arl1.

    Science.gov (United States)

    Houghton, Fiona J; Chew, Pau Ling; Lodeho, Sylvain; Goud, Bruno; Gleeson, Paul A

    2009-08-21

    Mammalian golgins of the trans-Golgi network (TGN) are small G protein effectors that are required for membrane transport and contain a Golgi targeting C-terminal GRIP domain. The localization of two TGN golgins, p230/golgin-245 and golgin-97, is mediated by the small GTPase Arl1, whereas recruitment of the TGN golgin GCC185 is controversial. Recently, GCC185 was proposed to localize to the Golgi by the co-operation of two small GTPases, Rab6A/A' and Arl1 (Burguete et al., 2008), a model based predominantly on in vitro interactions. Here we demonstrate that Golgi recruitment of endogenous GCC185 does not involve Rab6A/A' and Arl1. We find minimal colocalization between Rab6A/A' and endogenous GCC185 on Golgi membranes and failed to detect an interaction between Rab6A/A' and C-terminal domains of GCC185 by yeast two-hybrid analyses. Moreover, depletion of both Rab6A/A' and Arl1 also had no effect on the localization of endogenous GCC185 or the isolated GRIP domain of GCC185.

  7. Multiple Roles of VARP in Endosomal Trafficking: Rabs, Retromer Components and R-SNARE VAMP7 Meet on VARP.

    Science.gov (United States)

    Fukuda, Mitsunori

    2016-07-01

    VARP (VPS9-ankyrin-repeat protein, also known as ANKRD27) was originally identified as an N-terminal VPS9 (vacuolar protein sorting 9)-domain-containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP-interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R-SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer-mediated endosome-to-plasma membrane sorting of transmembrane proteins.

  8. A Rab32-dependent pathway contributes to Salmonella typhi host restriction.

    Science.gov (United States)

    Spanò, Stefania; Galán, Jorge E

    2012-11-16

    Unlike other Salmonellae, the intracellular bacterial human pathogen Salmonella Typhi exhibits strict host specificity. The molecular bases for this restriction are unknown. Here we found that the expression of a single type III secretion system effector protein from broad-host Salmonella Typhimurium allowed Salmonella Typhi to survive and replicate within macrophages and tissues from mice, a nonpermissive host. This effector proteolytically targeted Rab32, which controls traffic to lysosome-related organelles in conjunction with components of the biogenesis of lysosome-related organelle complexes (BLOCs). RNA interference-mediated depletion of Rab32 or of an essential component of a BLOC complex was sufficient to allow S. Typhi to survive within mouse macrophages. Furthermore, S. Typhi was able to survive in macrophages from mice defective in BLOC components.

  9. 32 CFR 202.10 - RAB adjournment and dissolution.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false RAB adjournment and dissolution. 202.10 Section... dissolution. (a) RAB adjournment—(1) Requirements for RAB adjournment. An Installation Commander may adjourn a... Commander decides to adjourn the RAB. (b) RAB dissolution—(1) Requirements for RAB dissolution. An...

  10. TRPML and lysosomal function.

    Science.gov (United States)

    Zeevi, David A; Frumkin, Ayala; Bach, Gideon

    2007-08-01

    Mucolipin 1 (MLN1), also known as TRPML1, is a member of the mucolipin family. The mucolipins are the only lysosomal proteins within the TRP superfamily. Mutations in the gene coding for TRPML1 result in a lysosomal storage disorder (LSD). This review summarizes the current knowledge related to this protein and the rest of the mucolipin family.

  11. The proteome of lysosomes.

    Science.gov (United States)

    Schröder, Bernd A; Wrocklage, Christian; Hasilik, Andrej; Saftig, Paul

    2010-11-01

    Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

  12. Increased neuronal Rab5 immunoreactive endosomes do not colocalize with TDP-43 in motor neuron disease.

    Science.gov (United States)

    Matej, Radoslav; Botond, Gergö; László, Lajos; Kopitar-Jerala, Natasa; Rusina, Robert; Budka, Herbert; Kovacs, Gabor G

    2010-09-01

    Sporadic motor neuron disease (MND) is characterized by progressive degeneration of motor neurons and intraneuronal cytoplasmic translocation and deposition of the nuclear protein TDP-43. There is a paucity of data on the subcellular mechanisms of the nuclear-cytoplasmic trafficking of TDP-43, particularly about the precise role of the endosomal-lysosomal system (ELS). In the present study, using a neuron-specific morphometric approach, we examined the expression of the early endosomal marker Rab5 and lysosomal cathepsins B, D, F, and L as well as PAS-stained structures in the anterior horn cells in 11 individuals affected by sporadic MND and 5 age-matched controls. This was compared with the expression of ubiquitin, p62 and TDP-43 and its phosphorylated form. The principal finding was the increased expression of the endosomal marker Rab5 and lysosomal cathepsin D, and of PAS-positive structures in motor neurons of MND cases. Furthermore, the area-portion of Rab5 immunoreactivity correlated well with the intracellular accumulation of ubiquitin, p62 and (phosphorylated) TDP-43. However, double immunolabelling and immunogold electron microscopy excluded colocalization of phosphorylated TDP-43 with the ELS. These data contrast with observations on neuronal cytopathology in Alzheimer's or prion diseases where the disease-specific proteins are processed within endosomes, and suggest a distinct role of the ELS in MND.

  13. Rab1b overexpression modifies Golgi size and gene expression in HeLa cells and modulates the thyrotrophin response in thyroid cells in culture

    Science.gov (United States)

    Romero, Nahuel; Dumur, Catherine I.; Martinez, Hernán; García, Iris A.; Monetta, Pablo; Slavin, Ileana; Sampieri, Luciana; Koritschoner, Nicolas; Mironov, Alexander A.; De Matteis, Maria Antonietta; Alvarez, Cecilia

    2013-01-01

    Rab1b belongs to the Rab-GTPase family that regulates membrane trafficking and signal transduction systems able to control diverse cellular activities, including gene expression. Rab1b is essential for endoplasmic reticulum–Golgi transport. Although it is ubiquitously expressed, its mRNA levels vary among different tissues. This work aims to characterize the role of the high Rab1b levels detected in some secretory tissues. We report that, in HeLa cells, an increase in Rab1b levels induces changes in Golgi size and gene expression. Significantly, analyses applied to selected genes, KDELR3, GM130 (involved in membrane transport), and the proto-oncogene JUN, indicate that the Rab1b increase acts as a molecular switch to control the expression of these genes at the transcriptional level, resulting in changes at the protein level. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element-binding protein consensus binding site in those target promoter regions. Moreover, our results reveal that, in a secretory thyroid cell line (FRTL5), Rab1b expression increases in response to thyroid-stimulating hormone (TSH). Additionally, changes in Rab1b expression in FRTL5 cells modify the specific TSH response. Our results show, for the first time, that changes in Rab1b levels modulate gene transcription and strongly suggest that a Rab1b increase is required to elicit a secretory response. PMID:23325787

  14. Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering.

    Science.gov (United States)

    Peurois, François; Veyron, Simon; Ferrandez, Yann; Ladid, Ilham; Benabdi, Sarah; Zeghouf, Mahel; Peyroche, Gérald; Cherfils, Jacqueline

    2017-02-14

    Attachment of active, GTP-bound small GTPases to membranes by post-translational lipid modifications is pivotal for their ability to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by GEFs and their termination by GAPs. Here we replaced the lipid modification by a histidine tag in eleven full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel-lipid containing membranes and characterize the kinetics of their activation by GEFs. Remarkably this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio towards RhoG by ≈30 fold and Rac1 by ≈10 fold, and uncovered a previously unknown broader specificity towards RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine tag strategy as a potent and simple means to mimic small GTPases lipidation, which opens broad perspectives of applications to uncover regulations brought about by membranes.

  15. Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion.

    Science.gov (United States)

    Gundry, Christine; Marco, Sergi; Rainero, Elena; Miller, Bryan; Dornier, Emmanuel; Mitchell, Louise; Caswell, Patrick T; Campbell, Andrew D; Hogeweg, Anna; Sansom, Owen J; Morton, Jennifer P; Norman, Jim C

    2017-03-15

    The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser(435) by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser(897) by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma-whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis-indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo.

  16. Rab18 is required for viral assembly of hepatitis C virus through trafficking of the core protein to lipid droplets.

    Science.gov (United States)

    Dansako, Hiromichi; Hiramoto, Hiroki; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2014-08-01

    During persistent infection of HCV, the HCV core protein (HCV-JFH-1 strain of genotype 2a) is recruited to lipid droplets (LDs) for viral assembly, but the mechanism of recruitment of the HCV core protein is uncertain. Here, we demonstrated that one of the Ras-related small GTPases, Rab18, was required for trafficking of the core protein around LDs. The knockdown of Rab18 reduced intracellular and extracellular viral infectivity, but not intracellular viral replication in HCV-JFH-1-infected RSc cells (an HuH-7-derived cell line). Exogenous expression of Rab18 increased extracellular viral infectivity almost two-fold. Furthermore, Rab18 was co-localized with the core protein in HCV-JFH-1-infected RSc cells, and the knockdown of Rab18 blocked recruitment of the HCV-JFH-1 core protein to LDs. These results suggest that Rab18 has an important role in viral assembly through the trafficking of the core protein to LDs.

  17. Tumor susceptibility gene 101 (TSG101 is a novel binding-partner for the class II Rab11-FIPs.

    Directory of Open Access Journals (Sweden)

    Conor P Horgan

    Full Text Available The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101, a component of the ESCRT-I (endosomal sorting complex required for transport complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway.

  18. Atypical GTPases as drug targets.

    Science.gov (United States)

    Soundararajan, Meera; Eswaran, Jeyanthy

    2012-01-01

    The Ras GTPases are the founding members of large Ras superfamily, which constitutes more than 150 of these important class of enzymes. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. There are a number of GTPases that have been identified recently, which do not confine to this prototype termed as "atypical GTPases" but have proved to play a remarkable role in vital cellular functions. In this review, we provide an overview of the crucial physiological functions mediated by RGK and Centaurin class of multi domain atypical GTPases. Moreover, the recently available atypical GTPase structures of the two families, regulation, physiological functions and their critical roles in various diseases will be discussed. In summary, this review will highlight the emerging atypical GTPase family which allows us to understand novel regulatory mechanisms and thus providing new avenues for drug discovery programs.

  19. AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes.

    Directory of Open Access Journals (Sweden)

    Viorica Ivan

    Full Text Available The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.

  20. Rab33b and Rab6 are functionally overlapping regulators of Golgi homeostasis and trafficking.

    Science.gov (United States)

    Starr, Tregei; Sun, Yi; Wilkins, Ngozi; Storrie, Brian

    2010-05-01

    We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra-Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra-Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex- or Zeste White 10 (ZW10)-depletion induced disruption of the Golgi ribbon in HeLa cells. Moreover, efficient GTP-restricted Rab6 induced relocation of Golgi enzymes to the endoplasmic reticulum (ER) was Rab33b-dependent, but not vice versa, suggesting that the two Rabs act sequentially in an intra-Golgi Rab cascade. In support of this hypothesis, we found that overexpression of GTP-Rab33b induced the dissociation of Rab6 from Golgi membranes in vivo. In addition, the transport of Shiga-like toxin B fragment (SLTB) from the trans to cis Golgi and ER required Rab33b. Surprisingly, depletion of Rab33b had little, if any, immediate effect on cell growth and multiplication. Furthermore, anterograde trafficking of tsO45G protein through the Golgi apparatus was normal. We suggest that the Rab33b/Rab6 regulated intra-Golgi retrograde trafficking pathway must coexist with other Golgi trafficking pathways. In conclusion, we provide the first evidence that Rab33b and Rab6 act to coordinate a major intra-Golgi retrograde trafficking pathway. This coordination may have parallels with Rab conversion/cascade events that regulate endosome, phagosome and exocytic processes.

  1. Lysosome Biogenesis and Autophagy

    NARCIS (Netherlands)

    Reggiori, Fulvio; Klumperman, Judith|info:eu-repo/dai/nl/075097273

    2016-01-01

    Lysosomes degrade biological components acquired by endocytosis, the major cellular pathway for internalization of extracellular material, and macroautophagy. This chapter presents an overview of these two major degradative intracellular pathways, and highlights the emerging cross talks between

  2. RHO GTPase in plants

    Science.gov (United States)

    2010-01-01

    Plants possess a single subfamily of Rho GTPases, ROP, which does usual things as do Rho-family GTPases in animal and fungal systems, namely participating in the spatial control of cellular processes by signaling to the cytoskeleton and vesicular trafficking. As one would expect, ROPs are modulated by conserved regulators such as DHR2-type GEFs, RhoGAPs and Rho GDIs. What is surprising is that plants have invented new regulators such as PRONE-type GEFs (known as RopGEFs) and effectors such as RICs and ICRs/RIPs in the regulation of the cytoskeleton and vesicular trafficking. This review will discuss recent work on characterizing ROP regulators and effectors as well as addressing why and how a mixture of conserved and novel Rho signaling mechanisms is utilized to modulate fundamental cellular processes such as cytoskeletal dynamics/reorganization and vesicular trafficking. PMID:21686259

  3. Characterization of the role Rab25 in energy metabolism and cancer using extracellular flux analysis and material balance.

    Science.gov (United States)

    Mitra, Shreya; Molina, Jennifer; Mills, Gordon B; Dennison, Jennifer B

    2015-01-01

    Rab25, by altering trafficking of critical cellular resources, influences cell metabolism and survival during stress conditions. Overall, perturbations in the vesicular trafficking machinery change cellular bioenergetics that can be directly measured in real time as Oxygen Consumption Rate, OCR (mitochondrial respiration) and Extracellular Acidification Rate, ECAR (glycolysis) by an extracellular flux analyzer (XF96, Seahorse Biosciences, MA). Additionally, overall turnover of glucose, lactate, as well as glutamine and glutamate can be measured biochemically using the YSI2900 Biochemistry Analyzer (YSI Incorporated, Life Sciences, OH). A combination of these two methods allows a precise and quantitative approach to interrogate the role of Rab25 as well as other Rab GTPases in central carbon energy metabolism.

  4. Structural basis for Rab1 de-AMPylation by the Legionella pneumophila effector SidD.

    Directory of Open Access Journals (Sweden)

    Yang Chen

    Full Text Available The covalent attachment of adenosine monophosphate (AMP to proteins, a process called AMPylation (adenylylation, has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

  5. Zebrafish Rab5 proteins and a role for Rab5ab in nodal signalling.

    Science.gov (United States)

    Kenyon, Emma J; Campos, Isabel; Bull, James C; Williams, P Huw; Stemple, Derek L; Clark, Matthew D

    2015-01-15

    The RAB5 gene family is the best characterised of all human RAB families and is essential for in vitro homotypic fusion of early endosomes. In recent years, the disruption or activation of Rab5 family proteins has been used as a tool to understand growth factor signal transduction in whole animal systems such as Drosophila melanogaster and zebrafish. In this study we have examined the functions for four rab5 genes in zebrafish. Disruption of rab5ab expression by antisense morpholino oligonucleotide (MO) knockdown abolishes nodal signalling in early zebrafish embryos, whereas overexpression of rab5ab mRNA leads to ectopic expression of markers that are normally downstream of nodal signalling. By contrast MO disruption of other zebrafish rab5 genes shows little or no effect on expression of markers of dorsal organiser development. We conclude that rab5ab is essential for nodal signalling and organizer specification in the developing zebrafish embryo.

  6. Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

    Science.gov (United States)

    Cromm, Philipp M; Schaubach, Sebastian; Spiegel, Jochen; Fürstner, Alois; Grossmann, Tom N; Waldmann, Herbert

    2016-04-14

    Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

  7. Rho GTPases in platelet function.

    Science.gov (United States)

    Aslan, J E; McCarty, O J T

    2013-01-01

    The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low-molecular-weight or 'small' GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non-transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho-driven cellular processes. © 2012 International Society on Thrombosis and Haemostasis.

  8. The universally conserved prokaryotic GTPases.

    Science.gov (United States)

    Verstraeten, Natalie; Fauvart, Maarten; Versées, Wim; Michiels, Jan

    2011-09-01

    Members of the large superclass of P-loop GTPases share a core domain with a conserved three-dimensional structure. In eukaryotes, these proteins are implicated in various crucial cellular processes, including translation, membrane trafficking, cell cycle progression, and membrane signaling. As targets of mutation and toxins, GTPases are involved in the pathogenesis of cancer and infectious diseases. In prokaryotes also, it is hard to overestimate the importance of GTPases in cell physiology. Numerous papers have shed new light on the role of bacterial GTPases in cell cycle regulation, ribosome assembly, the stress response, and other cellular processes. Moreover, bacterial GTPases have been identified as high-potential drug targets. A key paper published over 2 decades ago stated that, "It may never again be possible to capture [GTPases] in a family portrait" (H. R. Bourne, D. A. Sanders, and F. McCormick, Nature 348:125-132, 1990) and indeed, the last 20 years have seen a tremendous increase in publications on the subject. Sequence analysis identified 13 bacterial GTPases that are conserved in at least 75% of all bacterial species. We here provide an overview of these 13 protein subfamilies, covering their cellular functions as well as cellular localization and expression levels, three-dimensional structures, biochemical properties, and gene organization. Conserved roles in eukaryotic homologs will be discussed as well. A comprehensive overview summarizing current knowledge on prokaryotic GTPases will aid in further elucidating the function of these important proteins.

  9. Bacterial Cytotoxins Target Rho GTPases

    Science.gov (United States)

    Schmidt, Gudula; Aktories, Klaus

    1998-06-01

    Low molecular mass GTPases of the Rho family, which are involved in the regulation of the actin cytoskeleton and in various signal transduction processes, are the eukaryotic targets of bacterial protein toxins. The toxins covalently modify Rho proteins by ADP ribosylation, glucosylation, and deamidation, thereby inactivating and activating the GTPases.

  10. RAB23 Mutations in Carpenter Syndrome Imply an Unexpected Role for Hedgehog Signaling in Cranial-Suture Development and Obesity

    Science.gov (United States)

    Jenkins, Dagan ; Seelow, Dominik ; Jehee, Fernanda S. ; Perlyn, Chad A. ; Alonso, Luís G. ; Bueno, Daniela F. ; Donnai, Dian ; Josifiova, Dragana ; Mathijssen, Irene M. J. ; Morton, Jenny E. V. ; Ørstavik, Karen Helene ; Sweeney, Elizabeth ; Wall, Steven A. ; Marsh, Jeffrey L. ; Nürnberg, Peter ; Passos-Bueno, Maria Rita ; Wilkie, Andrew O. M. 

    2007-01-01

    Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis—an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components—and provides a new molecular target for studies of obesity. PMID:17503333

  11. Carpenter syndrome: extended RAB23 mutation spectrum and analysis of nonsense-mediated mRNA decay.

    Science.gov (United States)

    Jenkins, Dagan; Baynam, Gareth; De Catte, Luc; Elcioglu, Nursel; Gabbett, Michael T; Hudgins, Louanne; Hurst, Jane A; Jehee, Fernanda Sarquis; Oley, Christine; Wilkie, Andrew O M

    2011-04-01

    Carpenter syndrome, a rare autosomal recessive disorder characterized by a combination of craniosynostosis, polysyndactyly, obesity, and other congenital malformations, is caused by mutations in RAB23, encoding a member of the Rab-family of small GTPases. In 15 out of 16 families previously reported, the disease was caused by homozygosity for truncating mutations, and currently only a single missense mutation has been identified in a compound heterozygote. Here, we describe a further 8 independent families comprising 10 affected individuals with Carpenter syndrome, who were positive for mutations in RAB23. We report the first homozygous missense mutation and in-frame deletion, highlighting key residues for RAB23 function, as well as the first splice-site mutation. Multi-suture craniosynostosis and polysyndactyly have been present in all patients described to date, and abnormal external genitalia have been universal in boys. High birth weight was not evident in the current group of patients, but further evidence for laterality defects is reported. No genotype-phenotype correlations are apparent. We provide experimental evidence that transcripts encoding truncating mutations are subject to nonsense-mediated decay, and that this plays an important role in the pathogenesis of many RAB23 mutations. These observations refine the phenotypic spectrum of Carpenter syndrome and offer new insights into molecular pathogenesis.

  12. RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity.

    Science.gov (United States)

    Jenkins, Dagan; Seelow, Dominik; Jehee, Fernanda S; Perlyn, Chad A; Alonso, Luis G; Bueno, Daniela F; Donnai, Dian; Josifova, Dragana; Josifiova, Dragana; Mathijssen, Irene M J; Morton, Jenny E V; Orstavik, Karen Helene; Sweeney, Elizabeth; Wall, Steven A; Marsh, Jeffrey L; Nurnberg, Peter; Passos-Bueno, Maria Rita; Wilkie, Andrew O M

    2007-06-01

    Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.

  13. Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells.

    Science.gov (United States)

    Cebrian, Ignacio; Croce, Cristina; Guerrero, Néstor A; Blanchard, Nicolas; Mayorga, Luis S

    2016-12-01

    Cross-presentation by MHC class I molecules allows the detection of exogenous antigens by CD8(+) T lymphocytes. This process is crucial to initiate cytotoxic immune responses against many pathogens (i.e., Toxoplasma gondii) and tumors. To achieve efficient cross-presentation, dendritic cells (DCs) have specialized endocytic pathways; however, the molecular effectors involved are poorly understood. In this work, we identify the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. Our results demonstrate that Rab22a is recruited to DC endosomes and phagosomes, as well as to the vacuole containing T. gondii parasites. The silencing of Rab22a expression did not affect the uptake of exogenous antigens or parasite invasion, but it drastically reduced the intracellular pool and the recycling of MHC-I molecules. The knockdown of Rab22a also hampered the cross-presentation of soluble, particulate and T. gondii-associated antigens, but not the endogenous MHC-I antigen presentation through the classical secretory pathway. Our findings provide compelling evidence that Rab22a plays a central role in the MHC-I endocytic trafficking, which is crucial for efficient cross-presentation by DCs.

  14. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Directory of Open Access Journals (Sweden)

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  15. RAB13 — EDRN Public Portal

    Science.gov (United States)

    RAB13 is a member of the Rab family of small G proteins and plays a role in regulating membrane trafficking between trans-Golgi network (TGN) and recycling endosomes (RE). RAB13 is thought to participate in polarized transport, in the assembly and/or the activity of tight junctions, which are components of the apical junctional complex (AJC) of epithelial cells. There are several alternatively spliced transcript variants for this gene, and a pseudogene located on chromosome 12.

  16. Rho GTPase function in tumorigenesis

    DEFF Research Database (Denmark)

    Karlsson, R; Pedersen, Esben Ditlev Kølle; Wang, Zhipeng;

    2009-01-01

    Malignant tumor cells display uncontrolled proliferation, loss of epithelial cell polarity, altered interactions with neighboring cells and the surrounding extracellular matrix, and enhanced migratory properties. Proteins of the Rho GTPase family regulate all these processes in cell culture and......, for that reason, Rho GTPases, their regulators, and their effectors have been suggested to control tumor formation and progression in humans. However, while the tumor-relevant functions of Rho GTPases are very well documented in vitro, we are only now beginning to assess their contribution to cancer in human...... patients and in animal models. This review will give a very brief overview of Rho GTPase function in general and then focus on in vivo evidence for a role of Rho GTPases in malignant tumors, both in human patients and in genetically modified mice....

  17. Putative Vitis vinifera Rop- and Rab-GAP-, GEF-, and GDI-interacting proteins uncovered with novel methods for public genomic and EST database analysis.

    Science.gov (United States)

    Abbal, Philippe; Tesniere, Catherine

    2010-01-01

    To understand how grapevine Rop and Rab proteins achieve their functional versatility in signalling, identification of the putative VvRop- and VvRab-interacting proteins was performed using newly designed tools. In this study, sequences encoding eight full-length proteins for VvRop GTPase-activating proteins (GAPs), five for VvRabGAPs, six for VvRop guanine nucleotide exchange factors (GEFs), one for VvRabGEF, five for VvRop GDP dissociation inhibitors (GDIs), and three for VvRabGDIs were identified. These proteins had a CRIB motif or PH domain, a TBC domain, a PRONE domain, a DENN domain, or GDI signatures, respectively. By bootstrap analysis, an unrooted consensus phylogenetic tree was constructed which indicated that VvRopGDIs and VvRopGEFs--but not VvRopGAP--belonged to the same clade, and that VvRabGEF1 protein was more closely related to VvRopGAPs than to the other putative VvRab-interacting proteins. Twenty-two genes out of 28 encoding putative VvRop- and VvRab-interacting proteins could be located on identified grapevine chromosomes. Generally one gene was anchored on one chromosome, but in some cases up to four genes were located on the same chromosome. Expression patterns of the genes encoding putative VvRop- and VvRab-interacting proteins were also examined using a newly developed tool based on public expressed sequence tag (EST) database analysis. Expression patterns were sometimes found to be specific to an organ or a developmental stage. Although some limitations exist, the use of EST database analysis is stressed, in particular in the case of species where expression data are obtained at high costs in terms of time and effort.

  18. Charcot-Marie-Tooth 2b associated Rab7 mutations cause axon growth and guidance defects during vertebrate sensory neuron development.

    Science.gov (United States)

    Ponomareva, Olga Y; Eliceiri, Kevin W; Halloran, Mary C

    2016-01-20

    Charcot-Marie-Tooth2b (CMT2b) is an axonal form of a human neurodegenerative disease that preferentially affects sensory neurons. CMT2b is dominantly inherited and is characterized by unusually early onset, presenting in the second or third decade of life. Five missense mutations in the gene encoding Rab7 GTPase have been identified as causative in human CMT2b disease. Although several studies have modeled CMT2b disease in cultured neurons and in Drosophila, the mechanisms by which defective Rab7 leads to disease remain poorly understood. We used zebrafish to investigate the effects of CMT2b-associated Rab7 mutations in a vertebrate model. We generated transgenic animals expressing the CMT2b-associated mutant forms of Rab7 in sensory neurons, and show that these Rab7 variants cause neurodevelopmental defects, including defects in sensory axon growth, branching and pathfinding at early developmental stages. We also find reduced axon growth and branching in neurons expressing a constitutively active form of Rab7, suggesting these defects may be caused by Rab7 gain-of-function. Further, we use high-speed, high-resolution imaging of endosome transport in vivo and find that CMT2b-associated Rab7 variants cause reduced vesicle speeds, suggesting altered transport may underlie axon development defects. Our data provide new insight into how disease-associated alterations in Rab7 protein disrupt cellular function in vertebrate sensory neurons. Moreover, our findings suggest that defects in axon development may be a previously unrecognized component of CMT2b disease.

  19. Interaction of LRRK2 with kinase and GTPase signaling cascades

    Directory of Open Access Journals (Sweden)

    Joon Y Boon

    2014-07-01

    Full Text Available LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease. Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in C. elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the MAPK pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GAPs and GEFs are another strong theme in LRRK2 biology, with LRRK2 binding to Rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1 and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term Parkinson’s disease.

  20. Entamoeba histolytica: the over expression of a mutated EhRabB protein produces a decrease of in vitro and in vivo virulence.

    Science.gov (United States)

    Juárez-Hernández, L J; García-Pérez, R M; Salas-Casas, A; García-Rivera, G; Orozco, E; Rodríguez, M A

    2013-03-01

    Vesicular trafficking, which is implicated in secretion of cytolytic molecules as well as in phagocytosis, plays an important role in the pathogenic mechanism of Entamoeba histolytica, the protozoan parasite causative of human amoebiasis. Thus, Rab GTPases, that are key regulators of vesicle trafficking, should be considered as molecules involved in the parasite virulence. EhRabB is a Rab protein located in cytoplasmic vesicles that are translocated to phagocytic mouths during ingestion of target cells, suggesting that this Rab protein is involved in phagocytosis. To prove this hypothesis, we over expressed the wild type EhrabB gene and a mutant gene encoding for a protein (RabBN118I) unable to bind guanine nucleotides and therefore constitutively inactive. The over expression of the mutated protein in E. histolytica trophozoites provoked a dominant negative effect, reflected in a significant decrease of both phagocytosis and cytopathic effect as well as in a failure to produce hepatic abscesses in hamsters. These results confirm that EhRabB is involved in phagocytosis and virulence of E. histolytica.

  1. Role of EhRab7A in phagocytosis of type 1 fimbriated E. coli by Entamoeba histolytica.

    Science.gov (United States)

    Verma, Kuldeep; Nozaki, Tomoyoshi; Datta, Sunando

    2016-12-01

    Entamoeba histolytica, the causative agent of amoebic colitis and liver abscess in human, ingests the intestinal bacteria and variety of host cells. Phagocytosis of bacteria by the amebic trophozoite has been reported to be important for the virulence of the parasite. Here, we set out to characterize different stages of phagocytosis of type 1 E. coli and investigated the role of a set of amoebic Rab GTPases in the process. The localizations of the Rab GTPases during different stages of the phagocytosis were investigated using laser scanning confocal microscopy and their functional relevance were determined using fluorescence activated cell sorter based assay as well as colony forming unit assay. Our results demonstrate that EhRab7A is localized on the phagosomes and involved in both early and late stages of type 1 E. coli phagocytosis. We further showed that the E. coli or RBC containing phagosomes are distinct from the large endocytic vacuoles in the parasite which are exclusively used to transport human holotransferrin and low density lipoprotein. Remarkably, type 1 E. coli uptake was found to be insensitive to cytochalasin D treatment, suggesting that the initial stage of E. coli phagocytosis is independent of the formation of actin filaments. © 2016 John Wiley & Sons Ltd.

  2. Involvement of the endosomal-lysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease

    DEFF Research Database (Denmark)

    Kovács, Gábor G; Gelpi, Ellen; Ströbel, Thomas

    2007-01-01

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes....... Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated......-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrP. Our results indicate that effects in the ELS...

  3. TMF is a golgin that binds Rab6 and influences Golgi morphology

    Directory of Open Access Journals (Sweden)

    Siniossoglou Symeon

    2004-05-01

    Full Text Available Abstract Background Golgins are coiled-coil proteins associated with the Golgi apparatus, that are believed to be involved in the tethering of vesicles and the stacking of cisternae, as well as other functions such as cytoskeletal association. Many are peripheral membrane proteins recruited by GTPases. Several have been described in animal cells, and some in yeast, but the relationships between golgins from different species can be hard to define because although they share structural features, their sequences are not well conserved. Results We show here that the yeast protein Sgm1, previously shown to be recruited to the Golgi by the GTPase Ypt6, binds to Ypt6:GTP via a conserved 100-residue coiled-coil motif that can be identified in a wide range of eukaryotes. The mammalian equivalent of Sgm1 is TMF/ARA160, a protein previously identified in various screens as a putative transcription or chromatin remodelling factor. We show that it is a Golgi protein, and that it binds to the three known isoforms of the Ypt6 homologue Rab6. Depletion of the protein by RNA interference in rat NRK cells results in a modest dispersal of Golgi membranes around the cell, suggesting a role for TMF in the movement or adherence of Golgi stacks. Conclusion We have identified TMF as an evolutionarily conserved golgin that binds Rab6 and contributes to Golgi organisation in animal cells.

  4. A complex of Rab13 with MICAL-L2 and α-actinin-4 is essential for insulin-dependent GLUT4 exocytosis.

    Science.gov (United States)

    Sun, Yi; Jaldin-Fincati, Javier; Liu, Zhi; Bilan, Philip J; Klip, Amira

    2016-01-01

    Insulin promotes glucose uptake into skeletal muscle through recruitment of glucose transporter 4 (GLUT4) to the plasma membrane. Rab GTPases are molecular switches mobilizing intracellular vesicles, and Rab13 is necessary for insulin-regulated GLUT4-vesicle exocytic translocation in muscle cells. We show that Rab13 engages the scaffold protein MICAL-L2 in this process. RNA interference-mediated knockdown of MICAL-L2 or truncated MICAL-L2 (MICAL-L2-CT) impaired insulin-stimulated GLUT4 translocation. Insulin increased Rab13 binding to MICAL-L2, assessed by pull down and colocalization under confocal fluorescence and structured illumination microscopies. Association was also visualized at the cell periphery using TIRF microscopy. Insulin further increased binding of MICAL-L2 to α-actinin-4 (ACTN4), a protein involved in GLUT4 translocation. Rab13, MICAL-L2, and ACTN4 formed an insulin-dependent complex assessed by pull down and confocal fluorescence imaging. Of note, GLUT4 associated with the complex in response to insulin, requiring the ACTN4-binding domain in MICAL-L2. This was demonstrated by pull down with distinct fragments of MICAL-L2 and confocal and structured illumination microscopies. Finally, expression of MICAL-L2-CT abrogated the insulin-dependent colocalization of Rab13 with ACTN4 or Rab13 with GLUT4. Our findings suggest that MICAL-L2 is an effector of insulin-activated Rab13, which links to GLUT4 through ACTN4, localizing GLUT4 vesicles at the muscle cell periphery to enable their fusion with the membrane.

  5. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

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    Amber L. Jolly

    2016-01-01

    Full Text Available Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo” occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

  6. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Directory of Open Access Journals (Sweden)

    Cecilia Bucci

    2014-10-01

    Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

  7. Harnessing membrane trafficking to promote cancer spreading and invasion: The case of RAB2A.

    Science.gov (United States)

    Kajiho, Hiroaki; Kajiho, Yuko; Scita, Giorgio

    2017-01-06

    How cancer disseminates and metastasizes remains an outstanding open question. Emerging evidence indicates that membrane trafficking is frequently harnessed by tumors of epithelial origin to acquire a mesenchymal program of invasiveness. However, the critical molecular hubs used by cancer cells this context have only began to be elucidated. Here, we discussed the results of a recent phenotypic screening that led to the identification of the small GTPase RAB2A, not previously involved in cancer dissemination, as pivotal for the acquisition of pericellular proteolysis, cell dissemination and distant metastatic spreading of human breast cancer. At the cellular levels, RAB2A controls both canonical polarized Golgi-to-Plasma membrane trafficking of the junctional protein E-cadherin, and post-endocytic trafficking of the membrane-bound metalloprotease, MT1-MMP. This finding reveals an unexpected plasticity in the control of diverse trafficking routes exerted by RAB2A through canonical (Golgi stacking) and non-canonical (late endosome recycling) functional interactions, contributing to break established membrane trafficking dogma on the rigorous molecular distinction between polarized Golgi and post endocytic routes. Finally, they suggest that epithelial cancers may specifically select for those molecules that enable them to control multiple trafficking routes, in turn essential for the regulation of activities necessary for acquisition of mesenchymal traits.

  8. Endocytosis of adiponectin receptor 1 through a clathrin-and Rab5-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    Qiurong Ding; Zhenzhen Wang; Yan Chen

    2009-01-01

    In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogen-esis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Epsl5 mutants or depleting K+ trapped AdipoRl at the plasma membrane, and K+ depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoRl and adiponectin is clathrin-dependent. Depletion of K+ and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might down-regulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoRl is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.

  9. Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

    Science.gov (United States)

    Herrera-Martínez, M; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Chávez-Munguía, B; Talamás-Rohana, P

    2013-01-01

    In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

  10. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.

    Science.gov (United States)

    Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G

    2012-11-21

    Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

  11. The lysosome and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Lisha Zhang; Rui Sheng; Zhenghong Qin

    2009-01-01

    It has long been believed that the lysosome is an important digestive organelle. There is increasing evidence that the lysosome is also involved in pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Abnormal protein degradation and deposition induced by lysosoreal dysfunction may be the primary contributor to age-related neurodegeneration. In this review, the possible relationship between lysosome and various neurodegenerative diseases is described.

  12. Small GTPases and Stress Responses of vvran1 in the Straw Mushroom Volvariella volvacea

    Directory of Open Access Journals (Sweden)

    Jun-Jie Yan

    2016-09-01

    Full Text Available Small GTPases play important roles in the growth, development and environmental responses of eukaryotes. Based on the genomic sequence of the straw mushroom Volvariella volvacea, 44 small GTPases were identified. A clustering analysis using human small GTPases as the references revealed that V. volvacea small GTPases can be grouped into five families: nine are in the Ras family, 10 are in the Rho family, 15 are in the Rab family, one is in the Ran family and nine are in the Arf family. The transcription of vvran1 was up-regulated upon hydrogen peroxide (H2O2 stress, and could be repressed by diphenyleneiodonium chloride (DPI, a NADPH oxidase-specific inhibitor. The number of vvran1 transcripts also increased upon cold stress. Diphenyleneiodonium chloride, but not the superoxide dismutase (SOD inhibitor diethy dithiocarbamate (DDC, could suppress the up-regulation of vvran1 gene expression to cold stress. These results combined with the high correlations between gene expression and superoxide anion (O2− generation indicated that vvran1 could be one of the candidate genes in the downstream of O2− mediated pathways that are generated by NADPH oxidase under low temperature and oxidative stresses.

  13. What vibrations tell us about GTPases.

    Science.gov (United States)

    Kötting, Carsten; Gerwert, Klaus

    2015-02-01

    In this review, we discuss how time-resolved Fourier transform infrared (FTIR) spectroscopy is used to understand how GTP hydrolysis is catalyzed by small GTPases and their cognate GTPase-activating proteins (GAPs). By interaction with small GTPases, GAPs regulate important signal transduction pathways and transport mechanisms in cells. The GTPase reaction terminates signaling and controls transport. Dysfunctions of GTP hydrolysis in these proteins are linked to serious diseases including cancer. Using FTIR, we resolved both the intrinsic and GAP-catalyzed GTPase reaction of the small GTPase Ras with high spatiotemporal resolution and atomic detail. This provided detailed insight into the order of events and how the active site is completed for catalysis. Comparisons of Ras with other small GTPases revealed conservation and variation in the catalytic mechanisms. The approach was extended to more nearly physiological conditions at a membrane. Interactions of membrane-anchored GTPases and their extraction from the membrane are studied using the attenuated total reflection (ATR) technique.

  14. A RabGAP regulates life-cycle duration via trimeric G-protein cascades in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Hidekazu Kuwayama

    Full Text Available BACKGROUND: The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3-deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.

  15. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

    Kallunki, T; Olsen, O D; Jaattela, Marja

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-asso......:10.1038/onc.2012.292....

  16. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    Science.gov (United States)

    Bernheimer, Alan W.; Schwartz, Lois L.

    1964-01-01

    Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100–1104. 1964.—Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of β-glucuronidase and acid phosphatase. Staphylococcal α-toxin, Clostridium perfringens α-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal β-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens ε-toxin were not active in either system. Staphylococcal δ-toxin, C. histolyticum collagenase, crude C. perfringens β-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes. PMID:5874534

  17. Small GTPases : emerging targets in rheumatoid arthritis

    NARCIS (Netherlands)

    Ferreira de Abreu, J.R.

    2009-01-01

    The studies provided in this thesis show that modulation of small GTPase function regulates cellular activation and inflammation in RA and animal models of the disease. Small GTPases are therefore emerging targets in RA and further studies should explore the development of novel small GTPase modulat

  18. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

    Science.gov (United States)

    Rojas, Raul; van Vlijmen, Thijs; Mardones, Gonzalo A.; Prabhu, Yogikala; Rojas, Adriana L.; Mohammed, Shabaz; Heck, Albert J.R.; Raposo, Graça; van der Sluijs, Peter; Bonifacino, Juan S.

    2008-01-01

    The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes. PMID:18981234

  19. Modulation of RAB5A early endosome trafficking in response to KRas mediated macropinocytic fluxes in pancreatic cancer cells.

    Science.gov (United States)

    Teske, Christian; Schweitzer, Christine; Palamidessi, Andrea; Aust, Daniela E; Scita, Giorgio; Weitz, Jürgen; Welsch, Thilo

    2017-09-01

    KRAS is the key mutated gene in pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that KRas modulates endocytic uptake. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression in PDAC. Surprisingly, PANC-1 cells lacking KRas exhibited significantly enlarged early and late endosomes containing internalized dextran and epidermal growth factor. Endosome enlargement was accompanied by reduced endosomal degradation. Both KRas silencing and lysosomal blockade caused an upregulation of the master regulator of early endosome biogenesis, RAB5A, which is likely responsible for the expansion of the early endosomal compartment, because simultaneous KRAS/RAB5A knockdown abolished endosome enlargement. In contrast, early endosome shrinkage was seen in MIA PaCa-2 cells despite RAB5A upregulation, indicating that distinct KRas-modulated responses operate in different metabolic subtypes of PDAC. In conclusion, mutant KRAS promotes endosomal degradation in PDAC cell lines, which is impaired by KRAS silencing. Moreover, KRAS silencing activates RAB5A upregulation and drives PDAC subtype-dependent modulation of endosome trafficking. Copyright © 2017. Published by Elsevier Inc.

  20. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-11-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

  1. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

    Kallunki, T; Olsen, O D; Jaattela, Marja

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-associated......-targeting anti-cancer drugs. In this review we compile our current knowledge on cancer-associated changes in lysosomal composition and discuss the consequences of these alterations to cancer progression and the possibilities they can bring to cancer therapy.Oncogene advance online publication, 9 July 2012; doi...

  2. Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

    Science.gov (United States)

    Sakamori, Ryotaro; Das, Soumyashree; Yu, Shiyan; Feng, Shanshan; Stypulkowski, Ewa; Guan, Yinzheng; Douard, Veronique; Tang, Waixing; Ferraris, Ronaldo P.; Harada, Akihiro; Brakebusch, Cord; Guo, Wei; Gao, Nan

    2012-01-01

    The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell–based approaches could be beneficial to infants with this often lethal condition. PMID:22354172

  3. Topological and functional properties of the small GTPases protein interaction network.

    Directory of Open Access Journals (Sweden)

    Anna Delprato

    Full Text Available Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.

  4. Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

    Science.gov (United States)

    Cano, Victoria; March, Catalina; Insua, Jose Luis; Aguiló, Nacho; Llobet, Enrique; Moranta, David; Regueiro, Verónica; Brennan, Gerard P; Millán-Lou, Maria Isabel; Martín, Carlos; Garmendia, Junkal; Bengoechea, José A

    2015-11-01

    Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

  5. A Novel Aberrant Splice Site Mutation in RAB23 Leads to an Eight Nucleotide Deletion in the mRNA and Is Responsible for Carpenter Syndrome in a Consanguineous Emirati Family.

    Science.gov (United States)

    Ben-Salem, S; Begum, M A; Ali, B R; Al-Gazali, L

    2013-01-01

    Carpenter syndrome is caused by mutations in the RAB23 gene that encodes a small GTPase of the Rab subfamily of proteins. Rab proteins are known to be involved in the regulation of cellular trafficking and signal transduction. Currently, only few mutations in RAB23 have been reported in patients with Carpenter syndrome. In this paper, we report the clinical features, molecular and functional analysis of 2 children from an Emirati consanguineous family with this syndrome. The affected children exhibit the typical features including craniosynostosis, typical facial appearance, polysyndactyly, and obesity. Molecular analysis of the RAB23 gene revealed a homozygous mutation affecting the first nucleotide of the acceptor splice site of exon 5 (c.482-1G>A). This mutation affects the authentic mRNA splicing and activates a cryptic acceptor site within exon 5. Thus, the erroneous splicing results in an eight nucleotide deletion, followed by a frameshift and premature termination codon at position 161 (p.V161fsX3). Due to the loss of the C-terminally prenylatable cysteine residue, the truncated protein will probably fail to associate with the target cellular membranes due to the absence of the necessary lipid modification. The p.V161fsX3 extends the spectrum of RAB23 mutations and points to the crucial role of prenylation in the pathogenesis of Carpenter syndrome within this family.

  6. The stress hormone corticosterone increases synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- and glucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complex.

    Science.gov (United States)

    Liu, Wenhua; Yuen, Eunice Y; Yan, Zhen

    2010-02-26

    Corticosterone, the major stress hormone, plays an important role in regulating neuronal functions of the limbic system, although the cellular targets and molecular mechanisms of corticosteroid signaling are largely unknown. Here we show that a short treatment of corticosterone significantly increases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission and AMPAR membrane trafficking in pyramidal neurons of prefrontal cortex, a key region involved in cognition and emotion. This enhancing effect of corticosterone is through a mechanism dependent on Rab4, the small GTPase-controlling receptor recycling between early endosome and plasma membrane. Guanosine nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab proteins between membrane and cytosol, forms an increased complex with Rab4 after corticosterone treatment. Corticosterone also triggers an increased GDI phosphorylation at Ser-213 by the serum- and glucocorticoid-inducible kinase (SGK). Moreover, AMPAR synaptic currents and surface expression and their regulation by corticosterone are altered by mutating Ser-213 on GDI. These results suggest that corticosterone, via SGK phosphorylation of GDI at Ser-213, increases the formation of GDI-Rab4 complex, facilitating the functional cycle of Rab4 and Rab4-mediated recycling of AMPARs to the synaptic membrane. It provides a potential mechanism underlying the role of corticosteroid stress hormone in up-regulating excitatory synaptic efficacy in cortical neurons.

  7. Lysosomal cell death at a glance

    DEFF Research Database (Denmark)

    Aits, Sonja; Jaattela, Marja

    2013-01-01

    Lysosomes serve as the cellular recycling centre and are filled with numerous hydrolases that can degrade most cellular macromolecules. Lysosomal membrane permeabilization and the consequent leakage of the lysosomal content into the cytosol leads to so-called "lysosomal cell death". This form...... of cell death is mainly carried out by the lysosomal cathepsin proteases and can have necrotic, apoptotic or apoptosis-like features depending on the extent of the leakage and the cellular context. This article summarizes our current knowledge on lysosomal cell death with an emphasis on the upstream...... mechanisms that lead to lysosomal membrane permeabilization....

  8. Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase

    OpenAIRE

    Buggia-Prévot, Virginie; Fernandez, Celia G; Riordan, Sean; Vetrivel, Kulandaivelu S.; Roseman, Jelita; Waters, Jack; Bindokas, Vytautas P.; Vassar, Robert; Thinakaran, Gopal

    2014-01-01

    Background BACE1 is one of the two enzymes that cleave amyloid precursor protein to generate Alzheimer's disease (AD) beta amyloid peptides. It is widely believed that BACE1 initiates APP processing in endosomes, and in the brain this cleavage is known to occur during axonal transport of APP. In addition, BACE1 accumulates in dystrophic neurites surrounding brain senile plaques in individuals with AD, suggesting that abnormal accumulation of BACE1 at presynaptic terminals contributes to patho...

  9. Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases

    OpenAIRE

    Elias, Marek; Brighouse, Andrew; Gabernet-Castello, Carme; Field, Mark C.; Dacks, Joel B.

    2012-01-01

    The presence of a nucleus and other membrane-bounded intracellular compartments is the defining feature of eukaryotic cells. Endosymbiosis accounts for the origins of mitochondria and plastids, but the evolutionary ancestry of the remaining cellular compartments is incompletely documented. Resolving the evolutionary history of organelle-identity encoding proteins within the endomembrane system is a necessity for unravelling the origins and diversification of the endogenously derived organelle...

  10. Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases

    National Research Council Canada - National Science Library

    Elias, Marek; Brighouse, Andrew; Gabernet-Castello, Carme; Field, Mark C; Dacks, Joel B

    2012-01-01

    .... Resolving the evolutionary history of organelle-identity encoding proteins within the endomembrane system is a necessity for unravelling the origins and diversification of the endogenously derived organelles...

  11. Sheep internal parasites on Rab and Pag

    Directory of Open Access Journals (Sweden)

    Relja Beck

    2010-01-01

    Full Text Available The purpose of our research was to determine which groups and species of internal parasites endanger the health of sheep on the islands of Rab and Pag. The research was carried out in 10 flocks on both islands taking the fresh dung out of 30% of the total number of sheep in each flock. It was ascertained that the gastrointestinal parasites and protozoa of Eimeria genus are present in most flocks on both islands. The presence of the fluke Dicrocoelium dendriticum was ascertained in considerably larger number of flocks on the island of Rab than on the island of Pag. On the other hand, the presence of parasites of Moniezia and Nematodirus genus was ascertained in larger number of flocks on the island of Pag. In two flocks on Rab parasites of Protostrongylus genus were ascertained while on the island of Pag they were not found in any flock.

  12. Rho GTPases in ameloblast differentiation

    Directory of Open Access Journals (Sweden)

    Keishi Otsu

    2016-05-01

    Full Text Available During tooth development, ameloblasts differentiate from inner enamel epithelial cells to enamel-forming cells by modulating the signal pathways mediating epithelial–mesenchymal interaction and a cell-autonomous gene network. The differentiation process of epithelial cells is characterized by marked changes in their morphology and polarity, accompanied by dynamic cytoskeletal reorganization and changes in cell–cell and cell–matrix adhesion over time. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete enamel-specific proteins. After deposition of the full thickness of enamel matrix, ameloblasts become smaller and regulate enamel maturation. Recent significant advances in the fields of molecular biology and genetics have improved our understanding of the regulatory mechanism of the ameloblast cell life cycle, mediated by the Rho family of small GTPases. They act as intracellular molecular switch that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. In our review, we summarize studies that provide current evidence for Rho GTPases and their involvement in ameloblast differentiation. In addition to the Rho GTPases themselves, their downstream effectors and upstream regulators have also been implicated in ameloblast differentiation.

  13. Identification of the privileged position in the imidazo[1,2-a]pyridine ring of phosphonocarboxylates for development of Rab geranylgeranyl transferase (RGGT) inhibitors.

    Science.gov (United States)

    Kaźmierczak, Aleksandra; Kusy, Damian; Niinivehmas, Sanna P; Gmach, Joanna; Joachimiak, Łukasz; Pentikäinen, Olli T; Gendaszewska-Darmach, Edyta; Blazewska, Katarzyna M

    2017-09-27

    Members of the Rab GTPase family are master regulators of vesicle trafficking. When disregulated, they are associated with a number of pathological states. The inhibition of RGGT, an enzyme responsible for post-translational geranylgeranylation of Rab GTPases represents one way to control the activity of these proteins. Since the number of molecules modulating RGGT is limited, we combined molecular modeling with biological assays to ascertain how modifications of phosphonocarboxylates, the first reported RGGT inhibitors, rationally improve understanding of their structure-activity relationship. We have identified the privileged position in the core scaffold of the imidazo[1,2-a]pyridine ring, which can be modified without compromising compounds' potency. Thus modified compounds are micromolar inhibitors of Rab11A prenylation, simultaneously being inactive against Rap1A/Rap1B modification, with the ability to inhibit proliferation of the HeLa cancer cell line. These findings were rationalized by molecular docking, which recognized interaction of phosphonic and carboxylic groups as decisive in phosphonocarboxylate localization in the RGGT binding site.

  14. Regulation of Rap GTPases in mammalian neurons.

    Science.gov (United States)

    Shah, Bhavin; Püschel, Andreas W

    2016-10-01

    Small GTPases are central regulators of many cellular processes. The highly conserved Rap GTPases perform essential functions in the mammalian nervous system during development and in mature neurons. During neocortical development, Rap1 is required to regulate cadherin- and integrin-mediated adhesion. In the adult nervous system Rap1 and Rap2 regulate the maturation and plasticity of dendritic spine and synapses. Although genetic studies have revealed important roles of Rap GTPases in neurons, their regulation by guanine nucleotide exchange factors (GEFs) that activate them and GTPase activating proteins (GAPs) that inactivate them by stimulating their intrinsic GTPase activity is just beginning to be explored in vivo. Here we review how GEFs and GAPs regulate Rap GTPases in the nervous system with a focus on their in vivo function.

  15. Rab proteins in the brain and corpus allatum of Bombyx mori.

    Science.gov (United States)

    Uno, Tomohide; Furutani, Masayuki; Watanabe, Chihiro; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Yamagata, Hiroshi; Mizoguchi, Akira; Takeda, Makio

    2016-07-01

    In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.

  16. GTPases in bacterial cell polarity and signalling.

    Science.gov (United States)

    Bulyha, Iryna; Hot, Edina; Huntley, Stuart; Søgaard-Andersen, Lotte

    2011-12-01

    In bacteria, large G domain GTPases have well-established functions in translation, protein translocation, tRNA modification and ribosome assembly. In addition, bacteria also contain small Ras-like GTPases consisting of stand-alone G domains. Recent data have revealed that small Ras-like GTPases as well as large G domain GTPases in bacteria function in the regulation of cell polarity, signal transduction and possibly also in cell division. The small Ras-like GTPase MglA together with its cognate GAP MglB regulates cell polarity in Myxococcus xanthus, and the small Ras-like GTPase CvnD9 in Streptomyces coelicolor is involved in signal transduction. Similarly, the large GTPase FlhF together with the ATPase FlhG regulates the localization and number of flagella in polarly flagellated bacteria. Moreover, large dynamin-like GTPases in bacteria may function in cell division. Thus, the function of GTPases in bacteria may be as pervasive as in eukaryotes.

  17. Role of Varp, a Rab21 exchange factor and TI-VAMP/VAMP7 partner, in neurite growth.

    Science.gov (United States)

    Burgo, Andrea; Sotirakis, Emmanuel; Simmler, Marie-Christine; Verraes, Agathe; Chamot, Christophe; Simpson, Jeremy C; Lanzetti, Letizia; Proux-Gillardeaux, Véronique; Galli, Thierry

    2009-10-01

    The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI-VAMP interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI-VAMP and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP.

  18. Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis.

    Science.gov (United States)

    Wang, Ting; Gilkes, Daniele M; Takano, Naoharu; Xiang, Lisha; Luo, Weibo; Bishop, Corey J; Chaturvedi, Pallavi; Green, Jordan J; Semenza, Gregg L

    2014-08-05

    Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxia-inducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.

  19. Rab27a is essential for the formation of neutrophil extracellular traps (NETs in neutrophil-like differentiated HL60 cells.

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    Tatsumi Kawakami

    Full Text Available Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs. NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an

  20. Probing E-cadherin endocytosis by morpholino-mediated Rab5 knockdown in zebrafish.

    Science.gov (United States)

    Ulrich, Florian; Heisenberg, Carl-Philipp

    2008-01-01

    The controlled internalization of membrane receptors and lipids is crucial for cells to control signaling pathways and interact with their environment. During clathrin-mediated endocytosis, membrane constituents are transported via endocytic vesicles into early endosomes, from which they are further distributed within the cell. The small guanosine triphosphatase (GTPase) Rab5 is both required and sufficient for the formation of these early endosomes and can be used to experimentally address endocytic processes. Recent evidence shows that endocytic turnover of E-cadherin regulates the migration of mesendodermal cells during zebrafish gastrulation by modulating their adhesive interactions with neighboring cells. This in turn leads to effective and synchronized movement within the embryo. In this review, we discuss techniques to manipulate E-cadherin endocytosis by morpholino-mediated knockdown of rab5 during zebrafish gastrulation. We describe the use of antibodies specifically directed against zebrafish E-cadherin to detect its intracellular localization and of in situ hybridization and primary cell culture to reveal patterns of cell migration and adhesion, respectively.

  1. Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility

    Science.gov (United States)

    McGrath, John A.; Stone, Kristina L.; Begum, Rumena; Simpson, Michael A.; Dopping-Hepenstal, Patricia J.; Liu, Lu; McMillan, James R.; South, Andrew P.; Pourreyron, Celine; McLean, W.H. Irwin; Martinez, Anna E.; Mellerio, Jemima E.; Parsons, Maddy

    2012-01-01

    The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.5786delC (p.Pro1929Leufs∗8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected person's skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and β4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins. PMID:23176819

  2. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells

    OpenAIRE

    Su Yeon Shim; Srinivasarao Karri; Sampson Law; Schatzl, Hermann M.; Sabine Gilch

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dy...

  3. Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis.

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    Xuefeng Li

    2016-01-01

    Full Text Available Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM, which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.

  4. Increased dosage of RAB39B affects neuronal development and could explain the cognitive impairment in male patients with distal Xq28 copy number gains.

    Science.gov (United States)

    Vanmarsenille, Lieselot; Giannandrea, Maila; Fieremans, Nathalie; Verbeeck, Jelle; Belet, Stefanie; Raynaud, Martine; Vogels, Annick; Männik, Katrin; Õunap, Katrin; Jacqueline, Vigneron; Briault, Sylvain; Van Esch, Hilde; D'Adamo, Patrizia; Froyen, Guy

    2014-03-01

    Copy number gains at Xq28 are a frequent cause of X-linked intellectual disability (XLID). Here, we report on a recurrent 0.5 Mb tandem copy number gain at distal Xq28 not including MECP2, in four male patients with nonsyndromic mild ID and behavioral problems. The genomic region is duplicated in two families and triplicated in a third reflected by more distinctive clinical features. The X-inactivation patterns in carrier females correspond well with their clinical symptoms. Our mapping data confirm that this recurrent gain is likely mediated by nonallelic homologous recombination between two directly oriented Int22h repeats. The affected region harbors eight genes of which RAB39B encoding a small GTPase, was the prime candidate since loss-of-function mutations had been linked to ID. RAB39B is expressed at stable levels in lymphocytes from control individuals, suggesting a tight regulation. mRNA levels in our patients were almost two-fold increased. Overexpression of Rab39b in mouse primary hippocampal neurons demonstrated a significant decrease in neuronal branching as well as in the number of synapses when compared with the control neurons. Taken together, we provide evidence that the increased dosage of RAB39B causes a disturbed neuronal development leading to cognitive impairment in patients with this recurrent copy number gain.

  5. PIKE GTPase Signaling and Function

    OpenAIRE

    Ahn, Jee-Yin; Ye, Keqiang

    2005-01-01

    PIKE (PI 3-Kinase Enhancer) is a recently identified brain specific nuclear GTPase, which binds PI 3-kinase and stimulates its lipid kinase activity. Nerve growth factor treatment leads to PIKE activation by triggering the nuclear translocation of phospholipase C-γ1 (PLC-γ1), which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. To date, three forms of PIKE have been characterized: PIKE-S, PIKE-L and PIKE-A. PIKE-S is initially identified shorte...

  6. Rho family and Rap GTPase activation assays.

    Science.gov (United States)

    Jennings, Richard T; Knaus, Ulla G

    2014-01-01

    The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione S-transferase (GST). Such domains bind preferentially to the GTP-bound form of the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.

  7. FGF21 promotes endothelial cell angiogenesis through a dynamin-2 and Rab5 dependent pathway.

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    Usman Yaqoob

    Full Text Available Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling.

  8. FGF21 Promotes Endothelial Cell Angiogenesis through a Dynamin-2 and Rab5 Dependent Pathway

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    Yaqoob, Usman; Jagavelu, Kumaravelu; Shergill, Uday; de Assuncao, Thiago; Cao, Sheng; Shah, Vijay H.

    2014-01-01

    Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling. PMID:24848261

  9. Lysosomal cell death mechanisms in aging.

    Science.gov (United States)

    Gómez-Sintes, Raquel; Ledesma, María Dolores; Boya, Patricia

    2016-12-01

    Lysosomes are degradative organelles essential for cell homeostasis that regulate a variety of processes, from calcium signaling and nutrient responses to autophagic degradation of intracellular components. Lysosomal cell death is mediated by the lethal effects of cathepsins, which are released into the cytoplasm following lysosomal damage. This process of lysosomal membrane permeabilization and cathepsin release is observed in several physiopathological conditions and plays a role in tissue remodeling, the immune response to intracellular pathogens and neurodegenerative diseases. Many evidences indicate that aging strongly influences lysosomal activity by altering the physical and chemical properties of these organelles, rendering them more sensitive to stress. In this review we focus on how aging alters lysosomal function and increases cell sensitivity to lysosomal membrane permeabilization and lysosomal cell death, both in physiological conditions and age-related pathologies. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Biomarkers in Lysosomal Storage Diseases

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    Joaquin Bobillo Lobato

    2016-12-01

    Full Text Available A biomarker is generally an analyte that indicates the presence and/or extent of a biological process, which is in itself usually directly linked to the clinical manifestations and outcome of a particular disease. The biomarkers in the field of lysosomal storage diseases (LSDs have particular relevance where spectacular therapeutic initiatives have been achieved, most notably with the introduction of enzyme replacement therapy (ERT. There are two main types of biomarkers. The first group is comprised of those molecules whose accumulation is directly enhanced as a result of defective lysosomal function. These molecules represent the storage of the principal macro-molecular substrate(s of a specific enzyme or protein, whose function is deficient in the given disease. In the second group of biomarkers, the relationship between the lysosomal defect and the biomarker is indirect. In this group, the biomarker reflects the effects of the primary lysosomal defect on cell, tissue, or organ functions. There is no “gold standard” among biomarkers used to diagnosis and/or monitor LSDs, but there are a number that exist that can be used to reasonably assess and monitor the state of certain organs or functions. A number of biomarkers have been proposed for the analysis of the most important LSDs. In this review, we will summarize the most promising biomarkers in major LSDs and discuss why these are the most promising candidates for screening systems.

  11. Papel de la proteína efectora Rab11-FIP2 en el transporte de receptores durante plasticidad sináptica

    OpenAIRE

    Royo Cantabrana, María

    2014-01-01

    Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-01-2014 Long Term Potentiation (LTP) is a well-known form of synaptic plasticity that relies on the capacity to regulate the insertion of AMPA-type glutamate receptors (AMPARs) into the postsynaptic membrane, in response to neuronal activity. This highly regulated process of intracellular membrane trafficking is controlled by the small GTPase Rab11...

  12. Regulation of cytokinesis by membrane trafficking involving small GTPases and the ESCRT machinery.

    Science.gov (United States)

    Nakayama, Kazuhisa

    2016-01-01

    During cell division, cells undergo membrane remodeling to achieve changes in their size and shape. In addition, cell division entails local delivery and retrieval of membranes and specific proteins as well as remodeling of cytoskeletons, in particular, upon cytokinetic abscission. Accumulating lines of evidence highlight that endocytic membrane removal from and subsequent membrane delivery to the plasma membrane are crucial for the changes in cell size and shape, and that trafficking of vesicles carrying specific proteins to the abscission site participate in local remodeling of membranes and cytoskeletons. Furthermore, the endosomal sorting complex required for transport (ESCRT) machinery has been shown to play crucial roles in cytokinetic abscission. Here, the author briefly overviews membrane-trafficking events early in cell division, and subsequently focus on regulation and functional significance of membrane trafficking involving Rab11 and Arf6 small GTPases in late cytokinesis phases and assembly of the ESCRT machinery in cytokinetic abscission.

  13. Insights into the classification of small GTPases

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    Dominik Heider

    2010-05-01

    Full Text Available Dominik Heider1, Sascha Hauke3, Martin Pyka4, Daniel Kessler21Department of Bioinformatics, Center for Medical Biotechnology, 2Institute of Cell Biology (Cancer Research, University of Duisburg-Essen, Essen, Germany; 3Institute of Computer Science, University of Münster, Münster, Germany; 4Interdisciplinary Center for Clinical Research, University Hospital of Münster, Münster, GermanyAbstract: In this study we used a Random Forest-based approach for an assignment of small guanosine triphosphate proteins (GTPases to specific subgroups. Small GTPases represent an important functional group of proteins that serve as molecular switches in a wide range of fundamental cellular processes, including intracellular transport, movement and signaling events. These proteins have further gained a special emphasis in cancer research, because within the last decades a huge variety of small GTPases from different subgroups could be related to the development of all types of tumors. Using a random forest approach, we were able to identify the most important amino acid positions for the classification process within the small GTPases superfamily and its subgroups. These positions are in line with the results of earlier studies and have been shown to be the essential elements for the different functionalities of the GTPase families. Furthermore, we provide an accurate and reliable software tool (GTPasePred to identify potential novel GTPases and demonstrate its application to genome sequences.Keywords: cancer, machine learning, classification, Random Forests, proteins

  14. PIKE GTPase Signaling and Function

    Directory of Open Access Journals (Sweden)

    2005-04-01

    Full Text Available PIKE (PI 3-Kinase Enhancer is a recently identified brain specific nuclear GTPase, which binds PI 3-kinase and stimulates its lipid kinase activity. Nerve growth factor treatment leads to PIKE activation by triggering the nuclear translocation of phospholipase C-γ1 (PLC-γ1, which acts as a physiologic guanine nucleotide exchange factor (GEF for PIKE through its SH3 domain. To date, three forms of PIKE have been characterized: PIKE-S, PIKE-L and PIKE-A. PIKE-S is initially identified shorter isoform. PIKE-L, a longer isoform of PIKE gene, differs from PIKE-S by C-terminal extension containing Arf-GAP (ADP ribosylation factor-GTPase Activating Protein and two ankyrin repeats domains. In contrast to the exclusive nuclear localization of PIKE-S, PIKE-L occurs in both the nucleus and the cytoplasm. PIKE-L physiologically associates with Homer 1, an mGluR I binding adaptor protein. The Homer/PIKE-L complex couples PI 3-kinase to mGluR I and regulates a major action of group I mGluRs, prevention of neuronal apoptosis. More recently, a third PIKE isoform, PIKE-A was identified in human glioblastoma multiforme brain cancers. Unlike the brain specific PIKE-L and -S isoforms, PIKE-A distributes in various tissues. PIKE-A contains the same domains present in PIKE-L but lacks N-terminal proline-rich domain (PRD, which binds PI 3-kinase and PLC-γ1. Instead, PIKE-A specifically binds to active Akt and upregulates its activity in a GTP-dependent manner, mediating human cancer cell invasion and preventing apoptosis. Thus, PIKE extends its roles from the nucleus to the cytoplasm, mediating cellular processes from cell invasion to programmed cell death.

  15. Brief exposure to copper activates lysosomal exocytosis.

    Science.gov (United States)

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

    Copper (Cu) is essential mineral, but its toxicity necessitates existence of powerful machinery responsible for the extraction of excess Cu from the cell. Cu exposure was recently shown to induce the translocation of Cu pump ATP7B to the lysosomes followed by lysosomal exocytosis. Here we sought to investigate the mechanisms underlying the effect of Cu on lysosomal exocytosis. We found that brief exposure to Cu activates lysosomal exocytosis, which was measured as a release of the lysosomal digestive enzyme β-hexosaminidase (β-hex) into the extracellular medium and by the presence lysosomal protein LAMP1 at the plasma membrane. Such release depends on calcium (Ca) and on the lysosomal SNARE VAMP7. ATP7B knockdown using RNAi suppressed the basal lysosomal exocytosis, but did not affect the ability of Cu to activate it. ATP7B knockdown was associated with sustained oxidative stress. The removal of Ca from the extracellular medium suppressed the Cu-dependent component of the lysosomal exocytosis. We propose that Cu promotes lysosomal exocytosis by facilitating a Ca-dependent step of the lysosomal exocytosis.

  16. How not to do kinetics: examples involving GTPases and guanine nucleotide exchange factors.

    Science.gov (United States)

    Goody, Roger S

    2014-01-01

    Guanine nucleotide exchange factors (GEFs) are crucial regulators of the action of GTPases in signal transduction and cellular regulation. Although their basic mechanism of action has been apparent for almost 20 years, there are still misconceptions concerning their properties, and these are confounded by superficial or incorrect interpretation of experimental results in individual cases. Here, an example is described in which an incorrect mechanism was derived because of an inadequate analysis of kinetic results. In a second example, a case is discussed where certain GTP analogs were erroneously described as being able to function as low molecular mass GEFs. In both cases, a lack of distinction between rates, rate constants, and apparent rate constants, together with a disregard of relative signal amplitudes, led to the misinterpretations. In a final example, it is shown how the lack of an appropriate kinetic investigation led to the false conclusion that a secreted protein from Legionella pneumophila can act not only as a GEF towards eukaryotic Rab1 but also as a factor that is able to actively dissociate the stable complex between Rab1 and GDP dissociation inhibitor.

  17. Ribosome-associated GTPases: the role of RNA for GTPase activation.

    Science.gov (United States)

    Clementi, Nina; Polacek, Norbert

    2010-01-01

    The GTPase super-family comprises a variety of G proteins found in all three domains of life. Although they are participating in completely different processes like signal transduction, protein biosynthesis and regulation of cell proliferation, they all share a highly conserved G domain and use a common mechanism for GTP hydrolysis. Exact timing in hydrolyzing the bound GTP serves as a molecular switch to initiate diverse cellular reactions. Classical GTPases depend on external proteins to fire GTP hydrolysis (GAPs), and following the GTPase reaction to exchange GDP for GTP (GEFs), converting the GTPase into the active state again. In recent years it became clear that there are many GTPases that do not follow this classical switch mode scheme. Certain ribosome-associated GTPases are not reliant on other GEF proteins to exchange GDP for GTP. Furthermore many of these G proteins are not activated by external GAPs, but by evolutionarily ancient molecules, namely by RNA.

  18. Inhibitors of lysosomal cysteine proteases

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    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  19. The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

    Institute of Scientific and Technical Information of China (English)

    Chiara Sandri; Guido Serini; Francesca Caccavari; Donatella Valdembri; Chiara Camillo; Stefan Veltel; Martina Santambrogio; Letizia Lanzetti; Fedenco Bussolino; Johanna Ivaska

    2012-01-01

    During developmental and tumor angiogenesis,semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases.R-Ras is mainly expressed in vascular cells,where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms.We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and-angiogenic activity of R-Ras.Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia.Upon binding,GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF)to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes.Here,the R-Ras/RIN2/Rab5 signaling module activates Racl-dependent cell adhesion via TIAM1,a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate.In conclusion,the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Racl.

  20. Identification and characterization of RBEL1 subfamily of GTPases in the Ras superfamily involved in cell growth regulation.

    Science.gov (United States)

    Montalbano, JoAnne; Lui, Ki; Sheikh, M Saeed; Huang, Ying

    2009-07-03

    Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236-302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.

  1. Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose.

    Science.gov (United States)

    Hatano, Tomoyuki; Morigasaki, Susumu; Tatebe, Hisashi; Ikeda, Kyoko; Shiozaki, Kazuhiro

    2015-01-01

    The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.

  2. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    Science.gov (United States)

    Wexselblatt, Ezequiel; Esko, Jeffrey D; Tor, Yitzhak

    2015-01-01

    GNeosomes, lysosomotropic lipid vesicles decorated with guanidinoneomycin, can encapsulate and facilitate the cellular internalization and lysosomal delivery of cargo ranging from small molecules to high molecular weight proteins, in a process that is exclusively dependent on cell surface glycosaminoglycans. Their cellular uptake mechanism and co-localization with lysosomes, as well as the delivery, release, and activity of internalized cargo, are quantified. GNeosomes are proposed as a universal platform for lysosomal delivery with potential as a basic research tool and a therapeutic vehicle.

  3. Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

    DEFF Research Database (Denmark)

    Sakamori, Ryotaro; Das, Soumyashree; Yu, Shiyan

    2012-01-01

    The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases...... reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells...... activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data...

  4. Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons.

    Science.gov (United States)

    Peled, Einat S; Isacoff, Ehud Y

    2011-04-01

    Synaptic transmission from a neuron to its target cells occurs via neurotransmitter release from dozens to thousands of presynaptic release sites whose strength and plasticity can vary considerably. We report an in vivo imaging method that monitors real-time synaptic transmission simultaneously at many release sites with quantal resolution. We applied this method to the model glutamatergic system of the Drosophila melanogaster larval neuromuscular junction. We find that, under basal conditions, about half of release sites have a very low release probability, but these are interspersed with sites with as much as a 50-fold higher probability. Paired-pulse stimulation depresses high-probability sites, facilitates low-probability sites, and recruits previously silent sites. Mutation of the small GTPase Rab3 substantially increases release probability but still leaves about half of the sites silent. Our findings suggest that basal synaptic strength and short-term plasticity are regulated at the level of release probability at individual sites.

  5. Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

    Science.gov (United States)

    Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-08-01

    Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

  6. A novel role of Rab11 in trafficking GPI-anchored trans-sialidase to the plasma membrane of Trypanosoma cruzi.

    Science.gov (United States)

    Niyogi, Sayantanee; Docampo, Roberto

    2015-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is a unicellular parasite that possesses a contractile vacuole complex (CVC). This organelle is usually present in free-living protists and is mainly involved in osmoregulation. However, in some organisms, like for example Dictyostelium discoideum, other roles include calcium homeostasis and transference of proteins to the plasma membrane. T. cruzi plasma membrane is very rich in glycosylphosphatidylinositol anchored proteins (GPI-AP) and a very important group of GPI-AP is that of the trans-sialidases. These enzymes catalyze the transfer of sialic acid from host glycoconjugates to mucins present in the surface of the parasite and are important for host cell invasion among other functions. We recently reported that a pathway dependent on the Rab GTPase Rab11 is involved in the traffic of trans-sialidases to the plasma membrane through the CVC of the infective stages of the parasite and that preventing this traffic results in considerable reduction in the ability of T. cruzi to infect host cells. We also found that traffic of other GPI-anchored proteins is also through the CVC but uses a Rab11-independent pathway. These represent unconventional pathways of GPI-anchored protein traffic to the plasma membrane.

  7. Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase

    Directory of Open Access Journals (Sweden)

    Isabelle Bouchez

    2015-07-01

    Full Text Available It has now been clearly shown that lipid droplets (LDs play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+ ATPase (V-ATPase. Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

  8. GTPases and the origin of the ribosome

    Directory of Open Access Journals (Sweden)

    Smith Temple F

    2010-05-01

    Full Text Available Abstract Background This paper is an attempt to trace the evolution of the ribosome through the evolution of the universal P-loop GTPases that are involved with the ribosome in translation and with the attachment of the ribosome to the membrane. The GTPases involved in translation in Bacteria/Archaea are the elongation factors EFTu/EF1, the initiation factors IF2/aeIF5b + aeIF2, and the elongation factors EFG/EF2. All of these GTPases also contain the OB fold also found in the non GTPase IF1 involved in initiation. The GTPase involved in the signal recognition particle in most Bacteria and Archaea is SRP54. Results 1 The Elongation Factors of the Archaea based on structural considerations of the domains have the following evolutionary path: EF1→ aeIF2 → EF2. The evolution of the aeIF5b was a later event; 2 the Elongation Factors of the Bacteria based on the topological considerations of the GTPase domain have a similar evolutionary path: EFTu→ IF→2→EFG. These evolutionary sequences reflect the evolution of the LSU followed by the SSU to form the ribosome; 3 the OB-fold IF1 is a mimic of an ancient tRNA minihelix. Conclusion The evolution of translational GTPases of both the Archaea and Bacteria point to the evolution of the ribosome. The elongation factors, EFTu/EF1, began as a Ras-like GTPase bringing the activated minihelix tRNA to the Large Subunit Unit. The initiation factors and elongation factor would then have evolved from the EFTu/EF1 as the small subunit was added to the evolving ribosome. The SRP has an SRP54 GTPase and a specific RNA fold in its RNA component similar to the PTC. We consider the SRP to be a remnant of an ancient form of an LSU bound to a membrane. Reviewers This article was reviewed by George Fox, Leonid Mirny and Chris Sander.

  9. PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

    Science.gov (United States)

    Yamaoka, Mami; Ando, Tomomi; Terabayashi, Takeshi; Okamoto, Mitsuhiro; Takei, Masahiro; Nishioka, Tomoki; Kaibuchi, Kozo; Matsunaga, Kohichi; Ishizaki, Ray; Izumi, Tetsuro; Niki, Ichiro; Ishizaki, Toshimasa; Kimura, Toshihide

    2016-02-01

    In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic β-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic β-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages.

  10. C9orf72’s Interaction with Rab GTPases—Modulation of Membrane Traffic and Autophagy

    Science.gov (United States)

    Tang, Bor L.

    2016-01-01

    Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 (C9orf72) is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF) complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1) autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings. PMID:27774051

  11. Rho GTPase expression in human myeloid cells.

    Directory of Open Access Journals (Sweden)

    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  12. Comparison of human and Drosophila atlastin GTPases.

    Science.gov (United States)

    Wu, Fuyun; Hu, Xiaoyu; Bian, Xin; Liu, Xinqi; Hu, Junjie

    2015-02-01

    Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.

  13. Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover.

    Science.gov (United States)

    Szíber, Zsófia; Liliom, Hanna; Morales, Carlos O Oueslati; Ignácz, Attila; Rátkai, Anikó Erika; Ellwanger, Kornelia; Link, Gisela; Szűcs, Attila; Hausser, Angelika; Schlett, Katalin

    2017-01-15

    Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase-dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity. © 2017 Szíber et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models.

    Science.gov (United States)

    Mazzulli, Joseph R; Zunke, Friederike; Isacson, Ole; Studer, Lorenz; Krainc, Dimitri

    2016-02-16

    Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the accumulation of protein aggregates comprised of α-synuclein (α-syn). A major barrier in treatment discovery for PD is the lack of identifiable therapeutic pathways capable of reducing aggregates in human neuronal model systems. Mutations in key components of protein trafficking and cellular degradation machinery represent important risk factors for PD; however, their precise role in disease progression and interaction with α-syn remains unclear. Here, we find that α-syn accumulation reduced lysosomal degradation capacity in human midbrain dopamine models of synucleinopathies through disrupting hydrolase trafficking. Accumulation of α-syn at the cell body resulted in aberrant association with cis-Golgi-tethering factor GM130 and disrupted the endoplasmic reticulum-Golgi localization of rab1a, a key mediator of vesicular transport. Overexpression of rab1a restored Golgi structure, improved hydrolase trafficking and activity, and reduced pathological α-syn in patient neurons. Our work suggests that enhancement of lysosomal hydrolase trafficking may prove beneficial in synucleinopathies and indicates that human midbrain disease models may be useful for identifying critical therapeutic pathways in PD and related disorders.

  15. Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation.

    Science.gov (United States)

    Wang, Wuyang; Gao, Qiong; Yang, Meimei; Zhang, Xiaoli; Yu, Lu; Lawas, Maria; Li, Xinran; Bryant-Genevier, Marthe; Southall, Noel T; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2015-03-17

    Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca(2+) channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca(2+) imaging and whole-lysosome patch clamping, lysosomal Ca(2+) release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na(+)-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca(2+) signaling.

  16. BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release.

    Science.gov (United States)

    Cao, Qi; Zhong, Xi Zoë; Zou, Yuanjie; Zhang, Zhu; Toro, Ligia; Dong, Xian-Ping

    2015-05-26

    Promoting lysosomal trafficking represents a promising therapeutic approach for lysosome storage diseases. Efficient Ca(2+) mobilization from lysosomes is important for lysosomal trafficking. Ca(2+) release from lysosomes could generate a negative potential in the lumen to disturb subsequent Ca(2+) release in the absence of counter ion flux. Here we report that lysosomes express big-conductance Ca(2+)-activated potassium (BK) channels that form physical and functional coupling with the lysosomal Ca(2+) release channel, TRPML1. Ca(2+) release via TRPML1 causes BK activation, which in turn facilitates further lysosomal Ca(2+) release and membrane trafficking. Importantly, BK overexpression rescues the impaired TRPML1-mediated Ca(2+) release and abnormal lysosomal storage in cells from Niemann-Pick C1 patients. Therefore, we have identified a lysosomal K(+) channel that provides a positive feedback mechanism to facilitate TRPML1-mediated Ca(2+) release and membrane trafficking. Our findings suggest that upregulating BK may be a potential therapeutic strategy for certain lysosomal storage diseases and common neurodegenerative disorders.

  17. Sensitivity to lysosome-dependent cell death is directly regulated by lysosomal cholesterol content.

    Directory of Open Access Journals (Sweden)

    Hanna Appelqvist

    Full Text Available Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1 protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2, which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

  18. RhoGTPases in stem cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    RhoGTPases are small molecules that control a wide variety of signal transduction pathways. Their profound function in regulating the actin cytoskeleton is well recognized. Stem cells are unique in their ability to self-renew and produce progenitor cells that can differentiate into specialized cells. RhoGT-Pases influence stem cell morphology and cell migration as well as stem cell self-renewal, proliferation, transplantation, homing and differentiation. In this review, the multiple roles of the RhoGTPases in stem cells are discussed.

  19. Endosome-lysosomes and neurodegeneration.

    Science.gov (United States)

    Mayer, R J; Tipler, C; Laszlo, L; Arnold, J; Lowe, J; Landon, M

    1994-01-01

    A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.

  20. Approaches of targeting Rho GTPases in cancer drug discovery

    Science.gov (United States)

    Lin, Yuan; Zheng, Yi

    2016-01-01

    Introduction Rho GTPases are master regulators of actomyosin structure and dynamics and play pivotal roles in a variety of cellular processes including cell morphology, gene transcription, cell cycle progression and cell adhesion. Because aberrant Rho GTPase signaling activities are widely associated with human cancer, key components of Rho GTPase signaling pathways have attracted increasing interest as potential therapeutic targets. Similar to Ras, Rho GTPases themselves were, until recently, deemed “undruggable” because of structure-function considerations. Several approaches to interfere with Rho GTPase signaling have been explored and show promise as new ways for tackling cancer cells. Areas covered This review focuses on the recent progress in targeting the signaling activities of three prototypical Rho GTPases, i.e. RhoA, Rac1, and Cdc42. The authors describe the involvement of these Rho GTPases, their key regulators and effectors in cancer. Furthermore, the authors discuss the current approaches for rationally targeting aberrant Rho GTPases along their signaling cascades, upstream and downstream of Rho GTPases and posttranslational modifications at a molecular level. Expert opinion To date, while no clinically effective drugs targeting Rho GTPase signaling for cancer treatment are available, tool compounds and lead drugs that pharmacologically inhibit Rho GTPase pathways have shown promise. Small molecule inhibitors targeting Rho GTPase signaling may add new treatment options for future precision cancer therapy, particularly in combination with other anti-cancer agents. PMID:26087073

  1. Rab8A regulates insulin-stimulated GLUT4 translocation in C2C12 myoblasts.

    Science.gov (United States)

    Li, Hanbing; Ou, Liting; Fan, Jiannan; Xiao, Mei; Kuang, Cuifang; Liu, Xu; Sun, Yonghong; Xu, Yingke

    2017-02-01

    Rab proteins are important regulators of GLUT4 trafficking in muscle and adipose cells. It is still unclear which Rabs are involved in insulin-stimulated GLUT4 translocation in C2C12 myoblasts. In this study, we detect the colocalization of Rab8A with GLUT4 and the presence of Rab8A at vesicle exocytic sites by TIRFM imaging. Overexpression of dominant-negative Rab8A (T22N) diminishes insulin-stimulated GLUT4 translocation, while constitutively active Rab8A (Q67L) augments it. In addition, knockdown of Rab8A inhibits insulin-stimulated GLUT4 translocation, which is rescued by replenishment of RNAi-resistant Rab8A. Together, these results indicate an indispensable role for Rab8A in insulin-regulated GLUT4 trafficking in C2C12 cells.

  2. 32 CFR 202.4 - Composition of a RAB.

    Science.gov (United States)

    2010-07-01

    ... appropriate. At closing installations where BRAC Cleanup Teams (BCT) exist, representatives of the BCT may... installation and the other the community. Co-chairs shall be responsible for directing and managing the RAB...

  3. DESAIN APLIKASI RENCANA ANGGARAN BIAYA (RAB) BERBASIS WEB

    OpenAIRE

    Dedet Wira Kusuma

    2012-01-01

    DESAIN APLIKASI RENCANA ANGGARAN BIAYA ( RAB ) BERBASIS WEB Nama Mahasiswa : Dedet Wira Kusuma NPM : 07.301010.024 Dosen Pembimbing : Dr.-Ing Daud Nawir, ST., MT ABSTRAK Perkembangan teknologi komputer saat ini sudah sangat maju, setiap orang harus mengikuti perkembangan teknologi komputer yang ada. Rencana anggaran biaya (RAB) dipergunakan untuk merencanakan jumlah biaya yang dibutuhkan dalam pekerjaan yang masih dilakukan secara manual yaitu dengan menggunakan Mic...

  4. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

    Science.gov (United States)

    Fujita, Naonobu; Huang, Wilson; Lin, Tzu-han; Groulx, Jean-Francois; Jean, Steve; Nguyen, Jen; Kuchitsu, Yoshihiko; Koyama-Honda, Ikuko; Mizushima, Noboru; Fukuda, Mitsunori; Kiger, Amy A

    2017-01-01

    Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23367.001 PMID:28063257

  5. Rho GTPases in collective cell migration

    NARCIS (Netherlands)

    Zegers, M.M.; Friedl, P.

    2014-01-01

    The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle traffickin

  6. Invited review: Small GTPases and their GAPs.

    Science.gov (United States)

    Mishra, Ashwini K; Lambright, David G

    2016-08-01

    Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 431-448, 2016.

  7. Rho GTPases in collective cell migration

    NARCIS (Netherlands)

    Zegers, M.M.; Friedl, P.

    2014-01-01

    The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle

  8. Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi

    Science.gov (United States)

    Niyogi, Sayantanee; Jimenez, Veronica; Girard-Dias, Wendell; de Souza, Wanderley; Miranda, Kildare; Docampo, Roberto

    2015-01-01

    ABSTRACT The contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease, collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress; it also has a role in cell shrinking after hyperosmotic stress. Here, we report that, in addition to its role in osmoregulation, the CVC of T. cruzi has a role in the biogenesis of acidocalcisomes. Expression of dominant-negative mutants of the CVC-located small GTPase Rab32 (TcCLB.506289.80) results in lower numbers of less-electron-dense acidocalcisomes, lower content of polyphosphate, lower capacity for acidocalcisome acidification and Ca2+ uptake that is driven by the vacuolar proton pyrophosphatase and the Ca2+-ATPase, respectively, as well as less-infective parasites, revealing the role of this organelle in parasite infectivity. By using fluorescence, electron microscopy and electron tomography analyses, we provide further evidence of the active contact of acidocalcisomes with the CVC, indicating an active exchange of proteins between the two organelles. PMID:25964650

  9. GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

    Science.gov (United States)

    Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

    2010-11-19

    Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

  10. GDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide Exchange in the Rab5·Vps9 System*

    Science.gov (United States)

    Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

    2010-01-01

    Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg2+ and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction. PMID:20833725

  11. Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi [Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Cell Biology, School of Sciences and Technology, University of the Basque Country, P.O. box 644, E-48080 Bilbo (Spain); Marigomez, Ionan, E-mail: ionan.marigomez@ehu.e [Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Cell Biology, School of Sciences and Technology, University of the Basque Country, P.O. box 644, E-48080 Bilbo (Spain)

    2009-05-15

    Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density - Vv - and labilisation period -LP) and combined (lysosomal response index - LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists. - Lysosomal responses to pollutants measured by an integrative index.

  12. Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

    Directory of Open Access Journals (Sweden)

    Chih-Peng Chang

    Full Text Available Interferon-gamma (IFN-γ, a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1 translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/- mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

  13. The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

    Science.gov (United States)

    Rofe, Adam P; Davis, Luther J; Whittingham, Jean L; Latimer-Bowman, Elizabeth C; Wilkinson, Anthony J; Pryor, Paul R

    2017-04-01

    Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  14. Lysosomal exoglycosidases in nasal polyps.

    Science.gov (United States)

    Chojnowska, Sylwia; Minarowska, Alina; Knaś, Małgorzata; Niemcunowicz-Janica, Anna; Kołodziejczyk, Paweł; Zalewska-Szajda, Beata; Kępka, Alina; Minarowski, Łukasz; Waszkiewicz, Napoleon; Zwierz, Krzysztof; Szajda, Sławomir Dariusz

    2013-01-01

    Nasal polyps are smooth outgrowths assuming a shape of grapes, formed from the nasal mucosa, limiting air flow by projecting into a lumen of a nasal cavity. Up to now the surgical resection is the best method of their treatment, but etiology and pathogenesis of the nasal polyps is not yet fully established. The aim of the study was the assessment of the selected lysosomal exoglycosidases activity in the nasal polyps. In this study the activity of β-galactosidase, α-mannosidase and α-fucosidase was determined in the tissue of the nasal polyps obtained from 40 patients (10F, 30M) and control tissues derived from mucosa of lower nasal conchas obtained during mucotomy from 20 patients (10F, 10M). We observed significant lower values of GAL, FUC and tendency to decrease of MAN and GLU concentration in nasal polyps (P) in comparison to control healthy nasal mucosa (C). In nasal polyp tissue (P) no differences of GAL, MAN and FUC specific activity in comparison to control mucosa (C) were found. Our research supports bioelectrical theory of the nasal polyps pathogenesis and directs attention at research on glycoconjugates and glycosidases of the nasal mucosa extracellular matrix. Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.

  15. Presenilin 1 maintains lysosomal Ca2+ homeostasis by regulating vATPase-mediated lysosome acidification

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M.; Haslett, Luke J.; Kumar, Asok; Sato, Yutaka; Lie, Pearl P. Y.; Mohan, Panaiyur; Coffey, Erin E.; Kompella, Uday; Mitchell, Claire H.; Lloyd-Evans, Emyr; Nixon, Ralph A.

    2015-01-01

    Summary Presenilin-1 (PS1) deletion or Alzheimer’s Disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss of function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. PMID:26299959

  16. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release.

    Science.gov (United States)

    Shen, Dongbiao; Wang, Xiang; Li, Xinran; Zhang, Xiaoli; Yao, Zepeng; Dibble, Shannon; Dong, Xian-ping; Yu, Ting; Lieberman, Andrew P; Showalter, Hollis D; Xu, Haoxing

    2012-03-13

    Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

  17. A tale of two GTPases in cotranslational protein targeting.

    Science.gov (United States)

    Saraogi, Ishu; Akopian, David; Shan, Shu-Ou

    2011-11-01

    Guanosine triphosphatases (GTPases) comprise a superfamily of proteins that provide molecular switches to regulate numerous cellular processes. The "GTPase switch" paradigm, in which a GTPase acts as a bimodal switch that is turned "on" and "off" by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases. Recent work on a pair of GTPases in the signal recognition particle (SRP) pathway has revealed a distinct mode of GTPase regulation. Instead of the classical GTPase switch, the two GTPases in the SRP and SRP receptor undergo a series of conformational changes during their dimerization and reciprocal activation. Each conformational rearrangement provides a point at which these GTPases can communicate with and respond to their upstream and downstream biological cues, thus ensuring the spatial and temporal precision of all the molecular events in the SRP pathway. We suggest that the SRP and SRP receptor represent an emerging class of "multistate" regulatory GTPases uniquely suited to provide exquisite control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.

  18. [Application of lysosomal detection in marine pollution monitoring: research progress].

    Science.gov (United States)

    Weng, You-Zhu; Fang, Yong-Qiang; Zhang, Yu-Sheng

    2013-11-01

    Lysosome is an important organelle existing in eukaryotic cells. With the development of the study on the structure and function of lysosome in recent years, lysosome is considered as a target of toxic substances on subcellular level, and has been widely applied abroad in marine pollution monitoring. This paper summarized the biological characteristics of lysosomal marker enzyme, lysosome-autophagy system, and lysosomal membrane, and introduced the principles and methods of applying lysosomal detection in marine pollution monitoring. Bivalve shellfish digestive gland and fish liver are the most sensitive organs for lysosomal detection. By adopting the lysosomal detection techniques such as lysosomal membrane stability (LMS) test, neutral red retention time (NRRT) assay, morphological measurement (MM) of lysosome, immunohistochemical (Ih) assay of lysosomal marker enzyme, and electron microscopy (EM), the status of marine pollution can be evaluated. It was suggested that the lysosome could be used as a biomarker for monitoring marine environmental pollution. The advantages and disadvantages of lysosomal detection and some problems worthy of attention were analyzed, and the application prospects of lysosomal detection were discussed.

  19. Lysosomal Storage Disorders and Malignancy

    Directory of Open Access Journals (Sweden)

    Gregory M. Pastores

    2017-02-01

    Full Text Available Lysosomal storage disorders (LSDs are infrequent to rare conditions caused by mutations that lead to a disruption in the usual sequential degradation of macromolecules or their transit within the cell. Gaucher disease (GD, a lipidosis, is among the most common LSD, with an estimated incidence of 1 in 40,000 among the Caucasian, non-Jewish population. Studies have indicated an increased frequency of polyclonal and monoclonal gammopathy among patients with GD. It has been shown that two major sphingolipids that accumulate in GD, namely, β-glucosylceramide 22:0 (βGL1-22 and glucosylsphingosine (LGL1, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT cells. Investigations undertaken in an affected mouse model revealed βGL1-22- and LGL1-specific NKT cells were present and constitutively promoted the expression of a T-follicular helper (TFH phenotype; injection of these lipids led to downstream induction of germinal center B cells, hypergammaglobulinemia, and the production of antilipid antibodies. Subsequent studies have found clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC. Furthermore, substrate reduction ameliorated GD-associated gammopathy in mice. It had been hypothesized that chronic antigenic stimulation by the abnormal lipid storage and associated immune dysregulation may be the underlying mechanism for the increased incidence of monoclonal and polyclonal gammopathies, as well as an increased incidence of multiple myeloma in patients with GD. Current observations support this proposition and illustrate the value of investigations into rare diseases, which as ‘experiments of nature’ may provide insights into conditions found in the general population that continue to remain incompletely understood.

  20. Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

    Directory of Open Access Journals (Sweden)

    David Butler

    Full Text Available Alzheimer's disease (AD is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42 sandwich ELISA measures in APP(SwInd mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38 occurs as Aβ(1-42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof

  1. Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

    Science.gov (United States)

    Butler, David; Hwang, Jeannie; Estick, Candice; Nishiyama, Akiko; Kumar, Saranya Santhosh; Baveghems, Clive; Young-Oxendine, Hollie B; Wisniewski, Meagan L; Charalambides, Ana; Bahr, Ben A

    2011-01-01

    Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof

  2. Variants of Rab GTPase-Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke.

    Science.gov (United States)

    Lucitti, Jennifer L; Sealock, Robert; Buckley, Brian K; Zhang, Hua; Xiao, Lin; Dudley, Andrew C; Faber, James E

    2016-12-01

    The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury after large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ≈80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine mapping of C57BL/6 (B6, high extent) and BALB/cByJ (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene. We used additional CNG mapping and knockout mice to narrow the number of candidate genes. Subsequent inspection identified a nonsynonymous single nucleotide polymorphism between B6 and BC within Rabep2 (rs33080487). We then created B6 mice with the BC single nucleotide polymorphism at this locus plus 3 other lines for predicted alteration or knockout of Rabep2 using gene editing. The single amino acid change caused by rs33080487 accounted for the difference in collateral extent and infarct volume between B6 and BC mice attributable to Dce1. Mechanistically, variants of Rabep2 altered collaterogenesis during embryogenesis but had no effect on angiogenesis examined in vivo and in vitro. Rabep2 deficiency altered endosome trafficking known to be involved in VEGF-A→VEGFR2 signaling required for collaterogenesis. Naturally occurring variants of Rabep2 are major determinants of variation in collateral extent and stroke severity in mice. © 2016 American Heart Association, Inc.

  3. Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

    DEFF Research Database (Denmark)

    Kirkegaard, Thomas; Roth, Anke G; Petersen, Nikolaj H T

    2010-01-01

    inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM...

  4. Crosstalk of small GTPases at the Golgi apparatus.

    Science.gov (United States)

    Baschieri, Francesco; Farhan, Hesso

    2012-01-01

    Small GTPases regulate a wide range of homeostatic processes such as cytoskeletal dynamics, organelle homeostasis, cell migration and vesicle trafficking, as well as in pathologic conditions such as carcinogenesis and metastatic spreading. Therefore, it is important to understand the regulation of small GTPase signaling, but this is complicated by the fact that crosstalk exists between different GTPase families and that we have to understand how they signal in time and space. The Golgi apparatus represents a hub for several signaling molecules and its importance in this field is constantly increasing. In this review we will discuss small GTPases signaling at the Golgi apparatus. Then, we will highlight recent work that contributed to a better understanding of crosstalk between different small GTPase families, with a special emphasis on their crosstalk at the Golgi apparatus. Finally, we will give a brief overview of available methods and tools to investigate spatio-temporal small GTPase crosstalk.

  5. ErbB2-associated changes in the lysosomal proteome

    DEFF Research Database (Denmark)

    Nylandsted, Jesper; Becker, Andrea C; Bunkenborg, Jakob

    2011-01-01

    Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic...... purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact...... lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal...

  6. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

    Full Text Available Lysosomal storage diseases (LSDs describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS, where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm, diminished ATP production and increased generation of reactive oxygen species (ROS. Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD, the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase. Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer and glucosylsphingosine (GlcSph in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  7. Role for Rab10 in Methamphetamine-Induced Behavior.

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    Scott M Vanderwerf

    Full Text Available Lipid rafts are specialized, cholesterol-rich membrane compartments that help to organize transmembrane signaling by restricting or promoting interactions with subsets of the cellular proteome. The hypothesis driving this study was that identifying proteins whose relative abundance in rafts is altered by the abused psychostimulant methamphetamine would contribute to fully describing the pathways involved in acute and chronic effects of the drug. Using a detergent-free method for preparing rafts from rat brain striatal membranes, we identified density gradient fractions enriched in the raft protein flotillin but deficient in calnexin and the transferrin receptor, markers of non-raft membranes. Dopamine D1- and D2-like receptor binding activity was highly enriched in the raft fractions, but pretreating rats with methamphetamine (2 mg/kg once or repeatedly for 11 days did not alter the distribution of the receptors. LC-MS analysis of the protein composition of raft fractions from rats treated once with methamphetamine or saline identified methamphetamine-induced changes in the relative abundance of 23 raft proteins, including the monomeric GTP-binding protein Rab10, whose abundance in rafts was decreased 2.1-fold by acute methamphetamine treatment. Decreased raft localization was associated with a selective decrease in the abundance of Rab10 in a membrane fraction that includes synaptic vesicles and endosomes. Inhibiting Rab10 activity by pan-neuronal expression of a dominant-negative Rab10 mutant in Drosophila melanogaster decreased methamphetamine-induced activity and mortality and decreased caffeine-stimulated activity but not mortality, whereas inhibiting Rab10 activity selectively in cholinergic neurons had no effect. These results suggest that activation and redistribution of Rab10 is critical for some of the behavioral effects of psychostimulants.

  8. Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

    Directory of Open Access Journals (Sweden)

    Klöpper Tobias H

    2012-08-01

    Full Text Available Abstract Background Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. Results Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA, consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. Conclusions Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types.

  9. Rice Rab11 is required for JA-mediated defense signaling

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Min Ji [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of); Lee, Yun mi [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Son, Young Sim [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Im, Chak Han [Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360 (Korea, Republic of); Yi, Young Byung [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Rim, Yeong Gil [Systems and Synthetic Agrobiotech Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Bahk, Jeong Dong, E-mail: jdbahk@gnu.ac.kr [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Heo, Jae Bok, E-mail: jbheo72@dau.ac.kr [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of)

    2013-05-17

    Highlights: •OsRab11 interacts with OsOPR8. •OsOPR8 is localized in the cytosol and peroxisome. •OsRab11 enhances the NADPH consumption by OsOPR8. •Transgenic Arabidopsis overexpressing OsRab11 represents a pathogen-resistant phenotype. -- Abstract: Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

  10. Posttranslational lipid modification of Rho family small GTPases.

    Science.gov (United States)

    Mitin, Natalia; Roberts, Patrick J; Chenette, Emily J; Der, Channing J

    2012-01-01

    The Rho family comprises a major branch of the Ras superfamily of small GTPases. A majority of Rho GTPases are synthesized as inactive, cytosolic proteins. They then undergo posttranslational modification by isoprenoid or fatty acid lipids, and together with additional carboxyl-terminal sequences target Rho GTPases to specific membrane and subcellular compartments essential for function. We summarize the use of biochemical and cellular assays and pharmacologic inhibitors instrumental for the study of the role of posttranslational lipid modifications and processing in Rho GTPase biology.

  11. Is Rab25 a tumor promoter or suppressor--context dependency on RCP status?

    Science.gov (United States)

    Tang, Bor Luen

    2010-08-01

    Conflicting reports in the literature suggest that Rab25 could either be a context dependent promoter or suppressor of tumorigenesis. We hypothesized that whether Rab25 acts as a promoter or suppressor in tumor progression depends on the expression status of its effector, the Rab coupling protein (RCP). An elevated expression of RCP resulting from genomic amplification may enhance Rab25's tumor progression activity. Elevation of Rab25 alone may sequester endogenous RCP, and attenuates its activating effect on other oncogenic products, such as mutant Ras.

  12. Identification of Rab41/6d Effectors Provides an Explanation for the Differential Effects of Rab41/6d and Rab6a/a' on Golgi Organization.

    Science.gov (United States)

    Liu, Shijie; Majeed, Waqar; Kudlyk, Tetyana; Lupashin, Vladimir; Storrie, Brian

    2016-01-01

    Unexpectedly, members of the Rab VI subfamily exhibit considerable variation in their effects on Golgi organization and trafficking. By fluorescence microscopy, neither depletion nor overexpression of the GDP-locked form of Rab6a/a', the first trans Golgi-associated Rab protein discovered, affects Golgi ribbon organization while, on the other hand, both Rab41/6d depletion and overexpression of GDP-locked form cause Golgi fragmentation into a cluster of punctate elements, suggesting that Rab41/6d has an active role in maintenance of Golgi ribbon organization. To establish a molecular basis for these differences, we screened for Rab41/6d interacting proteins by yeast two-hybrid assay. 155 non-repetitive hits were isolated and sequenced, and after searching in NCBI database, 102 different proteins and protein fragments were identified. None of these hits overlapped with any published Rab6a/a' effector. Eight putative Rab41 interactors involved in membrane trafficking were found. Significantly, these exhibited a preferential interaction with GTP- vs. GDP-locked Rab41/6d. Of the 8 hits, the dynactin 6, syntaxin 8, and Kif18A plasmids were the only ones expressing the full-length protein. Hence, these 3 proteins were selected for further study. We found that depletion of dynactin 6 or syntaxin 8, but not Kif18A, resulted in a fragmented Golgi apparatus that displayed a Rab41/6d knockdown phenotype, i.e., the Golgi apparatus was disrupted into a cluster of punctate Golgi elements. Co-immunoprecipation experiments verified that the interaction of dynactin 6 and syntaxin 8 with GTP-locked Rab41/6d was stronger than that with wild type Rab41/6d and least with the GDP-locked form. In contrast, co-immunoprecipitation interaction with Rab6a was greatest with the GDP-locked Rab6a, suggestive of a non-physiological interaction. In conclusion, we suggest that dynactin 6, a subunit of dynactin complex, the minus-end-directed, dynein motor, provides a sufficient molecular basis to

  13. [EoRab43 regulating vesicular transport around the macronucleus in Euplotes octocarinatus].

    Science.gov (United States)

    Li, Jiang Jiao; Nie, Yu; Liaing, Ai Hua; Dang, Xu Hong; Wang, Wei

    2009-02-01

    Rab family proteins play a crucial role in regulating vesicular traffic in eukaryotic cells. EoRab43 is an atypical Rab gene identified in Euplotes octocarinatus. In order to understand the function of EoRab43, the 153bp fragment of the 3'-end of EoRab43 gene was subcloned into expression vector pGEX-6P-1, and the recombinant plasmid pGEX-EoRab43(153bp) was transfered into E.coli BL21 (DE3) to express the fusion protein. The fusion protein GST-EoRab43C was expressed and purified by affinity chromatography. BALB/c mice were immunolized by purified GST-EoRab43C. The titer of anti-EoRab43C polyclonal antibody was detected by indirect ELISA assay and the specificity of the antibody was detected by Western blot. Immunofluorescence experiments were performed using anti-EoRab43C antibody in the cells of Euplotes. The results showed that EoRab43 displayed a punctuate pattern in the cytoplasm around the macronuclear chromosome of Euplotes.

  14. Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases.

    Science.gov (United States)

    Lloyd-Evans, Emyr; Platt, Frances M

    2011-08-01

    Disrupted cellular Ca(2+) signaling is believed to play a role in a number of human diseases including lysosomal storage diseases (LSD). LSDs are a group of ∼50 diseases caused predominantly by mutations in lysosomal proteins that result in accumulation of macromolecules within the lysosome. We recently reported that Niemann-Pick type C (NPC) is the first human disease to be associated with defective lysosomal Ca(2+) uptake and defective NAADP-mediated lysosomal Ca(2+) release. These defects in NPC cells leads to the disruption in endocytosis and subsequent lipid storage that is a feature of this disease. In contrast, Chediak-Higashi Syndrome cells have been reported to have enhanced lysosomal Ca(2+) uptake whilst the TRPML1 protein defective in mucolipidosis type IV is believed to function as a Ca(2+) channel. In this review we provide a summary of the current knowledge on the role of lysosomal Ca(2+) signaling in the pathogenesis of this group of diseases.

  15. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin.

    Directory of Open Access Journals (Sweden)

    Ekaterina A Ivanova

    Full Text Available Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.

  16. C11ORF24 is a novel type I membrane protein that cycles between the Golgi apparatus and the plasma membrane in Rab6-positive vesicles.

    Science.gov (United States)

    Fraisier, Vincent; Kasri, Amal; Miserey-Lenkei, Stéphanie; Sibarita, Jean-Baptiste; Nair, Deepak; Mayeux, Adeline; Bardin, Sabine; Toyoda, Yusuke; Poser, Ina; Poznyakovskiy, Andrei; Goud, Bruno; Hyman, Anthony A; Dimitrov, Ariane

    2013-01-01

    The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

  17. Tonoplast targeting of VHA-a3 relies on a Rab5-mediated but Rab7-independent vacuolar trafficking route.

    Science.gov (United States)

    Feng, Qiang-Nan; Zhang, Yan; Li, Sha

    2017-04-01

    Vacuolar trafficking routes and their regulators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER-to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V-ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells. © 2017 Institute of Botany, Chinese Academy of Sciences.

  18. GTPases involved in bacterial ribosome maturation.

    Science.gov (United States)

    Goto, Simon; Muto, Akira; Himeno, Hyouta

    2013-05-01

    The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

  19. Lysosomal stress: a new player in perturbed lipid metabolism

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

    Lysosomes are involved in many different essential cellular processes, among others organelle and molecule degradation, exocytosis, cell energy metabolism, cholesterol and sphingolipid level regulation. Lysosomal stress has a strong impact on the immune system, affecting specially macrophages as the

  20. Lysosomal stress: a new player in perturbed lipid metabolism

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

    Lysosomes are involved in many different essential cellular processes, among others organelle and molecule degradation, exocytosis, cell energy metabolism, cholesterol and sphingolipid level regulation. Lysosomal stress has a strong impact on the immune system, affecting specially macrophages as the

  1. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion

    Institute of Scientific and Technical Information of China (English)

    Jing Zhou; Shi-Hao Tan; Valérie Nicolas; Chantal Bauvy; Nai-Di Yang; Jianbin Zhang; Yuan Xue

    2013-01-01

    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy.In this study,we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torinl),but not by an allosteric inhibitor (rapamycin),leads to activation of lysosomal function.Second,we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1),but not mTORC2,and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function.Third,we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation.Finally,Atg5 or Atg7deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation,suggesting that lysosomal activation occurring in the course of autophagy is dependent on antophagosome-lysosome fusion.Taken together,this study demonstrates that in the course of autophagy,lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

  2. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.

    Science.gov (United States)

    Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

    2013-04-01

    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

  3. Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

    Directory of Open Access Journals (Sweden)

    Yoo Eunice

    2007-02-01

    Full Text Available Abstract Background Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits. Results Here, we show a phylogenetic analysis of the three TRAPPII-specific subunits. One copy of each of the two essential subunits, Trs120 and Trs130, is present in almost every fully sequenced eukaryotic genome. Moreover, the primary, as well as the predicted secondary, structure of the Trs120- and Trs130-related sequences are conserved from fungi to animals. The mammalian orthologs of Trs120 and Trs130, NIBP and TMEM1, respectively, are candidates for human disorders. Currently, NIBP is implicated in signaling, and TMEM1 is suggested to have trans-membrane domains (TMDs and to function as a membrane channel. However, we show here that the yeast Trs130 does not function as a trans-membrane protein, and the human TMEM1 does not contain putative TMDs. The non-essential subunit, Trs65, is conserved only among many fungi and some unicellular eukaryotes. Multiple alignment analysis of each TRAPPII-specific subunit revealed conserved domains that include highly conserved amino acids. Conclusion We suggest that the function of both NIBP and TMEM1 in the regulation of intra-cellular trafficking is conserved from yeast to man. The conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals.

  4. Mucolipidosis type IV protein TRPML1-dependent lysosome formation.

    Science.gov (United States)

    Miller, Austin; Schafer, Jessica; Upchurch, Cameron; Spooner, Ellen; Huynh, Julie; Hernandez, Sebastian; McLaughlin, Brooke; Oden, Liam; Fares, Hanna

    2015-03-01

    Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endosomes to form hybrid organelles, lysosomes are reformed as discrete organelles. This lysosome reformation or formation is a poorly understood process that has not been systematically analyzed and that lacks known regulators. In this study, we quantitatively define the multiple steps of lysosome formation and identify the first regulator of this process.

  5. Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.

    OpenAIRE

    Wilson, J S; Apte, M V; Thomas, M. C.; Haber, P S; Pirola, R C

    1992-01-01

    Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from cho...

  6. Lysosomal storage disease 2 - Pompe's disease

    NARCIS (Netherlands)

    van der Ploeg, Ans T.; Reuser, Arnold J. J.

    2008-01-01

    Pompe's disease, glycogen-storage disease type II, and acid maltase deficiency are alternative names for the same metabolic disorder. It is a pan-ethnic autosomal recessive trait characterised by acid alpha-glucosidase deficiency leading to lysosomal glycogen storage. Pompe's disease is also

  7. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is lar

  8. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is

  9. Lysosomal proteolysis: effects of aging and insulin.

    Science.gov (United States)

    Gromakova, I A; Konovalenko, O A

    2003-07-01

    Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.

  10. TRPML: transporters of metals in lysosomes essential for cell survival?

    Science.gov (United States)

    Kiselyov, Kirill; Colletti, Grace A; Terwilliger, Austen; Ketchum, Kathleen; Lyons, Christopher W P; Quinn, James; Muallem, Shmuel

    2011-09-01

    Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca(2+)-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe(2+), Ca(2+) and Zn(2+) transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.

  11. A non-conserved miRNA regulates lysosomal function and impacts on a human lysosomal storage disorder

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Di Malta, Chiara; Wen, Jiayu

    2014-01-01

    Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity...

  12. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

    Full Text Available Lysosomal storage diseases (LSDs include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss.

  13. Involvement of Rab28 in NF-κB nuclear transport in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Jun Jiang

    Full Text Available Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB. Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs through a paracrine control of vascular smooth muscles cells (VSMCs, which at least partly via angiotensin II (Ang II. Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.

  14. Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing.

    Science.gov (United States)

    Bartusch, Christina; Döring, Tatjana; Prange, Reinhild

    2017-06-21

    Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

  15. Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA in human epithelial cells.

    Directory of Open Access Journals (Sweden)

    Giuseppe Bozza

    Full Text Available Neisseria meningitidis adhesin A (NadA is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR. Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.

  16. RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

    Science.gov (United States)

    Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

    2011-02-17

    Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics.

  17. Rho GTPase-formin pairs in cytoskeletal remodelling.

    Science.gov (United States)

    Eisenmann, Kathryn M; Peng, Jun; Wallar, Bradley J; Alberts, Arthur S

    2005-01-01

    Diaphanous-related formins (Drfs) are members of a conserved formin family of actin-nucleating proteins and are thought to act as Rho GTPase effectors in signal transduction pathways that govern gene expression, cytoskeletal remodelling and cell division. In vitro evidence suggests that the three mammalian Drf proteins--mDia1, mDia2 and mDia3-have distinct GTPase-binding specificities. However, much of our current understanding of GTPase-Drf partnerships in mammalian cell signalling is based on expression studies using Drfs missing their unique GTPase-binding domains. We have employed fluorescence resonance energy transfer (FRET) and gene targeting approaches to identify the function of different GTPase-formin pairs in cell signalling. These studies have allowed us to uncover new roles for Drf proteins in cytoskeletal remodelling and novel regulatory mechanisms whereby GTPases influence formin function. Our genetic experiments strongly suggest that Drfs cooperate with other GTPase effector proteins, including the gene product of the Wiskott-Aldrich syndrome gene, WASP, during the regulation of cell proliferation. Further, the Drf gene knockout experiments indicate that this family of formins has a role in cancer pathophysiology.

  18. Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus).

    Science.gov (United States)

    Xinli, Xiao; Lei, Peng

    2015-03-04

    The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit.

  19. In vitro comparative kinetic analysis of the chloroplast Toc GTPases.

    Science.gov (United States)

    Reddick, L Evan; Vaughn, Michael D; Wright, Sarah J; Campbell, Ian M; Bruce, Barry D

    2007-04-13

    A unique aspect of protein transport into plastids is the coordinate involvement of two GTPases in the translocon of the outer chloroplast membrane (Toc). There are two subfamilies in Arabidopsis, the small GTPases (Toc33 and Toc34) and the large acidic GTPases (Toc90, Toc120, Toc132, and Toc159). In chloroplasts, Toc34 and Toc159 are implicated in precursor binding, yet mechanistic details are poorly understood. How the GTPase cycle is modulated by precursor binding is complex and in need of careful dissection. To this end, we have developed novel in vitro assays to quantitate nucleotide binding and hydrolysis of the Toc GTPases. Here we present the first systematic kinetic characterization of four Toc GTPases (cytosolic domains of atToc33, atToc34, psToc34, and the GTPase domain of atToc159) to permit their direct comparison. We report the KM, Vmax, and Ea values for GTP hydrolysis and the Kd value for nucleotide binding for each protein. We demonstrate that GTP hydrolysis by psToc34 is stimulated by chloroplast transit peptides; however, this activity is not stimulated by homodimerization and is abolished by the R133A mutation. Furthermore, we show peptide stimulation of hydrolytic rates are not because of accelerated nucleotide exchange, indicating that transit peptides function as GTPase-activating proteins and not guanine nucleotide exchange factors in modulating the activity of psToc34. Finally, by using the psToc34 structure, we have developed molecular models for atToc33, atToc34, and atToc159G. By combining these models with the measured enzymatic properties of the Toc GTPases, we provide new insights of how the chloroplast protein import cycle may be regulated.

  20. Activity of lysosomal exoglycosidases in human gliomas.

    Science.gov (United States)

    Wielgat, P; Walczuk, U; Szajda, S; Bień, M; Zimnoch, L; Mariak, Z; Zwierz, K

    2006-12-01

    There is a lot of data suggesting that modifications of cell glycoconjugates may be important in progression of cancer. In the present work we studied activities of lysosomal exoglycosidases: beta-hexosaminidase and its isoenzymes A and B, beta-galactosidase and alpha-mannosidase, in human gliomas. Enzyme activity was determined spectrophotometrically based on the release of p-nitrophenol from p-nitrophenyl-derivative of appropriate sugars. The activities of the exoglycosidases tested were significantly higher in malignant glial tumors than in control tissue (normal brain tissue) and non-glial tumors. The highest activities of exoglycosidases were observed in high-grade gliomas, and a positive correlation of enzyme activities and degree of malignancy was noted. Our results suggest that lysosomal exoglycosidases may participate in the progression and dynamical development of glial tumors.

  1. Ras-like family small GTPases genes in Nilaparvata lugens: Identification, phylogenetic analysis, gene expression and function in nymphal development

    Science.gov (United States)

    Wang, Weixia; Li, Kailong; Wan, Pinjun; Lai, Fengxiang; Fu, Qiang; Zhu, Tingheng

    2017-01-01

    Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRβ, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRβ could be served as a candidate target for dsRNA-based pesticides for N.lugens control. PMID:28241066

  2. Insulin regulates Rab3-Noc2 complex dissociation to promote GLUT4 translocation in rat adipocytes.

    Science.gov (United States)

    Koumanov, Francoise; Pereira, Vinit J; Richardson, Judith D; Sargent, Samantha L; Fazakerley, Daniel J; Holman, Geoffrey D

    2015-08-01

    The glucose transporter GLUT4 is present mainly in insulin-responsive tissues of fat, heart and skeletal muscle and is translocated from intracellular membrane compartments to the plasma membrane (PM) upon insulin stimulation. The transit of GLUT4 to the PM is known to be dependent on a series of Rab proteins. However, the extent to which the activity of these Rabs is regulated by the action of insulin action is still unknown. We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation. We developed a new photoaffinity reagent (Bio-ATB-GTP) that allows GTP-binding proteomes to be explored. Using this approach we screened for insulin-responsive GTP loading of Rabs in primary rat adipocytes. We identified Rab3B as a new candidate insulin-stimulated G-protein in adipocytes. Using constitutively active and dominant negative mutants and Rab3 knockdown we provide evidence that Rab3 isoforms are key regulators of GLUT4 translocation in adipocytes. Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2. Disruption of the Rab3-Noc2 complex leads to displacement of Noc2 from the PM. This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation. The discovery of the involvement of Rab3 and Noc2 in an insulin-regulated step in GLUT4 translocation suggests that the control of this translocation process is unexpectedly similar to regulated secretion and particularly pancreatic insulin-vesicle release.

  3. Rab3A deletion selectively reduces spontaneous neurotransmitter release at the mouse neuromuscular synapse.

    Science.gov (United States)

    Sons, Michèle S; Plomp, Jaap J

    2006-05-17

    Rab3A is a synaptic vesicle-associated GTP-binding protein thought to be involved in modulation of presynaptic transmitter release through regulation of vesicle trafficking and membrane fusion. Electrophysiological studies at central nervous system synapses of Rab3A null-mutant mice have indicated that nerve stimulation-evoked transmitter release and its short- and long-term modulation are partly dependent on Rab3A, whereas spontaneous uniquantal release is completely independent of it. Here, we studied the acetylcholine (ACh) release at the neuromuscular junction (NMJ) of diaphragm and soleus muscles from Rab3A-deficient mice with intracellular microelectrode methods. Surprisingly, we found 20-40% reduction of spontaneous ACh release but completely intact nerve action potential-evoked release at both high- and low-rate stimulation and during recovery from intense release. The ACh release induced by hypertonic medium was also unchanged, indicating that the pool of vesicles for immediate release is unaltered at the Rab3A-deficient NMJ. These results indicate a selective role of Rab3A in spontaneous transmitter release at the NMJ which cannot or only partly be taken over by the closely related Rab3B, Rab3C, or Rab3D isoforms when Rab3A is deleted. It has been hypothesized that Rab3A mutation underlies human presynaptic myasthenic syndromes, in which severely reduced nerve action potential-evoked ACh release at the NMJ causes paralysis. Our observation that Rab3A deletion does not reduce evoked ACh release at any stimulation rate at the mouse NMJ, argues against this hypothesis.

  4. TCP-Rab:a receiver advertisement based TCP protocol

    Institute of Scientific and Technical Information of China (English)

    汤旭红; 刘正蓝; 朱淼良

    2004-01-01

    With the evolution of network technologies,the deficiencies of TCP protocol are becoming more and more distinct. The new TCP implementation,called Receiver Advertisement Based TCP(TCP-Rab)proposed here to eliminate these deficiencies,adopts two basic mechanisms:(1)Bandwidth Estimation and(2)Immediate Recovery. Bandwidth estimation is carried out at the receiver,and the result is sent back to the sender via the acknowledgments. Immediate Recovery guarantees high performance even in lossy link. Rab can distinguish the reason for packet loss,and thus adopt appropriate recovery strategy. For loss by network congestion,it will back off its congestion window,and for loss by link errors,it will recover the congestion window immediately. Simulations indicated that Rah has superiority over other TCP implementations.

  5. Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Patricio; Soto, Nicolás [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Díaz, Jorge [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Mendoza, Pablo [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Díaz, Natalia [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Quest, Andrew F.G. [Center for Molecular Studies of the Cell, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Torres, Vicente A., E-mail: vatorres@med.uchile.cl [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago (Chile)

    2015-08-21

    The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.

  6. Regulation of lysosomal ion homeostasis by channels and transporters.

    Science.gov (United States)

    Xiong, Jian; Zhu, Michael X

    2016-08-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H(+), Ca(2+), Na(+), K(+), and Cl(-) across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease.

  7. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    Science.gov (United States)

    Ravi, Sreeram; Peña, Karina A; Chu, Charleen T; Kiselyov, Kirill

    2016-11-01

    Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca(2+). We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.

  8. A potentially dynamic lysosomal role for the endogenous TRPML proteins.

    Science.gov (United States)

    Zeevi, David A; Frumkin, Ayala; Offen-Glasner, Vered; Kogot-Levin, Aviram; Bach, Gideon

    2009-10-01

    Lysosomal storage disorders (LSDs) constitute a diverse group of inherited diseases that result from lysosomal storage of compounds occurring in direct consequence to deficiencies of proteins implicated in proper lysosomal function. Pathology in the LSD mucolipidosis type IV (MLIV), is characterized by lysosomal storage of lipids together with water-soluble materials in cells from every tissue and organ of affected patients. Mutations in the mucolipin 1 (TRPML1) protein cause MLIV and TRPML1 has also been shown to interact with two of its paralogous proteins, mucolipin 2 (TRPML2) and mucolipin 3 (TRPML3), in heterologous expression systems. Heterogeneous lysosomal storage is readily identified in electron micrographs of MLIV patient cells, suggesting that proper TRPML1 function is essential for the maintenance of lysosomal integrity. In order to investigate whether TRPML2 and TRPML3 also play a role in the maintenance of lysosomal integrity, we conducted gene-specific knockdown assays against these protein targets. Ultrastructural analysis revealed lysosomal inclusions in both TRPML2 and TRPML3 knockdown cells, suggestive of a common mechanism for these proteins, in parallel with TRPML1, in the regulation of lysosomal integrity. However, co-immunoprecipitation assays revealed that physical interactions between each of the endogenous TRPML proteins are quite limited. In addition, we found that all three endogenous proteins only partially co-localize with each other in lysosomal as well as extra-lysosomal compartments. This suggests that native TRPML2 and TRPML3 might participate with native TRPML1 in a dynamic form of lysosomal regulation. Given that depletion of TRPML2/3 led to lysosomal storage typical to an LSD, we propose that depletion of these proteins might also underlie novel LSD pathologies not described hitherto.

  9. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    Science.gov (United States)

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  10. Medicago truncatula Rop GTPases expression in young nodules

    Directory of Open Access Journals (Sweden)

    Lucia Kusu

    2012-02-01

    Full Text Available Small GTPases are monomeric guanine nucleotide binding proteins with molecular weight of 21 to 30 kD. Plants have plantspecific small GTPases, termed Rop (Rho GTPases from plants which serve as signaling proteins in plant signal transduction. To have better understanding on nodulation process in legume, which Rop proteins that play a dominant role in nodule formation would be determined. Therefore, the expression of five Medicago truncatula Rop GTPase genes (MtRops in young nodules were studied in this experiment. RT-PCR method was used to examine the expression of MtRops. The MtRops expression in inoculated M. truncatula plants were compared with those in uninoculated plants. Results of this studies showed that the expression of MtRop5 and MtRop6 were significantly induced in young nodules, it was suggesting that these Rops may have important role during nodule formation.

  11. Study on the chaperone properties of conserved GTPases.

    Science.gov (United States)

    Wang, Xiang; Xue, Jiaying; Sun, Zhe; Qin, Yan; Gong, Weimin

    2012-01-01

    As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.

  12. High Rab11-FIP4 expression predicts poor prognosis and exhibits tumor promotion in pancreatic cancer

    Science.gov (United States)

    He, Yun; Ye, Mengsi; Zhou, Lingling; Shan, Yunfeng; Lu, Guangrong; Zhou, Yuhui; Zhong, Jinwei; Zheng, Jihang; Xue, Zhanxiong; Cai, Zhenzhai

    2017-01-01

    Some studies have demonstrated that Rab11-family interacting proteins (Rab11-FIPs) are connected with the tumorigenesis, and they may act as tumor promoters in some cancers. The clinicopathological significance of Rab11-family interacting protein 4 (Rab11-FIP4) expression and its possible effects on pancreatic cancer (PC) are still undiscovered. In this study, Rab11-FIP4 protein expression level in 60 PC specimens and pair-matched non-cancerous samples were detected by immunohistochemistry analysis. The results were analysed and compared with each patients' clinical data. Rab11-FIP4 expression in PC tissues increased significantly more than that of adjacent non-cancerous tissues (P=0.0001). Overexpression of Rab11-FIP4 in the PC tissues was significantly related to tumor size (P=0.0001), histological grade (P=0.028), metastasis (P=0.001) and TNM stage (P=0.004) but not with age (P=0.832), gender (P=0.228) or tumor site (P=0.875). Kaplan-Meier survival analysis showed that overexpression of Rab11-FIP4 was significantly related to overall survival time (P=0.0036). In addition, Rab11-FIP4 in PANC-1 pancreatic cancer cells were successfully knocked-out using the CRISPR/Cas9 system. Rab11-FIP4 knockout in PANC-1 cells inhibited cell growth, invasion and metastasis, and arrested cell cycle progression, but did not alter apoptosis. Our findings suggest that overexpression of Rab11-FIP4 predicts poor clinical outcomes for pancreatic cancer and contributes to pancreatic tumor progression. PMID:28035375

  13. Reverse engineering GTPase programming languages with reconstituted signaling networks.

    Science.gov (United States)

    Coyle, Scott M

    2016-07-02

    The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors. However, we know less about how these different activities work together as a system to specify complex dynamic signaling outcomes. Decoding this molecular "programming language" would help us understand how different species and cell types have used the same GTPase machinery in different ways to accomplish different tasks, and would also provide new insights as to how mutations to these networks can cause disease. We recently developed a bead-based microscopy assay to watch reconstituted H-Ras signaling systems at work under arbitrary configurations of regulators and effectors. (5) Here we highlight key observations and insights from this study and propose extensions to our method to further study this and other GTPase signaling systems.

  14. A thirty-year quest for a role of R-Ras in cancer: from an oncogene to a multitasking GTPase.

    Science.gov (United States)

    Liu, Wai Nam; Yan, Mingfei; Chan, Andrew M

    2017-09-10

    Since the identification of R-Ras, which is the first Ras-related GTPase isolated based on sequence similarity to the classical RAS oncogene, more than 160 members of the Ras superfamily of GTPases have been identified and classified into the Ras, Rho, Rap, Rab, Ran, Arf, Rheb, RGK, Rad, Rit, and Miro subfamilies. R-Ras belongs to the Ras subfamily of small G-proteins, which are frequently implicated in cell growth and differentiation. Although the roles of R-Ras in cellular transformation and integrin-mediated cell adhesion have been extensively studied, the physiological function of this enigmatic G-protein was only revealed when a mouse strain deficient in R-Ras was generated. In parallel, a plethora of research findings also linked R-Ras with processes including tumor angiogenesis, axon guidance, and immune cell trafficking. Several upstream factors that modulate R-Ras GTP-binding were identified including Notch, semaphorin, and chemokine C-C motif ligand 21. A review of our evolving understanding of the role of R-Ras in oncogenesis is timely, as this year marks the 30th anniversary of the publication describing the cloning of R-Ras. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

    Trapp, Stefan; Rosania, G.; Horobin, R.W.;

    2008-01-01

    Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick–Nernst–Planck equation. The cell model considers....... This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions....

  16. Release and uptake of lysosomal enzymes : studied in cultured cells

    OpenAIRE

    1980-01-01

    textabstractThe purpose of the experimental work described in this thesiswas to investigate some aspects of the release and uptake of lysosomal enzymes. The experiments involved the use of normal human and animal fibroblasts and some other cell types such as hepatocytes and hepatoma cells as sources of hydrolytic enzymes, and fibroblasts from patients with lysosomal storage diseases associated with a single lysosomal enzyme deficiency and with "1-cell" disease as recipient cells. In a number ...

  17. Factors and processes modulating phenotypes in neuronopathic lysosomal storage diseases

    OpenAIRE

    Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Banecka-Majkutewicz, Zyta; Banecki, Bogdan; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2013-01-01

    Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70 %) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal stor...

  18. Rab41 is a novel regulator of Golgi apparatus organization that is needed for ER-to-Golgi trafficking and cell growth.

    Directory of Open Access Journals (Sweden)

    Shijie Liu

    Full Text Available BACKGROUND: The 60(+ members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a', Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a' has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization. METHODS/RESULTS: Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel. CONCLUSION: We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a' and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our

  19. Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

    Science.gov (United States)

    Sukhai, Mahadeo A.; Prabha, Swayam; Hurren, Rose; Rutledge, Angela C.; Lee, Anna Y.; Sriskanthadevan, Shrivani; Sun, Hong; Wang, Xiaoming; Skrtic, Marko; Seneviratne, Ayesh; Cusimano, Maria; Jhas, Bozhena; Gronda, Marcela; MacLean, Neil; Cho, Eunice E.; Spagnuolo, Paul A.; Sharmeen, Sumaiya; Gebbia, Marinella; Urbanus, Malene; Eppert, Kolja; Dissanayake, Dilan; Jonet, Alexia; Dassonville-Klimpt, Alexandra; Li, Xiaoming; Datti, Alessandro; Ohashi, Pamela S.; Wrana, Jeff; Rogers, Ian; Sonnet, Pascal; Ellis, William Y.; Corey, Seth J.; Eaves, Connie; Minden, Mark D.; Wang, Jean C.Y.; Dick, John E.; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D.

    2012-01-01

    Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML. PMID:23202731

  20. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

    Mony, Vinod K; Benjamin, Shawna; O'Rourke, Eyleen J

    2016-01-01

    Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromolecules for the regeneration of basic building blocks, cellular housekeeping, or pathogen degradation. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different soluble hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense molecules like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis.

  1. Cell biology in China: Focusing on the lysosome.

    Science.gov (United States)

    Yang, Chonglin; Wang, Xiaochen

    2017-06-01

    The view that lysosomes are merely the recycling bins of the cell has changed greatly during recent years. Lysosomes are now known to play a central role in signal transduction, cellular adaptation, plasma membrane repair, immune responses and many other fundamental cellular processes. In conjunction with the seminal discoveries made by international colleagues, many important questions regarding lysosomes are being addressed by Chinese scientists. In this review, we briefly summarize recent exciting findings in China on lysosomal signaling, biogenesis, integrity and physiological functions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations

    DEFF Research Database (Denmark)

    Meeths, Marie; Bryceson, Yenan T; Rudd, Eva

    2010-01-01

    Griscelli syndrome type 2 (GS2) is an autosomal-recessive immunodeficiency caused by mutations in RAB27A, clinically characterized by partial albinism and haemophagocytic lymphohistocytosis (HLH). We evaluated the frequency of RAB27A mutations in 21 unrelated patients with haemophagocytic syndrom...

  3. Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

    Directory of Open Access Journals (Sweden)

    Fumitaka Momose

    Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

  4. Rab10 delivers GLUT4 storage vesicles to the plasma membrane.

    Science.gov (United States)

    Chen, Yu; Lippincott-Schwartz, Jennifer

    2013-05-01

    The glucose transporter, GLUT4, redistributes to the plasma membrane (PM) upon insulin stimulation, but also recycles through endosomal compartments. Different Rab proteins control these transport itineraries of GLUT4. However, the specific roles played by different Rab proteins in GLUT4 trafficking has been difficult to assess, primarily due to the complexity of endomembrane organization and trafficking. To address this problem, we recently performed advanced live cell imaging using total internal reflection fluorescence (TIRF) microscopy, which images objects ~150 nm from the PM, directly visualizing GLUT4 trafficking in response to insulin stimulation. Using IRAP-pHluorin to selectively label GSVs undergoing PM fusion in response to insulin, we identified Rab10 as the only Rab protein that binds this compartment. Rab14 was found to label transferrin-positive, endosomal compartments containing GLUT4. These also could fuse with the PM in response to insulin, albeit more slowly. Several other Rab proteins, including Rab4A, 4B and 8A, were found to mediate GLUT4 intra-endosomal recycling, serving to internalize surface-bound GLUT4 into endosomal compartments for ultimate delivery to GSVs. Thus, multiple Rab proteins regulate the circulation of GLUT4 molecules within the endomembrane system, maintaining optimal insulin responsiveness within cells.

  5. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

    Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  6. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M; Haslett, Luke J; Kumar, Asok; Sato, Yutaka; Lie, Pearl P Y; Mohan, Panaiyur; Coffey, Erin E; Kompella, Uday; Mitchell, Claire H; Lloyd-Evans, Emyr; Nixon, Ralph A

    2015-09-01

    Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  7. The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

    Science.gov (United States)

    Weber, Mary M; Faris, Robert; van Schaik, Erin J; McLachlan, Juanita Thrasher; Wright, William U; Tellez, Andres; Roman, Victor A; Rowin, Kristina; Case, Elizabeth Di Russo; Luo, Zhao-Qing; Samuel, James E

    2016-09-01

    Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

  8. Gaucher disease: a lysosomal neurodegenerative disorder.

    Science.gov (United States)

    Huang, W J; Zhang, X; Chen, W W

    2015-04-01

    Gaucher disease is a multisystemic disorder that affects men and woman in equal numbers and occurs in all ethnic groups at any age with racial variations and an estimated worldwide incidence of 1/75,000. It is caused by a genetic deficient activity of the lysosomal enzyme glucocerebrosidase due to mutations in the β-glucocerebrosidase gene, and resulting in lack of glucocerebroside degradation. The subsequent accumulation of glucocerebroside in lysosomes of tissue macrophages primarily in the liver, bone marrow and spleen, causes damage in haematological, skeletal and nervous systems. The clinical manifestations show a high degree of variability with symptoms that varies according to organs involved. In many cases, these disorders do not correlate with mutations in the β-glucocerebrosidase gene. Although several mutations have been identified as responsible for the deficient activity of glucocerebrosidase, mechanisms by which this enzymatic defect leads to Gaucher disease remain poorly understood. Recent reports indicate the implication of complex mechanisms, including enzyme deficiency, substrate accumulation, unfolded protein response, and macrophage activation. Further elucidating these mechanisms will advance understanding of Gaucher disease and related disorders.

  9. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  10. V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

    Science.gov (United States)

    Meo-Evoli, Nathalie; Almacellas, Eugènia; Massucci, Francesco Alessandro; Gentilella, Antonio; Ambrosio, Santiago; Kozma, Sara C; Thomas, George; Tauler, Albert

    2015-09-29

    In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

  11. Rac and Rho GTPases in cancer cell motility control

    Directory of Open Access Journals (Sweden)

    Parri Matteo

    2010-09-01

    Full Text Available Abstract Rho GTPases represent a family of small GTP-binding proteins involved in cell cytoskeleton organization, migration, transcription, and proliferation. A common theme of these processes is a dynamic reorganization of actin cytoskeleton which has now emerged as a major switch control mainly carried out by Rho and Rac GTPase subfamilies, playing an acknowledged role in adaptation of cell motility to the microenvironment. Cells exhibit three distinct modes of migration when invading the 3 D environment. Collective motility leads to movement of cohorts of cells which maintain the adherens junctions and move by photolytic degradation of matrix barriers. Single cell mesenchymal-type movement is characterized by an elongated cellular shape and again requires extracellular proteolysis and integrin engagement. In addition it depends on Rac1-mediated cell polarization and lamellipodia formation. Conversely, in amoeboid movement cells have a rounded morphology, the movement is independent from proteases but requires high Rho GTPase to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible and several moving cells, including tumor cells, show an high degree of plasticity in motility styles shifting ad hoc between mesenchymal or amoeboid movements. This review will focus on the role of Rac and Rho small GTPases in cell motility and in the complex relationship driving the reciprocal control between Rac and Rho granting for the opportunistic motile behaviour of aggressive cancer cells. In addition we analyse the role of these GTPases in cancer progression and metastatic dissemination.

  12. Lysosome/lipid droplet interplay in metabolic diseases.

    Science.gov (United States)

    Dugail, Isabelle

    2014-01-01

    Lysosomes and lipid droplets are generally considered as intracellular compartments with divergent roles in cell metabolism, lipid droplets serving as lipid reservoirs in anabolic pathways, whereas lysosomes are specialized in the catabolism of intracellular components. During the last few years, new insights in the biology of lysosomes has challenged this view by providing evidence for the importance of lysosome recycling as a sparing mechanism to maintain cellular fitness. On the other hand the understanding of lipid droplets has evolved from an inert intracellular deposit toward the status of an intracellular organelle with dynamic roles in cellular homeostasis beyond storage. These unrelated aspects have also recently converged in the finding of unexpected lipid droplet/lysosome communication through autophagy, and the discovery of lysosome-mediated lipid droplet degradation called lipopagy. Furthermore, adipocytes which are professional cells for lipid droplet formation were also shown to be active in peptide antigen presentation a pathway requiring lysosomal activity. The potential importance of lipid droplet/lysosome interplay is discussed in the context of metabolic diseases and the setting of chronic inflammation.

  13. [The blood-brain barrier and neurodegenerative lysosomal storage diseases].

    Science.gov (United States)

    Urayama, Akihiko

    2013-02-01

    Enzyme replacement therapy has been a very effective treatment for several lysosomal storage diseases. However, correcting central nervous system (CNS) storage has been challenging due to the presence of the blood-brain barrier (BBB), which hampers the entry of circulating lysosomal enzymes into the brain. In our previous studies, we discovered that luminally expressed cation-independent mannose 6-phosphate (M6P) receptor is a universal transporter for lysosomal enzymes that contain M6P moieties on the enzyme molecule. This receptor-mediated transport of lysosomal enzymes showed developmental down-regulation that resulted in a failure of delivery of lysosomal enzymes across the BBB in the adult brain. Conceptually, if one can re-induce M6P receptor-mediated transport of lysosomal enzymes in adult BBB, this could provide a novel brain targeting approach for treating abnormal storage in the CNS, regardless of the age of subjects. We found that systemic adrenergic stimuli restored functional transport of β-glucuronidase across the adult BBB. The concept of manipulating BBB transport activity by endogenous characteristics has also been demonstrated by another group who showed effective treatment in a Pompe disease model animal in vivo. It is intriguing that lysosomal enzymes utilize multiple mechanisms for their transport across the BBB. This review explores pharmacological manipulations for the delivery of lysosomal enzymes into the CNS, and the mechanisms of their transport across the BBB, based on existing evidence from studies of β-glucuronidase, sulfamidase, acid α-glucosidase, and arylsulfatase A.

  14. Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

    NARCIS (Netherlands)

    G.T.J. van der Horst (Gijsbertus); U. Rose (Ursula); R. Brossmer (Reinhard); F.W. Verheijen (Frans)

    1990-01-01

    markdownabstractAbstract ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki ≈ 21 μM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuramin

  15. Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin.

    Science.gov (United States)

    Yang, Nai-Di; Tan, Shi-Hao; Ng, Shukie; Shi, Yin; Zhou, Jing; Tan, Kevin Shyong Wei; Wong, Wai-Shiu Fred; Shen, Han-Ming

    2014-11-28

    Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells.

  16. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    Science.gov (United States)

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

  17. Mitochondrial respiration controls lysosomal function during inflammatory T cell responses

    Science.gov (United States)

    Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Dolores Ledesma, Maria; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

    2016-01-01

    Summary The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4+ T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration-deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward pro-inflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD+ levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify novel strategies for intervention in mitochondrial-related diseases. PMID:26299452

  18. The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

    Science.gov (United States)

    Bierings, Ruben; Hellen, Nicola; Kiskin, Nikolai; Knipe, Laura; Fonseca, Ana-Violeta; Patel, Bijal; Meli, Athina; Rose, Marlene; Hannah, Matthew J; Carter, Tom

    2012-09-27

    Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

  19. Ral-GTPases: approaching their 15 minutes of fame.

    Science.gov (United States)

    Feig, Larry A

    2003-08-01

    Andy Warhol, the famous pop artist, once claimed that "in the future everyone will be famous for 15 minutes". The same, it seems, can be said of proteins, because at any given time some proteins become more "fashionable" to study than others. But most proteins have been highly conserved throughout millions of years of evolution, which implies that they all have essential roles in cell biology. Thus, each one will no doubt enter the limelight if the right experiment in the right cell type is done. A good example of this is the Ras-like GTPases (Ral-GTPases), which until recently existed in the shadow of their close cousins--the Ras proto-oncogenes. Recent studies have yielded insights into previously unappreciated roles for Ral-GTPases in intensively investigated disciplines such as vesicle trafficking, cell morphology, transcription and possibly even human oncogenesis.

  20. IFN-inducible GTPases in host cell defense.

    Science.gov (United States)

    Kim, Bae-Hoon; Shenoy, Avinash R; Kumar, Pradeep; Bradfield, Clinton J; MacMicking, John D

    2012-10-18

    From plants to humans, the ability to control infection at the level of an individual cell-a process termed cell-autonomous immunity-equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell's interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic, and inflammasome-related antimicrobial activities within the cytosol, as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of guanylate binding proteins (GBPs) with tuberculosis susceptibility and Crohn's colitis.