Rab35 GTPase and cancer: Linking membrane trafficking to tumorigenesis

Rab35 is a small GTPase that is involved in many cellular processes, including membrane trafficking, cell polarity, lipid homeostasis, immunity, phagocytosis and cytokinesis. Recent studies showed that activating mutations confer Rab35 with oncogenic properties. Conversely, downregulation of Rab35 inverts apico‐basal cell polarity and promotes cell migration. Here we review Rab35’s known functions in membrane trafficking and signaling, cell division and cell migration in cancer cells and discuss the importance of Rab35‐dependent membrane trafficking in cancer progression.      

Rab35: GEFs,GAPs and Effectors

Rabs are the largest family of small GTPases and are master regulators of membrane trafficking. Following activation by guanine‐nucleotide exchange factors (GEFs), each Rab binds a specific set of effector proteins that mediate the various downstream functions of that Rab. Then, with the help of GTPase‐activating proteins, the Rab converts GTP to GDP, terminating its function. There are over 60 Rabs in humans and only a subset has been analyzed in any detail. Recently, Rab35 has emerged as a key regulator of cargo recycling at endosomes, with an additional role in regulation of the actin cytoskeleton. Here, we will focus on the regulation of Rab35 activity by the connecdenn/DENND1 family of GEFs and the TBC1D10/EPI64 family of GTPase‐activating proteins. We will describe how analysis of these proteins, as well as a plethora of Rab35 effectors has provided insights into Rab35 function. Finally, we will describe how Rab35 provides a novel link between the Rab and Arf family of GTPases with implications for tumor formation and invasiveness .      

Molecular Insights into Rab7‐Mediated Endosomal Recruitment of Core Retromer: Deciphering the Role of Vps26 and Vps35

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans‐Golgi network. It consists of two distinct sub‐complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub‐complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET‐based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N‐terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7‐mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub‐complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7‐mediated recruitment of the core retromer complex.      

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

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.      

Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC‐CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri‐nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.      

KIF13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

Membrane blebbing‐dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D‐environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D‐collagen, while RhoB overexpression enhanced blebbing and 3D‐collagen migration in a manner dependent on its plasma membrane localization and down‐stream effectors ROCK and Myosin II. RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11‐positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D‐migration of ALL cells. In conclusion, KIF13A‐mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.     

Spatiotemporal Resolution of Rab9 and CI‐MPR Dynamics in the Endocytic Pathway

Rab9 is a small GTPase that localizes to the trans‐Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose‐6‐phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation‐independent (CI‐MPR) away from the Golgi yet, has no effect on the retrograde transport of CI‐MPR. We also show that CI‐MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5‐positive, and late, Rab7a‐positive, endosomes. CI‐MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI‐MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI‐MPR to the endosomal pathway, entering the maturing endosome at the early‐to‐late transition.      

Dysregulation of Rab5‐mediated endocytic pathways in Alzheimer's disease

Increasing evidence has pointed to that dysregulation of the endo‐lysosomal system is an early cellular phenotype of pathogenesis for Alzheimer's disease (AD). Rab5, a small GTPase, plays a critical role in mediating these processes. Abnormal overactivation of Rab5 has been observed in post‐mortem brain samples of Alzheimer's patients as well as brain samples of mouse models of AD. Recent genome‐wide association studies of AD have identified RIN3 (Ras and Rab interactor 3) as a novel risk factor for the disease. RIN3 that functions as a guanine nucleotide exchange factor for Rab5 may serve as an important activator for Rab5 in AD pathogenesis. In this review, we present recent research highlights on the possible roles of dysregulation of Rab5‐mediated endocytic pathways in contributing to early pathogenesis of AD.      

Rab‐mediated trafficking role in neurite formation

Neuronal cells are characterized by the presence of two confined domains, which are different in their cellular properties, biochemical functions and molecular identity. The generation of asymmetric domains in neurons should logically require specialized membrane trafficking to both promote neurite outgrowth and differential distribution of components. Members of the Rab family of small GTPases are key regulators of membrane trafficking involved in transport, tethering and docking of vesicles through their effectors. RabGTPases activity is coupled to the activity of guanine nucleotide exchange factors or GEFs, and GTPase‐activating proteins known as GAPs. Since the overall spatiotemporal distribution of GEFs, GAPs and Rabs governs trafficking through the secretory and endocytic pathways, affecting exocytosis, endocytosis and endosome recycling, it is likely that RabGTPases could have a major role in neurite outgrowth, elongation and polarization. In this review we summarize the evidence linking the functions of several RabGTPases to axonal and dendritic development in primary neurons, as well as neurite formation in neuronal cell lines. We focused on the role of RabGTPases from the trans‐Golgi network, early/late and recycling endosomes, as well as the function of some Rab effectors in neuritogenesis. Finally, we also discuss the participation of the ADP‐ribosylation factor 6, a member of the ArfGTPase family, in neurite formation since it seems to have an important cross‐talk with RabGTPases.

     

Cloning and characterization of a novel splice variant of human Rab18 gene (RAB18).

Rab GTPase proteins are a kind of small GTP-binding proteins, which functions mainly focus on regulating interacellular trafficking pathways during vesicular transport. To date, 60 distinct human RAB proteins have been identified. RAB18 gene is discovered from endothelial cells. Its function is considered as endosomes and plasma membrane recycling. Research indicates RAB18 may relate to inflammation and some kinds of tumor. Here we report a splice variant of RAB18, which is 2571 bp in length and has an open reading frame coding a predicted 235 amino-acids protein. RT-PCR shows that the cDNA has different expression pattern with RAB18 and is highly expressed in testis.     

Rerouting of Plant Late Endocytic Trafficking Toward a Pathogen Interface

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host‐derived membranes. The mechanisms underlying the biogenesis and functions of host–microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host–pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast‐localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole‐targeted late endosomes to a host–pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host–pathogen interface.      

Rab6 functions in polarized transport in Drosophila photoreceptors

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected.

The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs.     

Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.     

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.     

Spatio-Temporal Cellular Dynamics of the Arabidopsis Flagellin Receptor Reveal Activation Status-Dependent Endosomal Sorting

The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor FLAGELLIN SENSING2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)–sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b– and ARA6/Rab F1–positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.     

Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling

We have previously demonstrated that glycosphingolipids are internalized from the plasma membrane of human skin fibroblasts by a clathrin-independent, caveolar-related mechanism and are subsequently transported to the Golgi apparatus by a process that is dependent on microtubules, phosphatidylinositol 3-kinase, Rab7, and Rab9. Here we characterized the early steps of intracellular transport of a fluorescent glycosphingolipid analog, BODIPY-lactosylceramide (LacCer), and compared this to fluorescent transferrin (Tfn), a well established marker for the clathrin pathway. Although these two markers were initially internalized into separate vesicles by distinct mechanisms, they became co-localized in early endosomes within 5 min. These results demonstrate that glycosphingolipid-containing vesicles derived from caveolar-related endocytosis fuse with the classical endosomal system. However, in contrast to Tfn, internalization and trafficking of LacCer was independent of Rab5a, a key regulator of transport to early endosomes. By taking advantage of the monomer/excimer properties of the fluorescent lipid analog, we were also able to visualize LacCer segregation into distinct microdomains of high (red emission) and low (green emission) concentrations in the early endosomes of living cells. Interestingly, the high concentration "red" microdomains co-localized with fluorescent Tfn upon exit from early endosomes and passed through Rab11-positive "recycling endosomes" prior to being transported back to the plasma membrane. These results together with our previous studies suggest that glycosphingolipids internalized by caveolar endocytosis are rapidly delivered to early endosomes where they are fractionated into two major pools, one that is transported via late endosomes to the Golgi apparatus and the other that is returned to the plasma membrane via the recycling compartment.     

Comprehensive Screening for Novel Rab‐Binding Proteins by GST Pull‐Down Assay Using 60 Different Mammalian Rabs‡

The Rab family belongs to the Ras‐like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S‐transferase (GST) pull‐down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab‐binding proteins we identified, mKIAA1055/TBC1D2B (Rab22‐binding protein), GAPCenA/TBC1D11 (Rab36‐binding protein) and centaurin β2/ACAP2 (Rab35‐binding protein), are GTPase‐activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab–GAP (Tre‐2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin β2 binds GTP‐Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin β2 did not exhibit any Rab35–GAP activity in vitro, the Rab35‐binding ANKR domain of centaurin β2 was found to be required for its plasma membrane localization and regulation of Rab35‐dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.     

Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3'-phosphorylation of phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.     

A Highlights from MBoC Selection: Functional Cross-Talk between Rab14 and Rab4 through a Dual Effector,RUFY1/Rabip4

The small GTPase Rab14 localizes to early endosomes and the trans-Golgi network, but its cellular functions on endosomes and its functional relationship with other endosomal Rab proteins are poorly understood. Here, we report that Rab14 binds in a GTP-dependent manner to RUFY1/Rabip4, which had been originally identified as a Rab4 effector. Rab14 colocalizes well with Rab4 on peripheral endosomes. Depletion of Rab14, but not Rab4, causes dissociation of RUFY1 from endosomal membranes. Coexpression of RUFY1 with either Rab14 or Rab4 induces clustering and enlargement of endosomes, whereas a RUFY1 mutant lacking the Rab4-binding region does not induce a significant morphological change in the endosomal structures even when coexpressed with Rab14 or Rab4. These findings suggest that Rab14 and Rab4 act sequentially, together with RUFY1; Rab14 is required for recruitment of RUFY1 onto endosomal membranes, and subsequent RUFY1 interaction with Rab4 may allow endosomal tethering and fusion. Depletion of Rab14 or RUFY1, as well as Rab4, inhibits efficient recycling of endocytosed transferrin, suggesting that Rab14 and Rab4 regulate endosomal functions through cooperative interactions with their dual effector, RUFY1.     

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.