Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3f^T22N) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.     

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.     

Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.     

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1_(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H⁺-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.     

N‐Cadherin Regulates Cell Migration Through a Rab5‐Dependent Temporal Control of Macropinocytosis

Macropinocytosis is a clathrin‐independent endocytic pathway implicated in fluid uptake, pathogen invasion and cell migration. During collective cell migration, macropinocytosis occurs primarily at membrane ruffles arising from the leading edges of migrating cells. We report here that N‐cadherin (Ncad) regulates the tempo of macropinocytosis and thereby influences wound‐induced collective cell migration. Using live‐cell and super‐resolution imaging techniques, we observed that Ncad formed clusters at the membrane ruffles and macropinosomes. De‐clustering of Ncad by an interfering antibody impaired the recruitment of Rab5‐an early endosomal marker‐to the macropinosomes. Moreover, we demonstrated that Ncad interacts with Rab5, and laser ablation of Ncad caused Rab5 to dissociate from the macropinosomes. Although Rab5 detached from macropinosomes upon the de‐clustering of Ncad, the recruitment of late endosomal marker Rab7 occurred earlier. Consequently, both centripetal trafficking of macropinosomes and collective migration were accelerated due to de‐clustering of Ncad. Thus, our results suggest that Ncad is involved in the maturation of macropinocytosis through Rab5 recruitment, linking macropinocytosis and cell migration through a novel function of Ncad.      

The Arf GAP ASAP1 provides a platform to regulate Arf4‐ and Rab11–Rab8‐mediated ciliary receptor targeting

Dysfunctional trafficking to primary cilia is a frequent cause of human diseases known as ciliopathies, yet molecular mechanisms for specific targeting of sensory receptors to cilia are largely unknown. Here, we show that the targeting of ciliary cargo, represented by rhodopsin, is mediated by a specialized system, the principal component of which is the Arf GAP ASAP1. Ablation of ASAP1 abolishes ciliary targeting and causes formation of actin‐rich periciliary membrane projections that accumulate mislocalized rhodopsin. We find that ASAP1 serves as a scaffold that brings together the proteins necessary for transport to the cilia including the GTP‐binding protein Arf4 and the two G proteins of the Rab family—Rab11 and Rab8—linked by the Rab8 guanine nucleotide exchange factor Rabin8. ASAP1 recognizes the FR ciliary targeting signal of rhodopsin. Rhodopsin FR‐AA mutant, defective in ASAP1 binding, fails to interact with Rab8 and translocate across the periciliary diffusion barrier. Our study implies that other rhodopsin‐like sensory receptors may interact with this conserved system and reach the cilia using the same platform.     

The Nutrient Stress-induced Small GTPase Rab5 Contributes to the Activation of Vesicle Trafficking and Vacuolar Activity

Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.     

Rab11‐FIP2 Interaction with MYO5B Regulates Movement of Rab11a‐Containing Recycling Vesicles

A tripartite association of Rab11a with both Rab11‐FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11‐FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11‐FIP2 that caused loss of interaction with MYO5B in yeast two‐hybrid assays as well as loss of interaction of Rab11‐FIP2(129‐356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full‐length Rab11‐FIP2 with MYO5B tail in HeLa cells. While EGFP‐Rab11‐FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP‐Rab11‐FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a‐containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11‐FIP2 association is perturbed by mutation or by Rab11‐FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11‐FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.      

Rab11‐Family of Interacting Protein 2 associates with chlamydial inclusions through its Rab‐binding domain and promotes bacterial multiplication

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named ‘inclusion’ and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11‐Family of Interacting Proteins, presents at the C‐terminus a Rab‐binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP‐tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11‐Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab‐binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab‐binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion.     

GRAB is a binding partner for the Rab11a and Rab11b GTPases

Co-ordination of Rab GTPase function has emerged as a crucial mechanism in the control of intracellular trafficking processes in eukaryotic cells. Here, we show that GRAB/Rab3IL1 [guanine nucleotide exchange factor for Rab3A; RAB3A interacting protein (rabin3)-like 1], a protein that has previously be shown to act as a GEF (guanine nucleotide exchange factor) for Rab3a, Rab8a and Rab8b, is also a binding partner for Rab11a and Rab11b, but not the closely related Rab25 GTPase. We demonstrate that exogenous expression of Rab11a and Rab11b shift GRAB’s distribution from the cytoplasm onto membranes. We find that the Rab11a/Rab11b-binding region of GRAB lies within its carboxy-terminus, a region distinct from its GEF domain and Rab3a-binding region. Finally, we describe a GRAB deletion mutant (GRAB_Δ223–228) that is deficient in Rab11-binding ability. These data identify GRAB as a dual Rab-binding protein that could potentially link Rab3 and Rab11 and/or Rab8 and Rab11-mediated intracellular trafficking processes.     

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.     

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.     

Rab22a controls MHC‐I intracellular trafficking and antigen cross‐presentation by dendritic cells

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.     

Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo‐tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules.” However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome‐edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra‐/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo‐tubular organelles as Rab11‐Rab8‐positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium‐hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.      

Rha1, an Arabidopsis Rab5 homolog,plays a critical role in the vacuolar trafficking of soluble cargo proteins

Rab proteins are members of the Ras superfamily of small GTP binding proteins and play important roles in various intracellular trafficking steps. We investigated the role of Rha1, an Arabidopsis Rab5 homolog, in intracellular trafficking in Arabidopsis protoplasts. In the presence of a dominant-negative mutant of Rha1, soluble vacuolar cargo proteins such as sporamin:green fluorescent protein (Spo:GFP) and Arabidopsis aleurain like protein:GFP are not delivered to the central vacuole; instead, they accumulate as a diffuse or punctate staining pattern within the cell. Spo:GFP at the punctate stains observed in the presence of hemagglutinin:Rha1[S24N] is colocalized with endogenous vacuolar sorting receptor (VSR(At-1)), which is known to localize primarily to the prevacuolar compartment, whereas Spo:GFP in the diffuse pattern is associated with tonoplasts. Furthermore, expression of Rha1[S24N] causes the secretion of a portion of the vacuolar proteins into medium. However, the inhibitory effect of Rha1[S24N] on vacuolar trafficking is relieved partially by coexpressed wild-type Rha1. Based on these results, we propose that Rha1 plays a critical role in the trafficking of soluble cargoes from the prevacuolar compartment to the central vacuole.     

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking

The closely related Rab6 isoforms, Rab6A and Rab6A', have been shown to regulate vesicular trafficking within the Golgi and post-Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A' biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A' hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B-subunit along the endocytic pathway, and causes defects in Golgi- associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A' is required for cell cycle progression through mitosis and identify Ile(62) as a key residue for uncoupling Rab6A' functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A' perform different functions within the cell and suggests a novel role for Rab6A' as the major Rab6 isoform regulating previously described Rab6-dependent transport pathways.     

Post-Golgi trafficking of rice storage proteins requires the small GTPase Rab7 activation complex MON1–CCZ1

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kDa glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage prevacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of vacuolar protein sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1–CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5- and Rab7-dependent pathway.

The small GTPases Rab5- and Rab7-dependent pathway is involved in rice storage protein trafficking to vacuoles.     

Rab11b resides in a vesicular compartment distinct from Rab11a in parietal cells and other epithelial cells

The Rab11 family of small GTPases is composed of three members, Rab11a, Rab11b, and Rab25. While recent work on Rab11a and Rab25 has yielded some insights into their function, Rab11b has received little attention. Therefore, we sought to examine the distribution of endogenous Rab11b in epithelial cells. In rabbit gastric parietal cells, unlike Rab11a, Rab11b did not colocalize or coisolate with H(+)/K(+)-ATPase. In MDCK cells, endogenous Rab11b localized to an apical pericentrisomal region distinct from Rab11a. The microtubule agents nocodazole and taxol dramatically alter Rab11a's localization in the cell, while effects on Rab11b's distribution were less apparent. These results indicate that in contrast to Rab11a, the Rab11b compartment in the apical region is not as dependent upon microtubules. While Rab11a is known to regulate transferrin trafficking in nonpolarized cells and IgA trafficking in polarized cells, Rab11b exhibited little colocalization with either of these cargoes. Thus, while Rab11a and Rab11b share high sequence homology, they appear to reside within distinct vesicle compartments.     

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.      

Rab11-FIP3 localises to a Rab11-positive pericentrosomal compartment during interphase and to the cleavage furrow during cytokinesis

The Rab11-family interacting protein 3 (Rab11-FIP3), also known as Arfophilin and Eferin, is a Rab11 and ADP-ribosylation factor (ARF) binding protein of unknown function. Here, we sought to investigate the subcellular localisation and elucidate the function of Rab11-FIP3 in eukaryotic membrane trafficking. Utilising a polyclonal antibody specific for Rab11-FIP3, we have demonstrated by immunofluorescence microscopy that Rab11-FIP3 colocalises with Rab11 in a distinctive pericentrosomal location in A431 cells. Additionally, we found that Rab11-FIP3 localises to punctate vesicular structures dispersed throughout A431 cells. We have demonstrated that both Rab11 and Rab11-FIP3 localise to the cleavage furrow during cytokinesis, and that Rab11-FIP3 localisation is dependent on both microtubule and actin filament integrity. We show that Rab11-FIP3 does not enter brefeldin A (BFA) induced membrane tubules that are positive for the transferrin receptor (TfnR). Furthermore, we show that expression of an amino-terminally truncated mutant of Rab11-FIP3 (Rab11-FIP3((244-756))) does not inhibit transferrin (Tfn) recycling in HeLa cells. It is likely that Rab11-FIP3 is involved in trafficking events other than Tfn trafficking; these may include the transport of endosomally derived membrane to the cleavage furrow during cytokinesis.