Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis

Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes, and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2. We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division.     

The FIP3-Rab11 protein complex regulates recycling endosome targeting to the cleavage furrow during late cytokinesis

An integral part of cell division is the separation of daughter cells via cytokinesis. There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11- and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial spatial and temporal regulation. These data provide the first detailed analysis of recycling endosomes in cell division and provide a new model for membrane traffic to the furrow. We propose that the dynamic Rab11-FIP3 interaction controls the delivery, targeting, and fusion of recycling endosomes with furrow during late cytokinesis and abscission.     

Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis

The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.     

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.     

Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis

The dual Rab11/Arf binding proteins, family of Rab11-interacting proteins FIP3 and FIP4 function in the delivery of recycling endosomes to the cleavage furrow and are, together with Rab11, essential for completion of abscission, the terminal step of cytokinesis. Here, we report that both FIP3 and FIP4 bind Arf6 in a nucleotide-dependent manner but exhibit differential affinities for Rab11 and Arf6. Both FIP3 and FIP4 can form ternary complexes with Rab11 and Arf6. Arf6 is localised to the furrow and midbody and we show that Arf6-GTP functions to localise FIP3 and FIP4 to midbodies during cytokinesis. Exo70p, a component of the Exocyst complex, also localises to the furrow of dividing cells and interacts with Arf6. We show that depletion of Exo70p leads to cytokinesis failure and an impairment of FIP3 and Rab11 localisation to the furrow and midbody. Moreover, Exo70p co-immunoprecipitates FIP3 and FIP4. Hence, we propose that FIP3 and FIP4 serve to couple Rab11-positive vesicle traffic from recycling endosomes to the cleavage furrow/midbody where they are tethered prior to fusion events via interactions with Arf6 and the Exocyst.     

Rab24 is Required for Normal Cell Division

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co‐localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i. e. chromatin bridges). Furthermore, in CHO cells stably transfected with GFP‐Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.     

Rab11A-Controlled Assembly of the Inner Membrane Complex Is Required for Completion of Apicomplexan Cytokinesis

The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.     

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.     

Molecular characterization of Rab11-FIP3 binding to ARF GTPases

Rab11-FIP3 is a Rab11-binding protein that has been implicated in regulating cytokinesis in mammalian cells. FIP3 functions by simultaneously interacting with Rab11 as well as Arf GTPases. However, unlike the interaction between Rab11 and FIP3, the structural basis of FIP3 binding to Arf GTPases has not yet been determined. The specificity of interaction between FIP3 and Arf GTPases remains controversial. While it was reported that FIP3 preferentially binds to Arf6 some data suggest that FIP3 can also interact with Arf5 and even possibly Arf4. The Arf-interaction motif on FIP3 also remains to be determined. Finally, the importance of Arf binding to FIP3 in regulating cell division and other cellular functions remains unclear. Here we used a combination of various biochemical techniques to measure the affinity of FIP3 binding to various Arfs and to demonstrate that FIP3 predominantly interacts with Arf6 in vitro and in vivo. In addition, we identified the motifs mediating Arf6 and FIP3 interaction and demonstrated that FIP3 binds to the Arf6 C-terminus rather than switch motifs. Finally we show that FIP3 and Arf6 binding is required for the targeting of Arf6 to the cleavage furrow during cytokinesis. Thus, we propose that FIP3 is a scaffolding protein that, in addition to regulating endosome targeting to the cleavage furrow, also is required for Arf6 recruitment to the midbody during late telophase.     

Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males

Rab11 is a small GTPase that regulates several aspects of vesicular trafficking. Here, we show that Rab11 accumulates at the cleavage furrow of Drosophila spermatocytes and that it is essential for cytokinesis. Mutant spermatocytes form regular actomyosin rings, but these rings fail to constrict to completion, leading to cytokinesis failures. rab11 spermatocytes also exhibit an abnormal accumulation of Golgi-derived vesicles at the telophase equator, suggesting a defect in membrane-vesicle fusion. These cytokinesis phenotypes are identical to those elicited by mutations in giotto (gio) and four wheel drive (fwd) that encode a phosphatidylinositol transfer protein and a phosphatidylinositol 4-kinase, respectively. Double mutant analysis and immunostaining for Gio and Rab11 indicated that gio, fwd, and rab11 function in the same cytokinetic pathway, with Gio and Fwd acting upstream of Rab11. We propose that Gio and Fwd mediate Rab11 recruitment at the cleavage furrow and that Rab11 facilitates targeted membrane delivery to the advancing furrow.     

Rab14/MACF2 complex regulates endosomal targeting during cytokinesis

Abscission is a complex cellular process that is required for mitotic division. It is well established that coordinated and localized changes in actin and microtubule dynamics are vital for cytokinetic ring formation, as well as establishment of the abscission site. Actin cytoskeleton reorganization during abscission would not be possible without the interplay between Rab11- and Rab35-containing endosomes and their effector proteins, whose roles in regulating endocytic pathways at the cleavage furrow have now been studied extensively. Here, we identified Rab14 as a novel regulator of cytokinesis. We demonstrate that depletion of Rab14 causes either cytokinesis failure or significantly prolongs division time. We show that Rab14 contributes to the efficiency of recruiting Rab11-endosomes to the thin intracellular bridge (ICB) microtubules and that Rab14 knockout leads to inhibition of actin clearance at the abscission site. Finally, we demonstrate that Rab14 binds to microtubule minus-end interacting MACF2/CAMSAP3 complex and that this binding affects targeting of endosomes to the ICB microtubules. Collectively, our data identified Rab14 and MACF2/CAMSAP3 as proteins that regulate actin depolymerization and endosome targeting during cytokinesis.     

Role of endosomal Rab GTPases in cytokinesis

Completion of cytokinesis requires Rab 11-dependent membrane trafficking events to deliver new membrane to the furrow and for abscission. Many Rabs have overlapping endosomal distributions, hence, we examined whether these Rabs also function in cytokinesis. Analysis of the distribution of Rabs 4, 5, 7, 8, 9, 11, 21, and 22 revealed that only Rab 11 was enriched within the furrow of cells in telophase or present within the midbody. By contrast, Rabs 4, 5, 7, 8, and 9 were mainly localised within a peri-nuclear compartment facing away from the furrow. Using RNA interference and dominant negative Rab mutants, we evaluated the role of these Rabs in furrowing and abscission. Consistent with previous work, we find that Rab 11 is intimately involved in abscission. However, we further found that depletion of Rab 4 slowed but did not prevent abscission. Depletion of any other Rab species had little effect on furrowing or abscission. These data suggest that the membrane trafficking events required for completion of cytokinesis are largely controlled by Rab 11 and not other endosomal Rab proteins, and further suggest that the relocation of Rab 11-specific cargo is an integral facet of abscission. Arf6 knockdown was without effect on cytokinesis, but when both Rab 11 and Arf6 were knocked-down, we found the furrow rapidly regressed and the cells were unable to form a stable midbody. We suggest that Rab 11 and Arf6 function synergistically in the switch from furrowing to abscission, as well as in the terminal stage of abscission.     

Cytokinesis failure in clathrin-minus cells is caused by cleavage furrow instability

The role of membrane traffic during cell division has only recently begun to be investigated. A growing number of trafficking proteins seem to be involved in the successful completion of cytokinesis. Clathrin was the first trafficking protein to be shown to be essential for cytokinesis in Dictyostelium. Here we investigate the nature of the cytokinesis defect of Dictyostelium clathrin null cells. We found that adherent clathrin null cells do form cleavage furrows but cannot maintain a consistent rate of furrow ingression. Clathrin null cells are completely defective in cytokinesis when placed in suspension. In these conditions, the cells develop an abnormal division morphology that consists of two lateral "furrows" on either side of a bulging equatorial region. Cells expressing GFP-myosin II were examined at various stages of cytokinesis. Clathrin null cells show multiple defects in myosin organization and localization that parallel the striking failure in furrow morphology. We postulate that this morphology is the result of contraction at the rear of the presumptive daughter cells in concert with incomplete furrow ingression.     

GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis.     

Endocytosis resumes during late mitosis and is required for cytokinesis

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.     

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.     

Local change in phospholipid composition at the cleavage furrow is essential for completion of cytokinesis

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P(2) antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P(2) in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P(2) is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.     

MITOSIS, CELL WALL AND FLAGELLA SYNTHESIS IN SYNCHRONIZED CULTURES OF THE PRASINOPHYTE, SCHERFFELIA DUBIA

Cell division occurs within the parental cell wall, yielding two progeny cells. Since Scherffelia dubia sheds all four flagella prior to cell division, the maturing progeny cells must regenerate new cell walls and flagella during and/or after cytokinesis. To better understand these processes, we have synchronized cell division in cultures of S. dubia and observed all stages of mitosis, cytokinesis, and progeny cell maturation, including flagella and cell wall formation, via DAPI staining of fixed cells, DIC microscopy of live cells embedded in agarose and standard TEM. Microscopical observations revealed the following sequence of events: 1) Golgi stacks divide during late interphase and immediately begin producing theca scales; 2) deflagellation and release of the parental cell wall from the plasma membrane occurs during early prophase; 3) synthesis of theca and flagella scales within the Golgi and/or scale reticulum continues throughout mitosis; 4) during cytokinesis, a coalescence of vesicles containing theca scales at the posterior end of the cell results in a cleavage furrow slightly diagonal to the cells' longitudinal axis (40 min); 5) post-mitotic nascent basal body formation and flagella elongation at the inherited basal bodies (and later at the mature nascent basal bodies) occurs concurrently with continued cell wall synthesis; 6) the cleavage furrow rotates into a transverse position (35 min); 7) reorientation of the nuclei results in a "head to tail" orientation of the maturing progeny cells; and 8) matured progeny cells emerge from the posterior end of the parental theca not before 8 hrs after the onset of mitosis.     

The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation

Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. We demonstrate that Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, we demonstrate that microtubules are required for proper metaphase furrow formation. We propose that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows.     

Rab6-interacting protein 1 links Rab6 and Rab11 function

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.