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.     

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.     

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.     

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

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.     

Src signaling regulates completion of abscission in cytokinesis through ERK/MAPK activation at the midbody

Src family non-receptor-type tyrosine kinases regulate a wide variety of cellular events including cell cycle progression in G(2)/M phase. Here, we show that Src signaling regulates the terminal step in cytokinesis called abscission in HeLa cells. Abscission failure with an unusually elongated intercellular bridge containing the midbody is induced by treatment with the chemical Src inhibitors PP2 and SU6656 or expression of membrane-anchored Csk chimeras. By anti-phosphotyrosine immunofluorescence and live cell imaging, completion of abscission requires Src-mediated tyrosine phosphorylation during early stages of mitosis (before cleavage furrow formation), which is subsequently delivered to the midbody through Rab11-driven vesicle transport. Treatment with U0126, a MEK inhibitor, decreases tyrosine phosphorylation levels at the midbody, leading to abscission failure. Activated ERK by MEK-catalyzed dual phosphorylation on threonine and tyrosine residues in the TEY sequence, which is strongly detected by anti-phosphotyrosine antibody, is transported to the midbody in a Rab11-dependent manner. Src kinase activity during the early mitosis mediates ERK activation in late cytokinesis, indicating that Src-mediated signaling for abscission is spatially and temporally transmitted. Thus, these results suggest that recruitment of activated ERK, which is phosphorylated by MEK downstream of Src kinases, to the midbody plays an important role in completion of abscission.     

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β-sheet composed of 22 β-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.     

Syntaxin 2 and endobrevin are required for the terminal step of cytokinesis in mammalian cells

The terminal step of cytokinesis in animal cells is the abscission of the midbody, a cytoplasmic bridge that connects the two prospective daughter cells. Here we show that two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, specifically localize to the midbody during cytokinesis in mammalian cells. Inhibition of their function by overexpression of nonmembrane-anchored mutants causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission but not further upstream events, such as furrowing, are affected. These results indicate that successful completion of cytokinesis requires a SNARE-mediated membrane fusion event and that this requirement is distinct from exocytic events that may be involved in prior ingression of the plasma membrane.     

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

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.     

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.     

Both daughter cells traffic and exocytose membrane at the cleavage furrow during mammalian cytokinesis

Membrane trafficking during cytokinesis is not well understood. We used advanced live cell imaging techniques to track exocytosis of single vesicles to determine whether constitutively exocytosed membrane is focally delivered to the cleavage furrow. Ultrasensitive three-dimensional confocal time-lapse imaging of the temperature-sensitive membrane cargo protein vesicular stomatitis virus protein-yellow fluorescent protein revealed that vesicles from both daughter cells traffic out of the Golgi and into the furrow, following curvilinear paths. Immunolocalization and photobleaching experiments indicate that individual vesicles accumulate at the midbody and generate a reserve vesicle pool that is distinct from endosomal and lysosomal compartments. Total internal reflection fluorescence microscopy imaging provided direct evidence that Golgi-derived vesicles from both daughter cells not only traffic to the furrow region but dock and fuse there, supporting a symmetrically polarized exocytic delivery model. In contrast, quantitative analysis of midbody abscission showed inheritance of the midbody remnant by one daughter cell, indicating that cytokinesis is composed of both symmetrical and asymmetrical stages.     

Vesicle trafficking and membrane remodelling in cytokinesis

All cells complete cell division by the process of cytokinesis. At the end of mitosis, eukaryotic cells accurately mark the site of division between the replicated genetic material and assemble a contractile ring comprised of myosin II, actin filaments and other proteins, which is attached to the plasma membrane. The myosin-actin interaction drives constriction of the contractile ring, forming a cleavage furrow (the so-called 'purse-string' model of cytokinesis). After furrowing is completed, the cells remain attached by a thin cytoplasmic bridge, filled with two anti-parallel arrays of microtubules with their plus-ends interdigitating in the midbody region. The cell then assembles the abscission machinery required for cleavage of the intercellular bridge, and so forms two genetically identical daughter cells. We now know much of the molecular detail of cytokinesis, including a list of potential genes/proteins involved, analysis of the function of some of these proteins, and the temporal order of their arrival at the cleavage site. Such studies reveal that membrane trafficking and/or remodelling appears to play crucial roles in both furrowing and abscission. In the present review, we assess studies of vesicular trafficking during cytokinesis, discuss the role of the lipid components of the plasma membrane and endosomes and their role in cytokinesis, and describe some novel molecules implicated in cytokinesis. The present review covers experiments performed mainly on tissue culture cells. We will end by considering how this mechanistic insight may be related to cytokinesis in other systems, and how other forms of cytokinesis may utilize similar aspects of the same machinery.     

Cell migration regulates the kinetics of cytokinesis

Cytokinesis is the final stage of cell division in which the daughter cells separate. Although a growing body of evidence suggests that cell migration-induced traction forces may be required to provide physical assistance for daughter cells to dissociate during abscission, the role of cell migration in cytokinesis has not been directly elucidated. Recently, we have demonstrated that Crk and paxillin, which are pivotal components of the cell migration machinery, localize to the midbody and are essential for the abscission. These findings provided an important link between the cell migration and cytokinesis machineries and prompted us to dissect the role of cell migration in cytokinesis. We show that cell migration controls the kinetics of cleavage furrowing, midbody extension and abscission and coordinates proper subcellular redistribution of Crk and syntaxin-2 to the midbody after ingression.Key words: cell migration, cytokinesis, midbody, abscission, cleavage furrow, Crk, paxillin, syntaxin-2, ExoT     

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

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.     

EoRab11a在游仆虫及HEK293T细胞中的定位

Rab11是一种在真核生物细胞生命活动过程中发挥多种调控作用的小分子GTP酶.EoRab11a是八肋游仆虫中的Rab11蛋白同源物,为了解EoRab11a蛋白在细胞中的功能,本研究将EoRab11a基因克隆到哺乳动物表达载体pEGFP-C2中,构建重组表达质粒pEGFP-C2-EoRab11a,转染HEK293T细胞并观察其细胞定位.在间期HEK293T细胞中,EoRab11a定位于细胞核附近;在游仆虫细胞中,EoRab11a具有相似的分布模式.在HEK293T细胞的胞质分裂过程中,EoRab11a在分裂沟附近、分裂沟收缩区、以及最后形成的中间体处分布,提示EoRab11a可能参与了胞质分离过程中分裂沟及中间体处的膜泡运输事件.     

The Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis

The Rab6-binding kinesin, Rab6-KIFL, was identified in a two-hybrid screen for proteins that interact with Rab6, a small GTPase involved in membrane traffic through the Golgi apparatus. We find that Rab6-KIFL accumulates in mitotic cells where it localizes to the midzone of the spindle during anaphase, and to the cleavage furrow and midbody during telophase. Overexpression of Rab6-KIFL causes a cell division defect resulting in cell death. Microinjection of antibodies to Rab6-KIFL results in the cells becoming binucleate after one cell cycle, and time-lapse microscopy reveals that this is due to a defect in cleavage furrow formation and thus cytokinesis. These data show that endogenous Rab6-KIFL functions in cell division during cleavage furrow formation and cytokinesis, in addition to its previously described role in membrane traffic.     

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.     

TAXOL REDUCES THE RATE OF CYTOKINESIS IN PtK1CELLS

Mitotic PtK₁cells were treated both during mid-anaphase and at furrow initiation with the potent microtubule (MT) stabilizing agent, taxol, to determine the role of MTs in the rate of cytokinetic events. Rates of cytokinesis (μm/min) were measured by changes in furrow diameter. Incubation of PtK₁cells during mid-anaphase with 5 μg/ml taxol slows the rate of cytokinesis by an average of 43%. Instead of furrow initiation to midbody formation taking an average of 10.7 min (1.6 μm/min), furrowing to midbody formation was completed in an average of 19.0 min (0.9 μm/min), which does not include the 7-min period between taxol application in mid-anaphase and furrow initiation. Application of 5 μg/ml taxol to cells at furrow initiation had a reduced effect on decreasing the rate of cytokinesis and midbody formation; furrowing to midbody formation took an average of 14.6 min (1.2 μm/min). These data suggest that delays in the rate of cytokinesis is dependent on the mitotic stage at which taxol is applied. Ultrastructural analysis shows that taxol treatment of anaphase cells prevents midbody formation during early G₁, yet MT number and organization in the furrowed region is not significantly altered from untreated cells. There is little change in the organization and amount of contractile ring microfilaments, yet filaments are also found parallel to midbody MTs. Our results may be explained by the fact that taxol tends to stabilize MTs which probably affects the rate at which they depolymerize in the terminal phases of cytokinesis. Reduction in depolymerization rates of a stable population of MTs could serve to regulate the rate of cytokinesis.     

RalA-exocyst-dependent recycling endosome trafficking is required for the completion of cytokinesis

In eukaryotic cells, recycling endosome-mediated trafficking contributes to the completion of cytokinesis, in a manner under the control of the centrosome. We report that the exocyst complex and its interacting GTPase RalA play a critical role in this polarized trafficking process. RalA resides in the recycling endosome and relocates from the pericentrosomal region to key cytokinetic structures including the cleavage furrow, and later, the abscission site. This event is coupled to the dynamic redistribution of the exocyst proteins. These associate with the centrosome in interphase and concentrate on the central spindle/midbody during cytokinesis. Disruption of RalA-exocyst function leads to cytokinesis failure in late stages, particularly abscission, resembling the cytokinesis defects induced by loss of centrosome function. These data suggest that RalA and the exocyst may regulate vesicle delivery to the centrosome-related abscission site during the terminal stage of cytokinesis, implicating RalA as a critical regulator of cell cycle progression.     

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.