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.     

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.     

The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.     

Final stages of cytokinesis and midbody ring formation are controlled by BRUCE

Cytokinesis involves the formation of a cleavage furrow, followed by abscission, the cutting of the midbody channel, the final bridge between dividing cells. Recently, the midbody ring became known as central for abscission, but its regulation remains enigmatic. Here, we identify BRUCE, a 528 kDa multifunctional protein, which processes ubiquitin-conjugating activity, as a major regulator of abscission. During cytokinesis, BRUCE moves from the vesicular system to the midbody ring and serves as a platform for the membrane delivery machinery and mitotic regulators. Depletion of BRUCE in cell cultures causes defective abscission and cytokinesis-associated apoptosis, accompanied by a block of vesicular targeting and defective formation of the midbody and the midbody ring. Notably, ubiquitin relocalizes from midbody microtubules to the midbody ring during cytokinesis, and depletion of BRUCE disrupts this process. We propose that BRUCE coordinates multiple steps required for abscission and that ubiquitylation may be a crucial trigger.     

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     

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.     

WD Repeat-containing Protein 5 (WDR5) Localizes to the Midbody and Regulates Abscission

Cytokinesis partitions the cytoplasm of a parent cell into two daughter cells and is essential for the completion of cell division. The final step of cytokinesis in animal cells is abscission, which is a process leading to the physical separation of two daughter cells. Abscission requires membrane traffic and microtubule disassembly at a specific midbody region called the secondary ingression. Here, we report that WD repeat-containing protein 5 (WDR5), a core subunit of COMPASS/MLL family histone H3 lysine 4 methyltransferase (H3K4MT) complexes, resides at the midbody and associates with a subset of midbody regulatory proteins, including PRC1 and CYK4/MKLP1. Knockdown of WDR5 impairs abscission and increases the incidence of multinucleated cells. Further investigation revealed that the abscission delay is primarily due to slower formation of secondary ingressions in WDR5 knockdown cells. Consistent with these defects, midbody microtubules in WDR5 knockdown cells also display enhanced resistance to depolymerization by nocodazole. Recruitment of WDR5 to the midbody dark zone appears to require integrity of the WDR5 central arginine-binding cavity, as mutations that disrupt histone H3 and MLL1 binding to this pocket also abolish the midbody localization of WDR5. Taken together, these data suggest that WDR5 is specifically targeted to the midbody in the absence of chromatin and that it promotes abscission, perhaps by facilitating midbody microtubule disassembly.     

Conversion of midbodies into germ cell intercellular bridges

Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP) and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7 and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell-specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.     

Microtubules, membranes and cytokinesis

Proper division of the cell requires coordination between chromosome segregation by the mitotic spindle and cleavage of the cell by the cytokinetic apparatus. Interactions between the mitotic spindle, the contractile ring and the plasma membrane ensure that the cleavage furrow is properly placed between the segregating chromosomes and that new membrane compartments are formed to produce two daughter cells. The microtubule midzone is able to stimulate the cortex of the cell to ensure proper ingression and completion of the cleavage furrow. Specialized microtubule structures are responsible for directing membrane vesicles to the site of cell cleavage, and vesicle fusion is required for the proper completion of cytokinesis.     

ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.     

Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis

TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.     

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.     

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.     

Computational model of cytokinetic abscission driven by ESCRT-III polymerization and remodeling

The endosomal sorting complex required for transport (ESCRT)-III complex, capable of polymerization and remodeling, participates in abscission of the intercellular membrane bridge connecting two daughter cells at the end of cytokinesis. Here, we integrate quantitative imaging of ESCRT-III during cytokinetic abscission with biophysical properties of ESCRT-III complexes to formulate and test a computational model for ESCRT-mediated cytokinetic abscission. We propose that cytokinetic abscission is driven by an ESCRT-III fission complex, which arises from ESCRT-III polymerization at the edge of the cytokinetic midbody structure, located at the center of the intercellular bridge. Formation of the fission complex is completed by remodeling and breakage of the ESCRT-III polymer assisted by VPS4. Subsequent spontaneous constriction of the fission complex generates bending deformation of the intercellular bridge membrane. The related membrane elastic force propels the fission complex along the intercellular bridge away from the midbody until it reaches an equilibrium position, determining the scission site. Membrane attachment to the dome-like end-cap of the fission complex drives membrane fission, completing the abscission process. We substantiate the model by theoretical analysis of the membrane elastic energy and by experimental verification of the major model assumptions.     

Citron kinase controls abscission through RhoA and anillin

The small GTPase RhoA plays a crucial role in the different stages of cytokinesis, including contractile ring formation, cleavage furrow ingression, and midbody abscission. Citron kinase (CIT-K), a protein required for cytokinesis and conserved from insects to mammals, is currently considered a cytokinesis-specific effector of active RhoA. In agreement with previous observations, we show here that, as in Drosophila cells, CIT-K is specifically required for abscission in mammalian cells. However, in contrast with the current view, we provide evidence that CIT-K is an upstream regulator rather than a downstream effector of RhoA during late cytokinesis. In addition, we show that CIT-K is capable of physically and functionally interacting with the actin-binding protein anillin. Active RhoA and anillin are displaced from the midbody in CIT-K-depleted cells, while only anillin, but not CIT-K, is affected if RhoA is inactivated in late cytokinesis. The overexpression of CIT-K and of anillin leads to abscission delay. However, the delay produced by CIT-K overexpression can be reversed by RhoA inactivation, while the delay produced by anillin overexpression is RhoA-independent. Altogether, these results indicate that CIT-K is a crucial abscission regulator that may promote midbody stability through active RhoA and anillin.     

ESCRT machinery and cytokinesis: the road to daughter cell separation

The endosomal sorting complex required for transport (ESCRT) machinery is a set of cellular protein complexes required for at least three topologically equivalent membrane scission events, namely multivesicular body (MVB) formation, retroviral particle release and midbody abscission during cytokinesis. Recently, several studies have explored the mechanism by which the core ESCRT-III subunits mediate membrane scission and might be differentially required according to the functions of the pathway. In this review, we discuss the links between the ESCRT machinery and cytokinesis, with special focus on abscission initiation and regulation.     

Endocytic traffic in animal cell cytokinesis

Cytokinesis is the final step of mitosis whereby two daughter cells physically separate. It is initiated by the assembly of an actomyosin contractile ring at the mitotic cell equator, which constricts the cytoplasm between the two reforming nuclei resulting in the formation of a narrow intercellular bridge filled with central spindle microtubule bundles. Cytokinesis terminates with the cleavage of the intercellular bridge in a poorly understood process called abscission. Recent work has highlighted the importance of membrane trafficking events occurring from membrane compartments flanking the bridge to the central midbody region. In particular, polarized delivery of endocytic recycling membranes is essential for completion of animal cell cytokinesis. Why endocytic traffic occurs within the intercellular bridge remains largely mysterious and its significance for cytokinesis will be discussed.     

Beclin-1 knockdown shows abscission failure but not autophagy defect during oocyte meiotic maturation

Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes.     

Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis

Cytokinesis requires the coordination of many cellular complexes, particularly those involved in the constriction and reconstruction of the plasma membrane in the cleavage furrow. We have investigated the regulation and function of vesicle transport and fusion during cytokinesis in budding yeast. By using time-lapse confocal microscopy, we show that post-Golgi vesicles, as well as the exocyst, a complex required for the tethering and fusion of these vesicles, localize to the bud neck at a precise time just before spindle disassembly and actomyosin ring contraction. Using mutants affecting cyclin degradation and the mitotic exit network, we found that targeted secretion, in contrast to contractile ring activation, requires cyclin degradation but not the mitotic exit network. Analysis of cells in late anaphase bearing exocyst and myosin V mutations show that both vesicle transport and fusion machineries are required for the completion of cytokinesis, but this is not due to a delay in mitotic exit or assembly of the contractile ring. Further investigation of the dynamics of contractile rings in exocyst mutants shows these cells may be able to initiate contraction but often fail to complete the contraction due to premature disassembly during the contraction phase. This phenotype led us to identify Chs2, a transmembrane protein targeted to the bud neck through the exocytic pathway, as necessary for actomyosin ring stability during contraction. Chs2, as the chitin synthase that produces the primary septum, thus couples the assembly of the extracellular matrix with the dynamics of the contractile ring during cytokinesis.