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.     

Dynamic Phosphorylation of NudC by Aurora B in Cytokinesis

Nuclear distribution protein C (NudC) is a mitotic regulator that plays a role in cytokinesis. However, how NudC is regulated during cytokinesis remains unclear. Here, we show that NudC is phosphorylated by Aurora B, a kinase critical for cell abscission. NudC is co-localized with Aurora B at the midbody and co-immunoprecipitated with Aurora B in mitosis. Inhibition of Aurora B by ZM447439 reduced NudC phosphorylation, suggesting that NudC is an Aurora B substrate in vivo. We identified T40 on NudC as an Aurora B phosphorylation site. NudC depletion resulted in cytokinesis failure with a dramatic elongation of the intercellular bridge between daughter cells, sustained Aurora B activity at the midbody, and reduced cell abscission. These cytokinetic defects can be rescued by the ectopic expression of wild-type NudC. Reconstitution with T40A phospho-defective NudC was found to rescue the cytokinesis defect. In contrast, reconstitution with the T40D phospho-mimetic NudC was inefficient in supporting the completion of cytokinesis. These results suggest that that dynamic phosphorylation of NudC by Aurora B regulates cytokinesis.     

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.     

Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/CCdh1 Complex

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C^Cdh1 complex, but not by the APC/C^Cdc20 complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.     

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.     

ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration

In cytokinesis, there is a lengthy interval between cleavage furrow ingression and abscission, during which the midbody microtubule bundle provides both structural support for a narrow intercellular bridge and a platform that orchestrates the biochemical preparations for abscission. It is currently unclear how the midbody structure is stably maintained during this period. Here, we report a novel role for the ADP-ribosylation factor 6 (ARF6) GTPase in the post-mitotic stabilisation of midbody. Centralspindlin kinesin-6/RhoGAP complex, a midbody component critical for both the formation and function of the midbody, assembles in a sharp band at the centre of the structure in a manner antagonised by 14-3-3 protein. We show that ARF6 competes with 14-3-3 for binding to centralspindlin such that midbodies formed by centralspindlin mutants that can bind 14-3-3 but not ARF6 frequently collapse before abscission. These data indicate a novel mechanism for the regulation of midbody dynamics in which ARF6 protects the compacted centralspindlin assembly from dissipation by 14-3-3.     

Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission

The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.     

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.     

Midbody localization of vinexin recruits rhotekin to facilitate cytokinetic abscission

Vinexin is a SH3 domain-containing adaptor protein that has diverse roles in cell adhesion, signal transduction, gene regulation and stress granule assembly. In this study, we found that vinexin localizes at the midbody during cell division and facilitates cytokinesis. Knockdown of vinexin in HeLa cells delayed the mitotic cell cycle progression and increased the time of cell abscission and the failure to resolve the cytoplasmic bridge. Midbody-localized vinexin is essential for recruiting rhotekin to this structure for cytokinesis because overexpression of a vinexin mutant without a rhotekin-binding motif or knockdown of rhotekin also impaired cytokinetic abscission and increased the number of cells arrested at the midbody stage. Aberrant expression of vinexin and rhotekin in various cancers has been implicated to promote metastasis because of their functions in cell adhesion and signaling. Our findings reveal a novel role of vinexin and rhotekin in cytokinetic abscission and provide another perspective of how both molecules may affect oncogenic transformation via this fundamental cell cycle process.     

New signaling kid on the block: the role of the postmitotic midbody in polarity,stemness, and proliferation

To complete cytokinesis, abscission of the proteinaceous and microtubule-rich intercellular bridge needs to occur. The midbody (MB), a structure that forms on the intercellular bridge, is a key regulator of cytokinesis and appears to play a role in downstream signaling after abscission. Initially, it was thought that after abscission was completed, the MB was degraded. However, a new body of evidence has emerged suggesting that one daughter cell or a surrounding non–daughter cell can inherit or internalize the MB, thus leading to changes in cell proliferation and differentiation. In this review, we highlight the role that the MB has after mitosis. We will focus on the rising evidence that the MB plays an important role in establishment of cell polarity, such as apical lumen formation, neurite extension, and ciliation. Additionally, we will discuss the evidence suggesting that MBs can also serve the role of signaling organelles (MBsomes) that lead to cell proliferation, differentiation, and even tumorigenicity.     

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.     

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.     

Calmodulin protects Aurora B on the midbody to regulate the fidelity of cytokinesis

Aurora B kinase is an integral regulator of cytokinesis as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified an E3 ligase subunit, F box protein FBXL2, that by recognizing a calmodulin binding signature within Aurora B, ubiquitinates and removes the kinase from the midbody. Calmodulin, by competing with the F box protein for access to the calmodulin binding signature, protected Aurora B from FBXL2. Calmodulin co-localized with Aurora B on the midbody, preserved Aurora B levels in cells, and stabilized intercellular canals during delayed abscission. Genetic or pharmaceutical depletion of endogenous calmodulin significantly reduced Aurora B protein levels at the midbody resulting in tetraploidy and multi-spindle formation. The calmodulin inhibitor, calmidazolium, reduced Aurora B protein levels resulting in tetraploidy, mitotic arrest, and apoptosis of tumorigenic cells and profoundly inhibiting tumor formation in athymic nude mice. These observations indicate molecular interplay between Aurora B and calmodulin in telophase and suggest that calmodulin acts as a checkpoint sensor for chromosomal segregation errors during mitosis.     

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.     

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.     

ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters in cytokinetic abscission

Cytokinetic abscission, the final stage of cell division where the two daughter cells are separated, is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. The ESCRT-III subunit CHMP4B is a key effector in abscission, whereas its paralogue, CHMP4C, is a component in the abscission checkpoint that delays abscission until chromatin is cleared from the intercellular bridge. How recruitment of these components is mediated during cytokinesis remains poorly understood, although the ESCRT-binding protein ALIX has been implicated. Here, we show that ESCRT-II and the ESCRT-II–binding ESCRT-III subunit CHMP6 cooperate with ESCRT-I to recruit CHMP4B, with ALIX providing a parallel recruitment arm. In contrast to CHMP4B, we find that recruitment of CHMP4C relies predominantly on ALIX. Accordingly, ALIX depletion leads to furrow regression in cells with chromosome bridges, a phenotype associated with abscission checkpoint signaling failure. Collectively, our work reveals a two-pronged recruitment of ESCRT-III to the cytokinetic bridge and implicates ALIX in abscission checkpoint signaling.     

The large GTPase dynamin associates with the spindle midzone and is required for cytokinesis

Cytokinesis involves the concerted efforts of the microtubule and actin cytoskeletons as well as vesicle trafficking and membrane remodeling to form the cleavage furrow and complete daughter cell separation. The exact mechanisms that support membrane remodeling during cytokinesis remain largely undefined. In this study, we report that the large GTPase dynamin, a protein involved in membrane tubulation and vesiculation, is essential for successful cytokinesis. Using biochemical and morphological methods, we demonstrate that dynamin localizes to the spindle midzone and the subsequent intercellular bridge in mammalian cells and is also enriched in spindle midbody extracts. In Caenorhabditis elegans, dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells. Further, dynamin function appears necessary for cytokinesis, as C. elegans embryos from a dyn-1 ts strain, as well as dynamin RNAi-treated embryos, showed a marked defect in the late stages of cytokinesis. These findings indicate that, during mitosis, conventional dynamin is recruited to the spindle midzone and the subsequent intercellular bridge, where it plays an essential role in the final separation of dividing cells.     

Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K''s coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins.     

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.