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.     

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.     

Plant cytokinesis requires de novo secretory trafficking but not endocytosis

During plant cytokinesis membrane vesicles are efficiently delivered to the cell-division plane, where they fuse with one another to form a laterally expanding cell plate. These membrane vesicles were generally believed to originate from Golgi stacks. Recently, however, it was proposed that endocytosis contributes substantially to cell-plate formation. To determine the relative contributions of secretory and endocytic traffic to cytokinesis, we specifically inhibited either or both trafficking pathways in Arabidopsis. Blocking traffic to the division plane after the two pathways had converged at the trans-Golgi network disrupted cytokinesis and resulted in binucleate cells, whereas impairment of endocytosis alone did not interfere with cytokinesis. By contrast, inhibiting ER-Golgi traffic by eliminating the relevant BFA-resistant ARF-GEF caused retention of newly synthesized proteins, such as the cytokinesis-specific syntaxin KNOLLE in the ER, and prevented the formation of the partitioning membrane. Our results suggest that during plant cytokinesis, unlike animal cytokinesis, protein secretion is absolutely essential, whereas endocytosis is not.     

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.     

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.     

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.     

Cytokinesis in the C. elegans embryo: regulating contractile forces and a late role for the central spindle

Genetic and molecular studies in the nematode Caenorhabditis elegans have identified multiple essential pathways that regulate and execute cytokinesis in early embryonic cells. These pathways influence both the microfilament cytoskeleton and the microtubule cytoskeleton. Microfilaments are enriched throughout the cell cortex at all times during the cell cycle in embryonic cells. Cortical microfilaments are required for multiple processes in embryonic cells, including polar body extrusion during meiosis, anterior-posterior axis specification by the sperm-donated microtubule-organizing center, and cytokinesis during mitosis. In addition to contractile apparatus proteins that are required positively for cleavage furrow ingression, the Nedd8 ubiquitin-like protein modification pathway negatively regulates contractile forces outside the cleavage furrow during cytokinesis. Another pathway that acts positively during cytokinesis involves the mitotic spindle. The central spindle, where anti-parallel non-kinetochore microtubules overlap and are cross-linked, is required for a late step in cytokinesis, and other pathway(s) involved in membrane addition during cytokinesis may also require the central spindle. The amenability of C. elegans to classical genetics, the ease of reducing gene function with RNA interference, the completion of the genome sequence, and the availability of transgenic GFP fusion proteins that render the cytoskeleton fluorescent, all serve to make the early worm embryo an especially promising system for further advances in the identification of cytokinesis pathways, and in defining their interactions.     

Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.     

The inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis and is necessary for cytokinesis

During cytokinesis, constriction of an equatorial actomyosin ring physically separates the two daughter cells. At the cleavage furrow, the phosphoinositide PI(4,5)P2 plays an?important role by recruiting and regulating essential proteins of the cytokinesis machinery [1]. Accordingly, perturbation of PI(4,5)P2 regulation leads to abortive furrowing and binucleation [2-4]. To determine how PI(4,5)P2 is regulated during cytokinesis, we individually knocked down each of the enzymes controlling the phosphoinositide (PIP) cycle in Drosophila. We show that depletion of the Drosophila ortholog of human oculocerebrorenal syndrome of Lowe 1 (OCRL1), an inositol 5-phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome, triggers a high rate of cytokinesis failure. In absence of dOCRL, several essential components of the cleavage furrow were found to be incorrectly localized on giant cytoplasmic vacuoles rich in PI(4,5)P2 and in endocytic markers. We demonstrate that dOCRL is associated with endosomes and that it dephosphorylates PI(4,5)P2 on internal membranes to restrict this phosphoinositide at the plasma membrane and thereby regulates cleavage furrow formation and ingression. Identification of dOCRL as essential for cell division may be important to understand the molecular basis of the phenotypic manifestations of Lowe syndrome.     

Nir2, a human homolog of Drosophila melanogaster retinal degeneration B protein,is essential for cytokinesis

Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.     

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.     

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.     

Dynamic rearrangement of surface proteins is essential for cytokinesis

Cytokinesis is a complex process that involves dynamic cortical rearrangement. Our recent time-lapse recordings of the mouse egg unexpectedly revealed a high motility of the second polar body (2pb). Experiments to address its underlying mechanism show that neither mechanical compression by the zona pellucida nor the connection via the mid-body is required for the 2pb movement. Time-lapse recordings establish that the 2pb moves together with the cell membrane. These recordings, in which cell surface proteins are labeled with fluorescent latex-microbeads or monovalent antibodies against whole mouse proteins, indicate that the majority of the surface proteins dynamically accumulate in the cleavage furrow at every cell division. Comparable dynamics of the cell surface proteins, and specifically of E-cadherin, are also observed in cultured epithelial cells. The surface protein dynamics are closely correlated with, and dependent on, those of the underlying cortical actin. The cortical actin network may form a scaffold for membrane proteins and thereby transfer them during contractile ring formation toward the cleavage furrow. Immobilization of surface proteins by tetravalent lectin-mediated crosslinking results in the failure of cleavage, demonstrating that the observed protein dynamics are essential for cytokinesis. We propose that dynamic rearrangement of the cell surface proteins is a common feature of cytokinesis, playing a key role in modifying the mechanical properties of the cell membrane during cortical ingression.     

Myosin VI is required for targeted membrane transport during cytokinesis

Myosin VI plays important roles in endocytic and exocytic membrane-trafficking pathways in cells. Because recent work has highlighted the importance of targeted membrane transport during cytokinesis, we investigated whether myosin VI plays a role in this process during cell division. In dividing cells, myosin VI undergoes dramatic changes in localization: in prophase, myosin VI is recruited to the spindle poles; and in cytokinesis, myosin VI is targeted to the walls of the ingressing cleavage furrow, with a dramatic concentration in the midbody region. Furthermore, myosin VI is present on vesicles moving into and out of the cytoplasmic bridge connecting the two daughter cells. Inhibition of myosin VI activity by small interfering RNA (siRNA)-mediated knockdown or by overexpression of dominant-negative myosin VI tail leads to a delay in metaphase progression and a defect in cytokinesis. GAIP-interacting protein COOH terminus (GIPC), a myosin VI binding partner, is associated with the function(s) of myosin VI in dividing cells. Loss of GIPC in siRNA knockdown cells results in a more than fourfold increase in the number of multinucleated cells. Our results suggest that myosin VI has novel functions in mitosis and that it plays an essential role in targeted membrane transport during cytokinesis.     

KLIF-associated cytoskeletal proteins in Trypanosoma brucei regulate cytokinesis by promoting cleavage furrow positioning and ingression

Cytokinesis in the early divergent protozoan Trypanosoma brucei occurs from the anterior cell tip of the new-flagellum daughter toward the nascent posterior end of the old-flagellum daughter of a dividing biflagellated cell. The cleavage furrow ingresses unidirectionally along the preformed cell division fold and is regulated by an orphan kinesin named kinesin localized to the ingressing furrow (KLIF) that localizes to the leading edge of the ingressing furrow. Little is known about how furrow ingression is controlled by KLIF and whether KLIF interacts with and cooperates with other cytokinesis regulatory proteins to promote furrow ingression. Here, we investigated the roles of KLIF in cleavage furrow ingression and identified a cohort of KLIF-associated cytoskeletal proteins as essential cytokinesis regulators. By genetic complementation, we demonstrated the requirement of the kinesin motor activity, but not the putative tropomyosin domain, of KLIF in promoting furrow ingression. We further showed that depletion of KLIF impaired the resolution of the nascent posterior of the old-flagellar daughter cell, thereby stalking cleavage furrow ingression at late stages of cytokinesis. Through proximity biotinylation, we identified a subset of cytoskeleton-associated proteins (CAPs) as KLIF-proximal proteins, and functional characterization of these cytoskeletal proteins revealed the essential roles of CAP46 and CAP52 in positioning the cleavage furrow and the crucial roles of CAP42 and CAP50 in promoting cleavage furrow ingression. Together, these results identified multiple cytoskeletal proteins as cytokinesis regulators and uncovered their essential and distinct roles in cytokinesis.     

CAR-1, a protein that localizes with the mRNA decapping component DCAP-1, is required for cytokinesis and ER organization in Caenorhabditis elegans embryos

The division of one cell into two requires the coordination of multiple components. We describe a gene, car-1, whose product may provide a link between disparate cellular processes. Inhibition of car-1 expression in Caenorhabditis elegans embryos causes late cytokinesis failures: cleavage furrows ingress but subsequently regress and the spindle midzone fails to form, even though midzone components are present. The localized accumulation of membrane that normally develops at the apex of the cleavage furrow during the final phase of cytokinesis does not occur and organization of the endoplasmic reticulum is aberrant, indicative of a disruption in membrane trafficking. The car-1 gene has homologues in a number of species, including proteins that associate with RNA binding proteins. CAR-1 localizes to P-granules (germ-line specific ribonucleoprotein particles) and discrete, developmentally regulated cytoplasmic foci. These foci also contain DCAP-1, a protein involved in decapping mRNAs. Thus, CAR-1, a protein likely to be associated with RNA metabolism, plays an essential role in the late stage of cytokinesis, suggesting a novel link between RNA, membrane trafficking and cytokinesis in the C. elegans embryo.     

Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis

Plasma membrane subdomains enriched in sphingolipids, cholesterol, and signaling proteins are critical for organization of actin, membrane trafficking, and cell polarity, but the role of such domains in cytokinesis in animal cells is unknown. Here, we show that eggs form a plasma membrane domain enriched in ganglioside G(M1) and cholesterol where tyrosine phosphorylated proteins occur at late anaphase at the contractile ring. The equatorial membrane domain forms by movement-specific lipids and proteins and is dependent on anaphase onset, myosin light chain phosphorylation, actin, and microtubules. Isolated detergent-resistant membranes contain Src and PLCgamma, which become tyrosine phosphorylated at cytokinesis, and whose activation is required for furrow progression. These studies suggest that membrane domains at the cleavage furrow possess a signaling pathway that contributes to 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.     

Phospholipase C isoforms are localized at the cleavage furrow during cytokinesis

It has recently been demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2) is localized at the cleavage furrow in dividing cells and its hydrolysis is required for complete cytokinesis, suggesting a pivotal role of PIP2 in cytokinesis. Here, we report that at least three mammalian isoforms of phosphoinositide-specific phospholipase C (PLC), PLCdelta1, PLCdelta3 and PLCbeta1, are localized to the cleavage furrow during cytokinesis. Targeting of the delta1 isoform to the furrow depends on the specific interaction between the PH domain and PIP2 in the plasma membrane. The necessity of active PLC in animal cell cytokinesis was confirmed using the specific inhibitors for PIP2 hydrolysis. These results support the model that activation of selected PLC isoforms at the cleavage furrow controls progression of cytokinesis through regulation of PIP2 levels: induction of the cleavage furrow by a contractile ring consisting of actomyosin is regulated by PIP2-dependent actin-binding proteins and formation of specific lipid domains required for membrane separation is affected by alterations in the lipid composition of the furrow.     

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.