Genetic dissection of cytokinesis

Higher plants have evolved specific mechanisms for partitioning the cytoplasm of dividing cells. In the predominant mode of phragmoplast-assisted cytokinesis, a cell wall and flanking plasma membranes are made de novo from a transient membrane compartment, the cell plate, which in turn forms by vesicle fusion from the centre to the periphery of the dividing cell. Other modes of cytokinesis appear to occur in meiotic cells and developing gametophytes. Here we review recent progress in the analysis of plant cytokinesis, focusing on genetic studies in Arabidopsis which are beginning to identify structural and regulatory components of phragmoplast-assisted cytokinesis. Two classes of mutations have been described. In one class, the defects appear to be confined to cell plate formation, suggesting that the execution of cytokinesis is specifically affected. Mutations in the other class display more general defects in cell division. We also discuss possible roles of proteins that have been localised in cytokinetic cells but not characterised genetically. Finally, mutations affecting meiotic or gametophytic cell divisions suggest that mechanistically different modes of cytokinesis occur in higher plants.     

Cytokinesis in flowering plants: cellular process and developmental integration

In phragmoplast-assisted cytokinesis of somatic cells, vesicle fusion generates a cell plate that matures into a new cell wall and its flanking plasma membranes. Insight into this dynamic process has been gained in the past few years and additional molecular components of the basic machinery of cytokinesis have been identified. Specialized modes of cytokinesis occur in meiosis and gametophyte development, and recent studies indicate that they are genetically distinct from somatic cytokinesis.     

‘Life is a Highway’: Membrane Trafficking During Cytokinesis

Cytokinesis, the final stage of the cell cycle, is an essential step toward the formation of two viable daughter cells. In recent years, membrane trafficking has been shown to be important for the completion of cytokinesis. Vesicles originating from both the endocytic and secretory pathways are known to be shuttled to the plasma membrane of the ingressing cleavage furrow, delivering membrane and proteins to this dynamic region. Advances in cell imaging have led to exciting new discoveries regarding vesicle movement in living cells. Recent work has revealed a significant role for membrane trafficking, as controlled by regulatory proteins, during cytokinesis in animal cells. The endocytic and secretory pathways as well as motor proteins are revealed to be essential in the delivery of vesicles to the cleavage furrow during cytokinesis.     

How one becomes many: blastoderm cellularization in Drosophila melanogaster

Embryonic development in Drosophila melanogaster begins with a rapid series of mitotic nuclear divisions, unaccompanied by cytokinesis, to produce a multi-nucleated single cell embryo, the syncytial blastoderm. The syncytium then undergoes a process of cell formation, in which the individual nuclei become enclosed in individual cells. This process of cellularization involves integrating mechanisms of cell polarity, cell-cell adhesion and a specialized form of cytokinesis. The detailed molecular mechanism, however, is highly complex and, despite extensive analysis, remains poorly understood. Nevertheless, new insights are emerging from recent studies on aspects of membrane polarization and insertion, which show that membrane components from intracellular organelles are involved. In addition, actin and actin-associated proteins have been heavily implicated while new evidence shows that microtubule cytoskeletal elements are mechanistically involved in all aspects of cellularization. This review will draw on both the traditional models and the new data to provide a current perspective on the nature of cellular blastoderm formation in Drosophila melanogaster.     

SNAREs--engines for membrane fusion

Since the discovery of SNARE proteins in the late 1980s, SNAREs have been recognized as key components of protein complexes that drive membrane fusion. Despite considerable sequence divergence among SNARE proteins, their mechanism seems to be conserved and is adaptable for fusion reactions as diverse as those involved in cell growth, membrane repair, cytokinesis and synaptic transmission. A fascinating picture of these robust nanomachines is emerging.     

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.     

Membrane traffic: a driving force in cytokinesis

Dividing animal and plant cells maintain a constant chromosome content through temporally separated rounds of replication and segregation. Until recently, the mechanisms by which animal and plant cells maintain a constant surface area have been considered to be distinct. The prevailing view was that surface area was maintained in dividing animal cells through temporally separated rounds of membrane expansion and membrane invagination. The latter event, known as cytokinesis, produces two physically distinct daughter cells and has been thought to be primarily driven by actomyosin-based constriction. By contrast, membrane addition seems to be the primary mechanism that drives cytokinesis in plants and, thus, the two events are linked mechanistically and temporally. In this article (which is part of the Cytokinesis series), we discuss recent studies of a variety of organisms that have made a convincing case for membrane trafficking at the cleavage furrow being a key component of both animal and plant cytokinesis.     

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.     

Nuclear envelope fission is linked to cytokinesis in budding yeast

We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast. By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis. Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle. These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis.     

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.     

High lipid order of Arabidopsis cell‐plate membranes mediated by sterol and DYNAMIN‐RELATED PROTEIN1A function

Membranes of eukaryotic cells contain high lipid‐order sterol‐rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher‐plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell‐plate formation in Arabidopsis relies on sterol‐ and DYNAMIN‐RELATED PROTEIN1A (DRP1A)‐dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order‐sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid‐order membrane domain. The cell‐plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co‐localize with DRP1A at the cell plate, and DRP1A accumulates in detergent‐resistant membrane fractions. Modifications of sterol concentration or composition reduce cell‐plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid‐order domain, and pave the way to explore potential feedback between lipid order and function of dynamin‐related proteins during 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.     

Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells

Cytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number. When cytokinesis occurs in a polarized cell it can create daughters with distinct fates. In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition. Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis. To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features. Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features. Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent. To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches. Nevertheless, the three species discussed here provide substantial mechanistic diversity.     

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.     

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

The actin-binding and bundling protein,EPLIN, is required for cytokinesis

Cytokinesis involves two phases: 1) membrane ingression followed by 2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II, and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.     

The degradation of two mitotic cyclins contributes to the timing of cytokinesis

BACKGROUND: Cytokinesis occurs just as chromosomes complete segregation and reform nuclei. It has been proposed that cyclin/Cdk kinase inhibits cytokinesis until exit from mitosis; however, the timer of cytokinesis has not been experimentally defined. Whereas expression of a stable version of Drosophila cyclin B blocks cytokinesis along with numerous events of mitotic exit, stable cyclin B3 allows cytokinesis even though it blocks late events of mitotic exit. We examined the interface between mitotic cyclin destruction and the timing of cytokinesis. RESULTS: In embryonic mitosis 14, the cytokinesis furrow appeared 60 s after the metaphase/anaphase transition and closed 90 s later during telophase. In cyclin B or cyclin B3 mutant cells, the cytokinesis furrow appeared at an earlier stage of mitosis. Expression of stable cyclin B3 delayed and prolonged furrow invagination; nonetheless, cytokinesis completed during the extended mitosis. Reduced function of Pebble, a Rho GEF required for cytokinesis, also delayed and slowed furrow invagination, but incomplete furrows were aborted at the time of mitotic exit. In functional and genetic tests, cyclin B and cyclin B3 inhibited Pebble contributions to cytokinesis. CONCLUSIONS: Temporal coordination of mitotic events involves inhibition of cytokinesis by cyclin B and cyclin B3 and punctual relief of the inhibition by destruction of these cyclins. Both cyclins inhibit Pebble-dependent activation of cytokinesis, whereas cyclin B can inhibit cytokinesis by additional modes. Stable cyclin B3 also blocks the later return to interphase that otherwise appears to impose a deadline for the completion of cytokinesis.     

Cell biology of primary cell wall synthesis in plants

Building a complex structure such as the cell wall, with many individual parts that need to be assembled correctly from distinct sources within the cell, is a well-orchestrated process. Additional complexity is required to mediate dynamic responses to environmental and developmental cues. Enzymes, sugars, and other cell wall components are constantly and actively transported to and from the plasma membrane during diffuse growth. Cell wall components are transported in vesicles on cytoskeletal tracks composed of microtubules and actin filaments. Many of these components, and additional proteins, vesicles, and lipids are trafficked to and from the cell plate during cytokinesis. In this review, we first discuss how the cytoskeleton is initially organized to add new cell wall material or to build a new cell wall, focusing on similarities during these processes. Next, we discuss how polysaccharides and enzymes that build the cell wall are trafficked to the correct location by motor proteins and through other interactions with the cytoskeleton. Finally, we discuss some of the special features of newly formed cell walls generated during cytokinesis.

The cell wall is assembled via vesicle trafficking along cytoskeletal filaments during growth and division.