Cytokinesis in Eukaryotes

Cytokinesis is the final event of the cell division cycle, and its completion results in irreversible partition of a mother cell into two daughter cells. Cytokinesis was one of the first cell cycle events observed by simple cell biological techniques; however, molecular characterization of cytokinesis has been slowed by its particular resistance to in vitro biochemical approaches. In recent years, the use of genetic model organisms has greatly advanced our molecular understanding of cytokinesis. While the outcome of cytokinesis is conserved in all dividing organisms, the mechanism of division varies across the major eukaryotic kingdoms. Yeasts and animals, for instance, use a contractile ring that ingresses to the cell middle in order to divide, while plant cells build new cell wall outward to the cortex. As would be expected, there is considerable conservation of molecules involved in cytokinesis between yeast and animal cells, while at first glance, plant cells seem quite different. However, in recent years, it has become clear that some aspects of division are conserved between plant, yeast, and animal cells. In this review we discuss the major recent advances in defining cytokinesis, focusing on deciding where to divide, building the division apparatus, and dividing. In addition, we discuss the complex problem of coordinating the division cycle with the nuclear cycle, which has recently become an area of intense research. In conclusion, we discuss how certain cells have utilized cytokinesis to direct development.     

Cytokinesis in eukaryotes.

Cytokinesis is the final event of the cell division cycle, and its completion results in irreversible partition of a mother cell into two daughter cells. Cytokinesis was one of the first cell cycle events observed by simple cell biological techniques; however, molecular characterization of cytokinesis has been slowed by its particular resistance to in vitro biochemical approaches. In recent years, the use of genetic model organisms has greatly advanced our molecular understanding of cytokinesis. While the outcome of cytokinesis is conserved in all dividing organisms, the mechanism of division varies across the major eukaryotic kingdoms. Yeasts and animals, for instance, use a contractile ring that ingresses to the cell middle in order to divide, while plant cells build new cell wall outward to the cortex. As would be expected, there is considerable conservation of molecules involved in cytokinesis between yeast and animal cells, while at first glance, plant cells seem quite different. However, in recent years, it has become clear that some aspects of division are conserved between plant, yeast, and animal cells. In this review we discuss the major recent advances in defining cytokinesis, focusing on deciding where to divide, building the division apparatus, and dividing. In addition, we discuss the complex problem of coordinating the division cycle with the nuclear cycle, which has recently become an area of intense research. In conclusion, we discuss how certain cells have utilized cytokinesis to direct development.     

A unifying new model of cytokinesis for the dividing plant and animal cells

Cytokinesis ensures proper partitioning of the nucleocytoplasmic contents into two daughter cells. It has generally been thought that cytokinesis is accomplished differently in animals and plants because of the differences in the preparatory phases, into the centrosomal or acentrosomal nature of the process, the presence or absence of rigid cell walls, and on the basis of 'outside-in' or 'inside-out' mechanism. However, this long-standing paradigm needs further reevaluation based on new findings. Recent advances reveal that plant cells, similarly to animal cells, possess astral microtubules that regulate the cell division plane. Furthermore, endocytosis has been found to be important for cytokinesis in animal and plant cells: vesicles containing endocytosed cargo provide material for the cell plate formation in plants and for closure of the midbody channel in animals. Thus, although the preparatory phases of the cell division process differ between plant and animal cells, the later phases show similarities. We unify these findings in a model that suggests a conserved mode of cytokinesis.     

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation.     

The mammalian septin MSF localizes with microtubules and is required for completion of cytokinesis

Cytokinesis in animal cells involves the contraction of an actomyosin ring formed at the cleavage furrow. Nuclear division, or karyokinesis, must be precisely timed to occur before cytokinesis in order to prevent genetic anomalies that would result in either cell death or uncontrolled cell division. The septin family of GTPase proteins has been shown to be important for cytokinesis although little is known about their role during this process. Here we investigate the distribution and function of the mammalian septin MSF. We show that during interphase, MSF colocalizes with actin, microtubules, and another mammalian septin, Nedd5, and coprecipitates with six septin proteins. In addition, transfections of various MSF isoforms reveal that MSF-A specifically localizes with microtubules and that this localization is disrupted by nocodazole treatment. Furthermore, MSF isoforms localize primarily with tubulin at the central spindle during mitosis, whereas Nedd5 is mainly associated with actin. Microinjection of affinity-purified anti-MSF antibodies into synchronized cells, or depletion of MSF by small interfering RNAs, results in the accumulation of binucleated cells and in cells that have arrested during cytokinesis. These results reveal that MSF is required for the completion of cytokinesis and suggest a role that is distinct from that of Nedd5.     

Predetermination of cytokinesis and transition from holoblastic to partial superficial cleavage in early embryonic development of Tetrodontophora bielanensis (Collembola)

In the T. bielanensis embryo, only karyokinesis occurs during the first cleavage division, and a two-nuclear syncytial embryo forms. Then, two cytoplasmic concentrations in the form of elongated rolls perpendicular to each other develop below the periplasm at the animal pole of the egg. The second cleavage division is also associated with karyokineses only. After the embryo reaches the four-nuclear stage, cytokinesis occur at its animal pole, and two cleavage furrows perpendicular to each other develop in the periplasm above the cytoplasmic concentrations. The cell membranes forming within the furrows do not invade the cytoplasmic concentrations, but their growing tips push them into the egg interior, where they merge and form the central cytoplasmic concentration. The developing cell membranes do not invade the central cytoplasm; they band and grow above its surface. Four pyramidal blastomeres form as a result of this. The eight-blastomere embryo forms through both karyokinesis and cytokinesis, but the growing cell membranes now band below the previous ones and cut off anucleate parts of the mother blastomeres, which fuse with the central cytoplasm. Thus, during this phase of development the transition from holoblastic to partial superficial cleavage is initiated. Morphological analysis suggests that the formation of the first two cytokinesis is predetermined by and depends on factors connected with the animal pole periplasm. It also suggests that the central cytoplasm constitutes the morphological field, inducing the transition from holoblastic to partial superficial cleavage.     

Cell division: control of the chromosomal passenger complex in time and space

The ultimate goal of cell division is equal transmission of the duplicated genome to two new daughter cells. Multiple surveillance systems exist that monitor proper execution of the cell division program and as such ensure stability of our genome. One widely studied protein complex essential for proper chromosome segregation and execution of cytoplasmic division (cytokinesis) is the chromosomal passenger complex (CPC). This highly conserved complex consists of Borealin, Survivin, INCENP, and Aurora B kinase, and has a dynamic localization pattern during mitosis and cytokinesis. Not surprisingly, it also performs various functions during these phases of the cell cycle. In this review, we will give an overview of the latest insights into the regulation of CPC localization and discuss if and how specific localization impacts its diverse functions in the dividing cell.     

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.     

Characterization of AtCDC48. Evidence for multiple membrane fusion mechanisms at the plane of cell division in plants

The components of the cellular machinery that accomplish the various complex and dynamic membrane fusion events that occur at the division plane during plant cytokinesis, including assembly of the cell plate, are not fully understood. The most well-characterized component, KNOLLE, a cell plate-specific soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptor (SNARE), is a membrane fusion machine component required for plant cytokinesis. Here, we show the plant ortholog of Cdc48p/p97, AtCDC48, colocalizes at the division plane in dividing Arabidopsis cells with KNOLLE and another SNARE, the plant ortholog of syntaxin 5, SYP31. In contrast to KNOLLE, SYP31 resides in defined punctate membrane structures during interphase and is targeted during cytokinesis to the division plane. In vitro-binding studies demonstrate that AtCDC48 specifically interacts in an ATP-dependent manner with SYP31 but not with KNOLLE. In contrast, we show that KNOLLE assembles in vitro into a large approximately 20S complex in an Sec18p/NSF-dependent manner. These results suggest that there are at least two distinct membrane fusion pathways involving Cdc48p/p97 and Sec18p/NSF that operate at the division plane to mediate plant cytokinesis. Models for the role of AtCDC48 and SYP31 at the division plane will be discussed.     

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.     

Membrane trafficking during plant cytokinesis

Plant morphogenesis is regulated by cell division and expansion. Cytokinesis, the final stage of cell division, culminates in the construction of the cell plate, a unique cytokinetic membranous organelle that is assembled across the inside of the dividing cell. Both during cell-plate formation and cell expansion, the secretory pathway is highly active and is polarized toward the plane of division or toward the plasma membrane, respectively. In this review, we discuss results from recent genetic and biochemical research directed toward understanding the molecular events occurring during cytokinesis and cell expansion, including data supporting the idea that during cytokinesis one or more exocytic pathways are polarized toward the division plane. We will also highlight recent evidence for the roles of secretory vesicle transport and cytoskeletal machinery in cell-plate membrane trafficking and fusion.     

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.     

Quantitative Analysis of Cytokinesis In Situ during C. elegans Postembryonic Development

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.     

Cytokinesis in plant male meiosis

In somatic cell division, cytokinesis is the final step of the cell cycle and physically divides the mother cytoplasm into two daughter cells. In the meiotic cell division, however, pollen mother cells (PMCs) undergo two successive nuclear divisions without an intervening S-phase and consequently generate four haploid daughter nuclei out of one parental cell. In line with this, the physical separation of meiotic nuclei does not follow the conventional cytokinesis pathway, but instead is mediated by alternative processes, including polar-based phragmoplast outgrowth and RMA-mediated cell wall positioning. In this review, we outline the different cytological mechanisms of cell plate formation operating in different types of PMCs and additionally focus on some important features associated with male meiotic cytokinesis, including cytoskeletal dynamics and callose deposition. We also provide an up-to-date overview of the main molecular actors involved in PMC wall formation and additionally highlight some recent advances on the effect of cold stress on meiotic cytokinesis in plants.     

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.     

Multinucleation in mouse fibroblasts cultured in methocel medium

3T3-4E cells formed multinucleate cells with high frequency when incubated in methocel medium. The experiment with hydroxyurea and the cytological observation of mitoses showed that multinucleate cells were produced by nuclear division in the absence of cytokinesis. When transferred onto a solid substratum, most of the multinucleate cells divided within seven hours into mononucleate cells through the process of cytoplasmic division, indicating that cell spreading induced cytokinesis. Other mouse fibroblast lines examined so far showed only the low frequency of multinucleation. These findings indicate that in 3T3-4E cells cultivated in methocel, nuclear division occurred independently of cytokinesis, and that cytokinesis was also anchorage-dependent. This system will be available for studying cytoplasmic division of mammalian cells.     

Membrane trafficking in cytokinesis

Until recently, two distinct types of cytokinesis were thought to be responsible for the division of plant and animal cells. Plant cells divide through the formation of a membrane plate between the daughter cells, while animal cells divide by the constriction of a cortical actin-based ring around the cell. However, accumulating evidence suggests that the two mechanisms may have more in common than previously thought. In this review we will focus on recent developments that raise the possibility of unexpected similarities between the final steps in cytokinesis in animal and plant cells.     

Mechanisms controlling division-plane positioning

A critical and irreversible step in the cell division cycle is cytokinesis which physically separates the two daughter cells. This event is consequently subject to tight spatial and temporal regulation. This review focuses on the spatial regulatory mechanisms controlling the position of the division plane. Studies performed in prokaryotic and eukaryotic systems have revealed that various signal-emitting spatial cues – mitotic spindle, nucleus, nucleoid or cell tips – can favour or inhibit the assembly of the cytokinetic apparatus in their vicinity. Most often, several mechanisms operate in parallel to integrate spatial information and promote faithful genome segregation as well as proper cytoplasmic division. We primarily describe the spatial regulatory mechanisms operating in the fission yeast model system, where a detailed molecular understanding of cytokinesis has been achieved. In this system, spatial regulations target a major factor controlling the position of the division plane, the anillin-like protein Mid1. These mechanisms are then compared to spatial regulatory mechanisms prevailing in animal cells and rod-shaped bacteria.     

The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1-3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE (KN) and KEULE (KEU), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn, display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU, have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.