Cytokinesis In Animal Cells Research Articles

Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex

Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.

Germ fate determinants protect germ precursor cell division by reducing septin and anillin levels at the cell division plane.

Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formincyk-1(ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septinUNC-59 and anillinANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.

Anillin-related Mid1 as an adaptive and multimodal contractile ring anchoring protein: A simulation study

Cytokinesis of animal and fungi cells depends crucially on the anillin scaffold proteins. Fission yeast anillin-related Mid1 anchors cytokinetic ring precursor nodes to the membrane. However, it is unclear if both of its Pleckstrin Homology (PH) and C2 C-terminal domains bind to the membrane as monomers or dimers, and if one domain plays a dominant role. We studied Mid1 membrane binding with all-atom molecular dynamics near a membrane with yeast-like lipid composition. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the disordered L3 loop of C2 in a vertical orientation, with the PH away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. Simulations of C2-PH dimers show extensive asymmetric membrane contacts. These multiple modes of binding may reflect Mid1's multiple interactions with membranes, node proteins, and ability to sustain mechanical forces.

A Rab39-Klp98A-Rab35 endocytic recycling pathway is essential for rapid Golgi-dependent furrow ingression.

Ingression of the plasma membrane is an essential part of the cell topology-distorting repertoire and a key element in animal cell cytokinesis. Many embryos have rapid cleavage stages in which they are furrowing powerhouses, quickly forming and disassembling cleavage furrows on timescales of just minutes. Previous work has shown that cytoskeletal proteins and membrane trafficking coordinate to drive furrow ingression, but where these membrane stores are derived from and how they are directed to furrowing processes has been less clear. Here, we identify an extensive Rab35/Rab4>Rab39/Klp98A>trans-Golgi network (TGN) endocytic recycling pathway necessary for fast furrow ingression in the Drosophila embryo. Rab39 is present in vesiculotubular compartments at the TGN where it receives endocytically derived cargo through a Rab35/Rab4-dependent pathway. A Kinesin-3 family member, Klp98A, drives the movements and tubulation activities of Rab39, and disruption of this Rab39-Klp98A-Rab35 pathway causes deep furrow ingression defects and genomic instability. These data suggest that an endocytic recycling pathway rapidly remobilizes membrane cargo from the cell surface and directs it to the trans-Golgi network to permit the initiation of new cycles of cleavage furrow formation.

The Rho1 GTPase controls anillo-septin assembly to facilitate contractile ring closure during cytokinesis

Animal cell cytokinesis requires activation of the GTPase RhoA (Rho1 in Drosophila), which assembles an F-actin- and myosin II-dependent contractile ring (CR) at the equatorial plasma membrane. CR closure is poorly understood, but involves the multidomain scaffold protein, Anillin. Anillin binds many CR components including F-actin and myosin II (collectively actomyosin), RhoA and the septins. Anillin recruits septins to the CR but the mechanism is unclear. Live imaging of Drosophila S2 cells and HeLa cells revealed that the Anillin N-terminus, which scaffolds actomyosin, cannot recruit septins to the CR. Rather, septin recruitment required the ability of the Anillin C-terminus to bind Rho1-GTP and the presence of the Anillin PH domain, in a sequential mechanism occurring at the plasma membrane, independently of F-actin. Anillin mutations that blocked septin recruitment, but not actomyosin scaffolding, slowed CR closure and disrupted cytokinesis. Thus, CR closure requires coordination of two Rho1-dependent networks: actomyosin and anillo-septin.

Actomyosin-Driven Division of a Synthetic Cell.

One of the major challenges of bottom-up synthetic biology is rebuilding a minimal cell division machinery. From a reconstitution perspective, the animal cell division apparatus is mechanically the simplest and therefore attractive to rebuild. An actin-based ring produces contractile force to constrict the membrane. By contrast, microbes and plant cells have a cell wall, so division requires concerted membrane constriction and cell wall synthesis. Furthermore, reconstitution of the actin division machinery helps in understanding the physical and molecular mechanisms of cytokinesis in animal cells and thus our own cells. In this review, we describe the state-of-the-art research on reconstitution of minimal actin-mediated cytokinetic machineries. Based on the conceptual requirements that we obtained from the physics of the shape changes involved in cell division, we propose two major routes for building a minimal actin apparatus capable of division. Importantly, we acknowledge both the passive and active roles that the confining lipid membrane can play in synthetic cytokinesis. We conclude this review by identifying the most pressing challenges for future reconstitution work, thereby laying out a roadmap for building a synthetic cell equipped with a minimal actin division machinery.

Membrane area gain and loss during cytokinesis.

In cytokinesis of animal cells, the cell is symmetrically divided into two. Since the cell's volume is conserved, the projected area has to increase to allow for the change of shape. Here we aim to predict how membrane gain and loss adapt during cytokinesis. We work with a kinetic model in which membrane turnover depends on membrane tension and cell shape. We apply this model to a series of calculated vesicle shapes as a proxy for the shape of dividing cells. We find that the ratio of kinetic turnover parameters changes nonmonotonically with cell shape, determined by the dependence of exocytosis and endocytosis on membrane curvature. Our results imply that controlling membrane turnover will be crucial for the successful division of artificial cells.

Modeling area gain and loss during cytokinesis

One of the hallmarks of living systems is their ability to replicate and spread, which is achieved by division at the cellular level. During cytokinesis in animal cells, the cell is divided into two equal parts. Since division occurs while maintaining cellular volume, the membrane area must increase to allow the change in shape. However, how the ratio of membrane gain and loss adjusts during cell shape changes in cytokinesis is still unclear. To address this question, we developed a phenomenological model in which area gain and loss are balanced at the membrane level, controlled by membrane curvature and tension.

Mechanistic insights into central spindle assembly mediated by the centralspindlin complex

The central spindle spatially and temporally regulates the formation of division plane during cytokinesis in animal cells. The heterotetrameric centralspindlin complex bundles microtubules to assemble the central spindle, the mechanism of which is poorly understood. Here, we determined the crystal structures of the molecular backbone of ZEN-4/CYK-4 centralspindlin from Caenorhabditis elegans, which revealed the detailed mechanism of complex formation. The molecular backbone of centralspindlin has the intrinsic propensity to undergo liquid-liquid phase separation. The condensation of centralspindlin requires two patches of basic residues at ZEN-4 and multiple acidic residues at the intrinsically disordered region of CYK-4, explaining the synergy of the two subunits for the function. These complementary charged residues were critical for the microtubule bundling activity of centralspindlin in vitro and for the assembly of the central spindle in vivo. Together, our findings provide insights into the mechanism of central spindle assembly mediated by centralspindlin through charge-driven macromolecular condensation.

Encapsulated Actomyosin Patterns Drive Cell-Like Membrane Shape Changes

Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as α-actinin and fascin as well actin nucleator Arp2/3 complex largely determine the architecture and connectivity of actin network patterns; consequently, they regulate network remodeling and membrane shape changes. Membrane constriction in animal cell cytokinesis proceeds by assembly and contraction of a contractile ring pattern rich in α-actinin and myosin at the equator of the cell cortex, with which the ring is contiguous. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that, depending on vesicle size and concentrations of α-actinin and fascin, actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane enhances the interaction of the contractile networks with lipid membrane but does not change the architecture of the patterns. A membrane-bound actomyosin ring exerts force and constricts the membrane. An Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex and contribute to myosin-driven clustering of the cortex and consequently membrane deformation. An active gel theory unifies a model for the observed membrane constriction and protrusion induced by the membrane-bound actomyosin networks.

Comparative Analysis of the Roles of Non-muscle Myosin-IIs in Cytokinesis in Budding Yeast, Fission Yeast, and Mammalian Cells.

The contractile ring, which plays critical roles in cytokinesis in fungal and animal cells, has fascinated biologists for decades. However, the basic question of how the non-muscle myosin-II and actin filaments are assembled into a ring structure to drive cytokinesis remains poorly understood. It is even more mysterious why and how the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and humans construct the ring structure with one, two, and three myosin-II isoforms, respectively. Here, we provide a comparative analysis of the roles of the non-muscle myosin-IIs in cytokinesis in these three model systems, with the goal of defining the common and unique features and highlighting the major questions regarding this family of proteins.

A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila.

In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

Cell Division: Switching On ECT2 in a Non-Canonical Fashion

Determining the site of cell cleavage is crucial for cytokinesis and involves precise activation of the RhoGEF ECT2. A new study demonstrates how a non-canonical interaction of ECT2 with centralspindlin underlies cytokinesis in animal cells, solving a mechanistic conundrum.

Critical Roles of a RhoGEF-Anillin Module in Septin Architectural Remodeling during Cytokinesis.

How septin architecture is remodeled from an hourglass to a double ring during cytokinesis in fungal and animal cells remains unknown. Here, we show that during the hourglass-to-double-ring transition in budding yeast, septins acquire a "zonal architecture" in which paired septin filaments that are organized along the mother-bud axis associate with circumferential single septin filaments, the Rho guanine-nucleotide-exchange factor (RhoGEF) Bud3, and the anillin-like protein Bud4 exclusively at the outer zones and with myosin-II filaments in the middle zone. Deletion of Bud3 or its Bud4-interacting domain, but not its RhoGEF domain, leads to a complete loss of the single filaments, whereas deletion of Bud4 or its Bud3-interacting domain destabilizes the transitional hourglass, especially at the mother side, with partial loss of both filament types. Deletion of Bud3 and Bud4 together further weakens the transitional structure and abolishes the double ring formation while causing no obvious defect in actomyosin ring constriction. This and further analyses suggest that Bud3 stabilizes the single filaments, whereas Bud4 strengthens the interaction between the paired and single filaments at the outer zones of the transitional hourglass, as well as in the double ring. This study reveals a striking zonal architecture for the transitional hourglass that pre-patterns two cytokinetic structures-a septin double ring and an actomyosin ring-and also defines the essential roles of a RhoGEF-anillin module in septin architectural remodeling during cytokinesis at the filament level.

How cortical waves drive fission of motile cells

Cytokinesis-the division of a cell into two daughter cells-is a key step in cell growth and proliferation. It typically occurs in synchrony with the cell cycle to ensure that a complete copy of the genetic information is passed on to the next generation of daughter cells. In animal cells, cytokinesis commonly relies on an actomyosin contractile ring that drives equatorial furrowing and separation into the two daughter cells. However, also contractile ring-independent forms of cell division are known that depend on substrate-mediated traction forces. Here, we report evidence of an as yet unknown type of contractile ring-independent cytokinesis that we termed wave-mediated cytofission. It is driven by self-organized cortical actin waves that travel across the ventral membrane of oversized, multinucleated Dictyostelium discoideum cells. Upon collision with the cell border, waves may initiate the formation of protrusions that elongate and eventually pinch off to form separate daughter cells. They are composed of a stable elongated wave segment that is enclosed by a cell membrane and moves in a highly persistent fashion. We rationalize our observations based on a noisy excitable reaction-diffusion model in combination with a dynamic phase field to account for the cell shape and demonstrate that daughter cells emerging from wave-mediated cytofission exhibit a well-controlled size.

ZIP kinase phosphorylated and activated by Rho kinase/ROCK contributes to cytokinesis in mammalian cultured cells

Cytokinesis of animal cells requires contraction of a contractile ring, composed of actin filaments and myosin II filaments. Phosphorylation of myosin II regulatory light chain (MRLC) promotes contraction of the actomyosin ring by activating myosin II motor activity. Both Rho-associated coiled-coil kinase (Rho kinase/ROCK) and Zipper-interacting protein kinase (ZIP kinase/ZIPK) have been reported to phosphorylate MRLC at the contractile ring. However, it remains unclear whether these kinases function independently of each other. Here, we clarified that ROCK colocalizes and forms a complex with ZIPK at telophase. As ROCK is reported to phosphorylate and activate ZIPK in vitro, we hypothesized that ZIPK phosphorylated by ROCK contributes to control cytokinesis. To address this, we expressed EGFP-ZIPK wild type (WT), a non-phosphorylatable mutant (T265A) or a phosphorylation-mimicking mutant (T265D) in HeLa cells and treated these cells with a ROCK inhibitor. Decrease in phosphorylated MRLC and a delay of furrow ingression by the ROCK inhibitor were rescued by the expression of EGFP-ZIPK-T265D, but not EGFP-ZIPK-WT or -T265A. This suggests that ROCK regulates MRLC phosphorylation followed by furrow ingression, through ZIPK phosphorylation.

Critical Roles of a RhoGEF-Anillin Module in Septin Architectural Remodeling During Cytokinesis

How septin architecture is remodeled from an hourglass to a double ring during cytokinesis in fungal and animal cells remains unknown. Here, we show that during the hourglass-to-double ring transition in budding yeast, septins acquire a striking “zonal architecture” in which paired septin filaments that are organized along the mother-bud axis associate with circumferential single septin filaments, the RhoGEF Bud3, and the anillin-like protein Bud4 exclusively at the outer zones and with myosin-II filaments the middle zone. Deletion of Bud3 or its Bud4-interacting domain, but not its RhoGEF domain, leads to a complete loss of single filaments whereas deletion of Bud4 or its Bud3-interacting domain destabilizes the transitional hourglass, especially at the mother side, with partial loss of both filament types. This and further analysis indicate that Bud3 stabilizes the single filaments whereas Bud4 fastens the junction between the paired and single filaments. Deletion of BUD3 and BUD4 together abolishes transitional hourglass and prevents double ring formation. The absence of the septin structures in the double mutant presents a unique opportunity to examine the significance of the septin double ring in cytokinesis. We demonstrate that the septin double ring is dispensable for cytokinesis unless the actomyosin ring is also compromised. Thus, the two cytokinetic structures, which are pre-patterned by and simultaneously generated from the transitional hourglass, normally act in concert to ensure efficient cytokinesis. This study establishes a mechanism and function for the role of a RhoGEF-anillin module in septin architectural remodeling at the cell division site.

Classical and Emerging Regulatory Mechanisms of Cytokinesis in Animal Cells

The primary goal of cytokinesis is to produce two daughter cells, each having a full set of chromosomes. To achieve this, cells assemble a dynamic structure between segregated sister chromatids called the contractile ring, which is made up of filamentous actin, myosin-II, and other regulatory proteins. Constriction of the actomyosin ring generates a cleavage furrow that divides the cytoplasm to produce two daughter cells. Decades of research have identified key regulators and underlying molecular mechanisms; however, many fundamental questions remain unanswered and are still being actively investigated. This review summarizes the key findings, computational modeling, and recent advances in understanding of the molecular mechanisms that control the formation of the cleavage furrow and cytokinesis.

Unite to divide - how models and biological experimentation have come together to reveal mechanisms of cytokinesis.

Cytokinesis is the fundamental and ancient cellular process by which one cell physically divides into two. Cytokinesis in animal and fungal cells is achieved by contraction of an actomyosin cytoskeletal ring assembled in the cell cortex, typically at the cell equator. Cytokinesis is essential for the development of fertilized eggs into multicellular organisms and for homeostatic replenishment of cells. Correct execution of cytokinesis is also necessary for genome stability and the evasion of diseases including cancer. Cytokinesis has fascinated scientists for well over a century, but its speed and dynamics make experiments challenging to perform and interpret. The presence of redundant mechanisms is also a challenge to understand cytokinesis, leaving many fundamental questions unresolved. For example, how does a disordered cytoskeletal network transform into a coherent ring? What are the long-distance effects of localized contractility? Here, we provide a general introduction to 'modeling for biologists', and review how agent-based modeling and continuum mechanics modeling have helped to address these questions.

Molecular dissection of the actin-binding ability of the fission yeast α-actinin, Ain1, in vitro and in vivo.

A contractile ring (CR) is involved in cytokinesis in animal and yeast cells. Although several types of actin-bundling proteins associate with F-actin in the CR, their individual roles in the CR have not yet been elucidated in detail. Ain1 is the sole α-actinin homologue in the fission yeast Schizosaccharomyces pombe and specifically localizes to the CR with a high turnover rate. S. pombe cells lacking the ain1+ gene show defects in cytokinesis under stress conditions. We herein investigated the biochemical activity and cellular localization mechanisms of Ain1. Ain1 showed weaker affinity to F-actin in vitro than other actin-bundling proteins in S. pombe. We identified a mutation that presumably loosened the interaction between two calponin-homology domains constituting the single actin-binding domain (ABD) of Ain1, which strengthened the actin-binding activity of Ain1. This mutant protein induced a deformation in the ring shape of the CR. Neither a truncated protein consisting only of an N-terminal ABD nor a truncated protein lacking a C-terminal region containing an EF-hand motif localized to the CR, whereas the latter was involved in the bundling of F-actin in vitro. We herein propose detailed mechanisms for how each part of the molecule is involved in the proper cellular localization and function of Ain1.