Search

Epithelial folding is a common means to execute morphogenetic movements. The gastrulating Drosophila embryo offers many examples of epithelial folding events, including the ventral, cephalic, and dorsal furrows. Each of these folding events is associated with changes in intracellular contractility and/or cytoskeleton structures that autonomously promote epithelial folding. Here, we review accumulating evidence that suggests the progression and final form of ventral, cephalic, and dorsal furrows are also influenced by the behaviour of cells neighbouring these folds. We further discuss the prevalence and importance of junctional rearrangements during epithelial folding events, suggesting adherens junction components are prime candidates to modulate the transmission of the intercellular forces that influence folding events. Finally, we discuss how recently developed methods that enable precise spatial and/or temporal control of protein activity allow direct testing of molecular models of morphogenesis in vivo.

Epithelial folding is a common means to execute morphogenetic movements. The gastrulating Drosophila embryo offers many examples of epithelial folding events, including the ventral, cephalic, and dorsal furrows. Each of these folding events is associated with changes in intracellular contractility and/or cytoskeleton structures that autonomously promote epithelial folding. Here, we review accumulating evidence that suggests the progression and final form of ventral, cephalic, and dorsal furrows are also influenced by the behaviour of cells neighbouring these folds. We further discuss the prevalence and importance of junctional rearrangements during epithelial folding events, suggesting adherens junction components are prime candidates to modulate the transmission of the intercellular forces that influence folding events. Finally, we discuss how recently developed methods that enable precise spatial and/or temporal control of protein activity allow direct testing of molecular models of morphogenesis in vivo.

Haploid male gametes are produced through meiosis during gametogenesis. Whereas the cell biology of mitosis and meiosis is well studied in the nematode Caenorhabditis elegans, comparatively little is known regarding the physical division of primary spermatocytes during meiosis I. Here, we investigated this process using high-resolution time-lapse confocal microscopy and examined the spatiotemporal regulation of contractile ring assembly in C. elegans primary spermatocytes. We found that centralspindlin and RhoA effectors were recruited to the equatorial cortex of dividing primary spermatocytes for contractile ring assembly before segregation of homologous chromosomes. We also observed that perturbations shown to promote centralspindlin oligomerization regulated the cortical recruitment of NMY-2 and impacted the order in which primary spermatocytes along the proximal–distal axis of the gonad enter meiosis I. These results expand our understanding of the cellular division of primary spermatocytes into secondary spermatocytes during meiosis I. This article has an associated First Person interview with the first author of the paper.

Local accumulation of oskar (osk) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for osk mRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for osk mRNA. We demonstrate that posterior localization of osk/Staufen is determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes osk/Staufen from the cortex along microtubules, myosin-V anchors osk/Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density., eLife digest One of the most fundamental steps of embryonic development is deciding which end of the body should be the head, and which should be the tail. Known as 'axis specification', this process depends on the location of genetic material called mRNAs. In fruit flies, for example, the tail-end of the embryo accumulates an mRNA called oskar. If this mRNA is missing, the embryo will not develop an abdomen. The build-up of oskar mRNA happens before the egg is even fertilized and depends on two types of scaffold proteins in the egg cell called microtubules and microfilaments. These scaffolds act like ‘train tracks’ in the cell and have associated protein motors, which work a bit like trains, carrying cargo as they travel up and down along the scaffolds. For microtubules, one of the motors is a protein called kinesin-1, whereas for microfilaments, the motors are called myosins. Most microtubules in the egg cell are pointing away from the membrane, while microfilament tracks form a dense network of randomly oriented filaments just underneath the membrane. It was already known that kinesin-1 and a myosin called myosin-V are important for localizing oskar mRNA to the posterior of the egg. However, it was not clear why the mRNA only builds up in that area. To find out, Lu et al. used a probe to track oskar mRNA, while genetically manipulating each of the motors so that their ability to transport cargo changed. Modulating the balance of activity between the two motors revealed that kinesin-1 and myosin-V engage in a tug-of-war inside the egg: myosin-V tries to keep oskar mRNA underneath the membrane of the cell, while kinesin-1 tries to pull it away from the membrane along microtubules. The winner of this molecular battle depends on the number of microtubule tracks available in the local area of the cell. In most parts of the cell, there are abundant microtubules, so kinesin-1 wins and pulls oskar mRNA away from the membrane. But at the posterior end of the cell there are fewer microtubules, so myosin-V wins, allowing oskar mRNA to localize in this area. Artificially 'shaving' some microtubules in a local area immediately changed the outcome of this tug-of-war creating a build-up of oskar mRNA in the 'shaved' patch. This is the first time a molecular tug-of-war has been shown in an egg cell, but in other types of cell, such as neurons and pigment cells, myosins compete with kinesins to position other molecular cargoes. Understanding these processes more clearly sheds light not only on embryo development, but also on cell biology in general.

Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events., Cellular contractility is regulated by the GTPase RhoA, but how local signals are translated to a cell-level response is not known. Here the authors show that targeted RhoA activation results in propagation of force along stress fibres and actin flow, and identify zyxin as a regulator of stress fibre mechanics and homeostasis.

Local accumulation ofoskar (osk) mRNA in theDrosophilaoocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential foroskmRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes withoskmRNA, as a proxy for posterior determination. We demonstrate that posterior localization ofosk/Staufen is determined by competition between kinesin-1 and myosin-V. While kinesin-1 removesosk/Staufen from the cortex along microtubules, myosin-V anchorsosk/Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins it at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination ofDrosophilaoocyte is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density.

Using optogenetics to obtain spatiotemporal control of RhoA activation, Wagner and Glotzer show that local RhoA activation is sufficient to induce cytokinetic furrowing. Furrows can be induced at both the cell equator and cell poles during various stages of mitosis, and cell rounding allows active RhoA to induce furrows in interphase., The GTPase RhoA promotes contractile ring assembly and furrow ingression during cytokinesis. Although many factors that regulate RhoA during cytokinesis have been characterized, the spatiotemporal regulatory logic remains undefined. We have developed an optogenetic probe to gain tight spatial and temporal control of RhoA activity in mammalian cells and demonstrate that cytokinetic furrowing is primarily regulated at the level of RhoA activation. Light-mediated recruitment of a RhoGEF domain to the plasma membrane leads to rapid induction of RhoA activity, leading to assembly of cytokinetic furrows that partially ingress. Furthermore, furrow formation in response to RhoA activation is not temporally or spatially restricted. RhoA activation is sufficient to generate furrows at both the cell equator and cell poles, in both metaphase and anaphase. Remarkably, furrow formation can be initiated in rounded interphase cells, but not adherent cells. These results indicate that RhoA activation is sufficient to induce assembly of functional contractile rings and that cell rounding facilitates furrow formation.

Adriaans et al. provide evidence for the existence of two molecular pathways that can initiate cytokinesis in human cells: one depending on PLK1 and originating at the spindle midzone and the other depending on Aurora B activity at the equatorial cortex., Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin at the equatorial cortex and can operate independently of PLK1. We further show that PLK1 inhibition sequesters centralspindlin onto the spindle midzone, making it unavailable for Aurora B at the equatorial cortex. We propose that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex.

The active form of the small GTPase RhoA is necessary and sufficient for formation of a cytokinetic furrow in animal cells. Despite the conceptual simplicity of the process, the molecular mechanisms that control it are intricate and involve redundancy at multiple levels. Here, we discuss our current knowledge of the mechanisms underlying spatiotemporal regulation of RhoA during cytokinesis by upstream activators. The direct upstream activator, the RhoGEF Ect2, requires activation due to autoinhibition. Ect2 is primarily activated by the centralspindlin complex, which contains numerous domains that regulate its subcellular localization, oligomeric state, and Ect2 activation. We review the functions of these domains and how centralspindlin is regulated to ensure correctly timed, equatorial RhoA activation. Highlighting recent evidence, we propose that although centralspindlin does not always prominently accumulate on the plasma membrane, it is the site where it promotes RhoA activation during cytokinesis.

SUMMARYCell division-cytokinesis-involves large-scale rearrangements of the entire cell. Primarily driven by cytoskeletal proteins, cytokinesis also depends on topological rearrangements of the plasma membrane, which are coordinated with nuclear division in both space and time. Despite the fundamental nature of the process, different types of eukaryotic cells show variations in both the structural mechanisms of cytokinesis and the regulatory controls. In animal cells and fungi, a contractile actomyosin-based structure plays a central, albeit flexible, role. Here, the underlying molecular mechanisms are summarized and integrated and common themes are highlighted.

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment. DOI: http://dx.doi.org/10.7554/eLife.26722.001, eLife digest Living cells are not always symmetrical. Instead they are often polarized, with a distinct front and back or top and bottom. Cell polarization influences many processes, including how a cell moves and grows, and where it will divide. Breaking symmetry – in other words, making one part of a cell different from the rest – regularly involves a small protein called Cdc42, which can switch between an active and inactive form. This protein is found in a range of organisms from fungi to animals. Budding yeast is a valuable model to study cell polarization. This single-celled fungus polarizes in order to produce a daughter cell or ‘bud’ that emerges out of one end of the mother cell. To become polarized, the mother cell accumulates active Cdc42 in a small area of the cell membrane. This region then becomes the front of the cell, from where the future bud will emerge. However, it is not fully understood how active Cdc42 accumulates at only one place. One model proposed that some molecules of active Cdc42 that are already present on the membrane, recruit polarity proteins that in turn activate other, inactive Cdc42 molecules. This self-amplifying loop could eventually build up a local pool of active Cdc42. However, it has proved challenging to directly test this model. Optogenetics is a technique in which a beam of light is used to manipulate proteins inside cells in a precise manner. The method was first developed in the field of neuroscience over a decade ago, and has more recently been applied to cell biology. Now, Witte et al. have used optogenetics to move polarity proteins to defined places on the membrane of yeast cells and analyse how this change affected the pattern of Cdc42 activation. The experiments showed that polarity proteins not only activate Cdc42 but they also recruit more polarity proteins to the same place. The resulting positive feedback loop leads to active Cdc42 accumulating at one site on the membrane. Further work showed that this mechanism only operates in this manner just before the mother cell replicates its DNA, which is when a yeast cell will normally polarize. These results provide a new perspective on how cells can make one part of the cell different from the rest. Beyond yeast, cell polarization plays a major role when animals, including humans, are developing as embryos or healing wounds. These processes are often controlled by a protein that is equivalent to Cdc42 or by other highly related switch-like proteins. This means that yeast will continue to provide a useful model to study these processes in the laboratory. Lastly, the optogenetics approach developed Witte et al. will be useful to dissect other processes that involve molecules being located at specific place in a cell at specific time. DOI: http://dx.doi.org/10.7554/eLife.26722.002

A Caenorhabditis elegans mutant has been identified in which an ectopic myosin cap shifts the cleavage furrow relative to the spindle center. Surprisingly, the molecules that suppress this cap in wild-type embryos generate a cap in other asymmetrically dividing cells.

Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin. Background: Cytokinesis requires formation of the centralspindlin complex. Results: The neck linker regions in ZEN-4 are highly mobile when free, but their mobility is greatly restricted by CYK-4 binding. Conclusion: Central spindle assembly requires a binding event that modulates the structure of the kinesin ZEN-4. Significance: Structural changes in the centralspindlin complex contribute to cell division.

RhoA and the Rho guanine nucleotide exchange factor ECT-2 are involved in both polarization and cytokinesis. During cytokinesis, interactions of ECT-2 with the Rho GTPase-activating protein CYK-4 promote RhoA activation. A novel protein, NOP-1, acts in parallel with CYK-4 to promote RhoA activation during polarization and cytokinesis., The GTPase RhoA is a central regulator of cellular contractility in a wide variety of biological processes. During these events, RhoA is activated by guanine nucleotide exchange factors (GEFs). These molecules are highly regulated to ensure that RhoA activation occurs at the proper time and place. During cytokinesis, RhoA is activated by the RhoGEF ECT-2. In human cells, ECT-2 activity requires its association with CYK-4, which is a component of the centralspindlin complex. In contrast, in early Caenorhabditis elegans embryos, not all ECT-2–dependent functions require CYK-4. In this study, we identify a novel protein, NOP-1, that functions in parallel with CYK-4 to promote RhoA activation. We use mutations in nop-1 and cyk-4 to dissect cytokinesis and cell polarization. NOP-1 makes a significant, albeit largely redundant, contribution to cytokinesis. In contrast, NOP-1 is required for the preponderance of RhoA activation during the establishment phase of polarization.

The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the centralspindlin complex which is composed of two proteins, a kinesin-like protein, Mitotic kinesin-like protein 1, and a Rho GTPase activating protein (RhoGAP), CYK-4. Through its targeted localization to a narrow region of antiparallel microtubule overlap immediately following chromosome segregation, centralspindlin initiates central spindle assembly. Centralspindlin has several critical functions during cell division including positioning of the division plane, regulation of Rho family GTPases, as well as midbody assembly and abscission. In this review, we will examine the biochemistry of centralspindlin and its multiple functions during cell division. Remarkably, several of its critical functions are somewhat unexpected. Although endowed with motor domains, centralspindlin has an important role in generating stable, antiparallel microtubule bundles. Although it contains a Rho family GAP domain, it has a central role in the activation of RhoA during cytokinesis. Finally, centralspindlin functions as a motor protein complex, as a scaffold protein for key regulators of abscission and as a conventional RhoGAP. Because of these diverse functions, centralspindlin lies at the heart of the cytokinetic mechanism.

Light-inducible dimerization tags are engineered to rapidly recruit proteins to precise points in living yeast and mammalian cells. The affinities and response time of the interactions are tunable, and the authors used the system to activate cell signaling and to direct cell polarization in yeast. Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Summary Cytokinesis in animal cells is mediated by a cortical actomyosin-based contractile ring. The GTPase RhoA is a critical regulator of this process as it activates both nonmuscle myosin and a nucleator of actin filaments [1]. The site at which active RhoA and its effectors accumulate is controlled by the microtubule-based spindle during anaphase [2]. ECT-2, the guanine nucleotide exchange factor (GEF) that activates RhoA during cytokinesis, is regulated by phosphorylation and subcellular localization [3–5]. ECT2 localization depends on interactions with CYK-4/MgcRacGAP, a Rho GTPase-activating protein (GAP) domain containing protein [5, 6]. Here we show that, contrary to expectations, the Rho GTPase-activating protein (GAP) domain of CYK-4 promotes activation of RhoA during cytokinesis. Furthermore, we show that the primary phenotype caused by mutations in the GAP domain of CYK-4 is not caused by ectopic activation of CED-10/Rac1 and ARX-2/Arp2. However, inhibition of CED-10/Rac1 and ARX-2/Arp2 facilitates ingression of weak cleavage furrows. These results demonstrate that a GAP domain can contribute to activation of a small GTPase. Furthermore, cleavage furrow ingression is sensitive to the balance of contractile forces and cortical tension.

Cytokinesis is controlled by the central spindle and astral microtubules. In this paper, we show that the scaffold protein anillin contributes to aster-directed furrowing. We also demonstrate that anillin can associate with microtubules and that microtubule density correlates with the functional inhibition of anillin., Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.

Modulation of the microtubule and the actin cytoskeleton is crucial for proper cell division. Protein phosphorylation is known to be an important regulatory mechanism modulating these cytoskeletal networks. By contrast, there is a relative paucity of information regarding how protein phosphatases contribute to such modulation. Here, we characterize the requirements for protein phosphatase PPH-6 and its associated subunit SAPS-1 in one-cell stage C. elegans embryos. We establish that the complex of PPH-6 and SAPS-1 (PPH-6/SAPS-1) is required for contractility of the actomyosin network and proper spindle positioning. Our analysis demonstrates that PPH-6/SAPS-1 regulates the organization of cortical non-muscle myosin II (NMY-2). Accordingly, we uncover that PPH-6/SAPS-1 contributes to cytokinesis by stimulating actomyosin contractility. Furthermore, we demonstrate that PPH-6/SAPS-1 is required for the proper generation of pulling forces on spindle poles during anaphase. Our results indicate that this requirement is distinct from the role in organizing the cortical actomyosin network. Instead, we uncover that PPH-6/SAPS-1 contributes to the cortical localization of two positive regulators of pulling forces, GPR-1/2 and LIN-5. Our findings provide the first insights into the role of a member of the PP6 family of phosphatases in metazoan development.

Summary Cytokinesis in animal cells requires the central spindle and midbody, which contain prominent microtubule bundles [1]. Centralspindlin, a heterotetrameric complex consisting of kinesin-6 and RhoGAP (Rho-family GTPase-activating protein) subunits, is essential for the formation of these structures [2]. Centralspindlin becomes precisely localized to the central spindle, where it promotes the equatorial recruitment of important cytokinetic regulators. These include ECT2, the activator of the small GTPase RhoA, which controls cleavage furrow formation and ingression [3–6]. Centralspindlin's own RhoGAP domain also contributes to furrow ingression [7–10]. Finally, centralspindlin facilitates recruitment of the chromosome passenger complex [7, 8] and factors that control abscission [11, 12]. Despite the importance of localized accumulation of centralspindlin, the mechanism by which this motor protein complex suddenly concentrates to the center of interpolar microtubule bundles during anaphase is unclear. Here, we show that centralspindlin travels along central spindle microtubules as higher-order clusters. Clustering of centralspindlin is critical for microtubule bundling and motility along microtubules in vitro and for midbody formation in vivo. These data support a positive feedback loop of centralspindlin clustering and microtubule organization that may underlie its distinctive localization during cytokinesis.

Proper spatial and temporal regulation of the small GTPase RhoA at the equatorial cortex represents a critical step in the specification of the division plane in eukaryotes. Despite increased understanding of the mechanisms whereby RhoA becomes active following chromosome segregation, far less is known about how RhoA is spatially regulated so that it concentrates precisely at the division site. In the April 1, 2009, issue of Genes & Development, Yoshida and colleagues (pp. 810–823) uncovered two genetically separable mechanisms whereby Rho1 is recruited to the bud neck in the budding yeast Saccharomyces cerevisiae to facilitate cytokinesis.

Cleavage furrow formation in animal cells results from a local increase in cortical contractility. During anaphase, the spindle contains, in addition to astral arrays of microtubules, a set of bundled microtubules known as the central spindle. Each of these populations of microtubules, the astral arrays and the central spindle bundles, is sufficient to direct cleavage furrow formation, yet in wild-type situations these sets of microtubules co-operate to induce furrow formation at the same site, between the segregating chromosomes. These pathways have distinct genetic requirements that reflect their differential control of cortical actomyosin. We review our current understanding of the molecular mechanisms of furrow formation, with particular emphasis on the central spindle-independent pathway.

Summary Background After anaphase, the segregated chromosomes are sequestered by cytokinesis into two separate daughter cells by a cleavage furrow formed by the actomyosin-based contractile ring. The failure to properly position the contractile ring between the segregated chromosomes can result in aneuploidy. In both C. elegans embryos and human cells, the central spindle regulates division-plane positioning in parallel with a second pathway that involves astral microtubules. Results We combined genetic and pharmacological manipulations with live cell imaging to spatially separate the two division cues in a single cell. We demonstrate that the two pathways for furrow formation are mechanistically and genetically distinct. By following the distribution of green fluorescent protein (GFP)-tagged nonmuscle myosin, we have found that the astral pathway for furrow formation involves the negative regulation of cortical myosin recruitment. An asymmetrically positioned spindle induces the asymmetric cortical accumulation of myosin. This cortical myosin behaves as a coherent contractile network. If the cortical network is nonuniform over the cell, the cortical contractile elements coalesce into a single furrow. This coalescence requires interconnections among contractile elements. Conclusions We conclude that the two pathways of cleavage-furrow formation are mechanistically distinct. In particular, we conclude that the astral pathway for cleavage-furrow formation involves the negative regulation of myosin distribution by astral cues.

SummaryCytokinesis of animal cells requires ingression of the actomyosin-based contractile ring between segregated sister genomes. Localization of the RhoGEF Ect2 to the central spindle at anaphase promotes local activation of the RhoA GTPase, which induces assembly and ingression of the contractile ring. Here we have used BI 2536, an inhibitor of the mitotic kinase Plk1, to analyze the functions of this enzyme during late mitosis in human cells. We show that Plk1 acts after Cdk1 inactivation and independently from Aurora B to promote RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. Inhibition of Plk1 abolishes the interaction of Ect2 with its activator and midzone anchor, HsCyk-4, thereby preventing localization of Ect2 to the central spindle. We propose that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells.

The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times.

SummaryThe central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.

After anaphase onset, animal cells build an actomyosin contractile ring that constricts the plasma membrane to generate two daughter cells connected by a cytoplasmic bridge. The bridge is ultimately severed to complete cytokinesis. Myriad techniques have been used to identify proteins that participate in cytokinesis in vertebrates, insects, and nematodes. A conserved core of about 20 proteins are individually involved with cytokinesis in most animal cells. These components are found in the contractile ring, on the central spindle, within the RhoA pathway, and on vesicles that expand the membrane and sever the bridge. Cytokinesis involves additional proteins, but they, or their requirement in cytokinesis, are not conserved among animal cells.

SummaryThe hardest working complex in animal cell division has a new gig. This extraordinary machine, the centralspindlin complex, works overtime, contributing to nearly every step in cytokinesis. It has now been shown to stabilize an association between the plasma membrane and the midbody microtubules prior to abscission.

To complete the cell cycle, the cleavage furrow draws the plasma membrane toward the cell center, pinching the cytoplasm into two lobes that are subsequently separated into two cells. The position of the cleavage furrow is induced by the mitotic spindle during early anaphase. Although the mechanism of cleavage furrow positioning is not understood at a molecular level, recent results suggest that it might be mediated by local relief from the inhibitory effects of microtubules.

Cell multiplication requires sequestration of the duplicated and segregated genome into two daughter cells. The mitotic spindle is critical for orchestrating sister chromatid separation and division plane positioning. During anaphase, spindle microtubules become bundled to form the central spindle, which is essential for completion of cytokinesis. Central spindle assembly is mediated by a microtubule-associated protein and a kinesin-RhoGAP complex, both of which are regulated by phosphorylation/dephosphorylation. The central spindle also plays a role in cleavage furrow positioning, which appears to involve activation of RhoA. New results have provided some initial clues as to how furrow positioning is achieved. Particularly notable is the discovery that a protein activated by RhoA, formin, has actin nucleation activity.

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

The mitotic spindle provides the spatial cue that coordinates cytokinesis with nuclear division. However, the specific property of the mitotic spindle that mediates this spatial regulation remains obscure, in part because different aspects of the mitotic spindle appear to have furrow inducing activity in different systems. We show that in C. elegans embryos, although the central spindle is usually dispensable for furrow initiation, it becomes essential for furrow formation when the extent of centrosome separation during anaphase is reduced. Measurements of microtubule density demonstrate that furrow formation occurs in the vicinity of a local minimum of microtubule density. Reduction of the extent of spindle elongation or disruption of the central spindle causes delayed formation of the cleavage furrow. These data suggest that reduced microtubule density triggers cleavage furrow initiation and demonstrate that redundant mechanisms direct efficient formation of the cleavage furrow.

We describe a superfamily of eukaryotic and prokaryotic proteins (kleisins) that includes ScpA, Scc1, Rec8, and Barren. Scc1 interacts with SMC proteins through N- and C-terminal domains to form a ring-like structure. Since these are the only domains conserved among kleisins, we suggest that ring formation with SMC proteins may define this family.

In all eukaryotic organisms, the physical separation of two nascent cells must be coordinated with chromosome segregation and mitotic exit. In Saccharomyces cerevisiae and Schizosaccharomyces pombe this coordination depends on a number of genes that cooperate in intricate regulatory pathways termed mitotic exit network and septum initiation network, respectively. Here we have explored the function of potentially homologous genes in a metazoan organism, Caenorhabditis elegans, using RNA-mediated interference. Of all the genes tested, only depletion of CeCDC-14, the C. elegans homologue of the budding yeast dual-specificity phosphatase Cdc14p (Clp1/Flp1p in fission yeast), caused embryonic lethality. We show that CeCDC-14 is required for cytokinesis but may be dispensable for progression of the early embryonic cell cycles. In response to depletion of CeCDC-14, embryos fail to establish a central spindle, and several proteins normally found at this structure are mislocalized. CeCDC-14 itself localizes to the central spindle in anaphase and to the midbody in telophase. It colocalizes with the mitotic kinesin ZEN-4, and the two proteins depend on each other for correct localization. These findings identify the CDC14 phosphatase as an important regulator of central spindle formation and cytokinesis in a metazoan organism.