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We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

The Rho GTPases - RHOA, RAC1 and CDC42 - are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function., (© 2024. Springer Nature Limited.)

The cell cortex plays many critical roles, including interpreting and responding to internal and external signals. One behavior which supports a cell's ability to respond to both internal and externally-derived signaling is cortical excitability, wherein coupled positive and negative feedback loops generate waves of actin polymerization and depolymerization at the cortex. Cortical excitability is a highly conserved behavior, having been demonstrated in many cell types and organisms. One system well-suited to studying cortical excitability is Xenopus laevis , in which cortical excitability is easily monitored for many hours after fertilization. Indeed, recent investigations using X. laevis have furthered our understanding of the circuitry underlying cortical excitability and how it contributes to cytokinesis. Here, we describe the impact of wounding, which represents both a chemical and a physical signal, on cortical excitability. In early embryos (zygotes to early blastulae), we find that wounding results in a transient cessation ("freezing") of wave propagation followed by transport of frozen waves toward the wound site. We also find that wounding near cell-cell junctions results in the formation of an F-actin (actin filament)-based structure that pulls the junction toward the wound; at least part of this structure is based on frozen waves. In later embryos (late blastulae to gastrulae), we find that cortical excitability diminishes and is progressively replaced by epithelial excitability, a process in which wounded cells communicate with other cells via wave-like increases of calcium and apical F-actin. While the F-actin waves closely follow the calcium waves in space and time, under some conditions the actin wave can be uncoupled from the calcium wave, suggesting that they may be independently regulated by a common upstream signal. We conclude that as cortical excitability disappears from the level of the individual cell within the embryo, it is replaced by excitability at the level of the embryonic epithelium itself., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sepaniac, Davenport and Bement.)

The extent to which calcium signaling participates in specific events of animal cell meiosis or mitosis is a subject of enduring controversy. We have previously demonstrated that buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, a fast calcium chelator), but not ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA, a slow calcium chelator), rapidly depolymerizes spindle microtubules in Xenopus oocytes, suggesting that spindle assembly and/or stability requires calcium nanodomains-calcium transients at extremely restricted spatial-temporal scales. In this study, we have investigated the function of inositol-1,4,5-trisphosphate receptor (IP 3 R), an endoplasmic reticulum (ER) calcium channel, in spindle assembly using Trim21-mediated depletion of IP 3 R. Oocytes depleted of IP 3 R underwent germinal vesicle breakdown but failed to emit the first polar body and failed to assemble proper meiotic spindles. Further, we developed a cell-free spindle assembly assay in which cytoplasm was aspirated from single oocytes. Spindles assembled in this cell-free system were encased in ER membranes, with IP 3 R enriched at the poles, while disruption of either ER organization or calcium signaling resulted in rapid spindle disassembly. As in intact oocytes, formation of spindles in cell-free oocyte extracts also required IP 3 R. We conclude that intracellular calcium signaling involving IP 3 R-mediated calcium release is required for meiotic spindle assembly in Xenopus oocytes.

Polar body emission is a special form of cytokinesis in oocyte meiosis that ensures the correct number of chromosomes in reproduction-competent eggs. The molecular mechanism of the last step, polar body abscission, is poorly understood. While it has been proposed that Ca 2+ signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca 2+ have been difficult to document in oocyte meiosis except for the global Ca 2+ wave induced by sperm at fertilization. Here, we find that microinjection of the calcium chelator dibromo-BAPTA inhibits polar body abscission in Xenopus laevis oocytes. Using a novel, microtubule-targeted ratio-metric calcium sensor, we detected a calcium transient that is focused at the contractile ring-associated plasma membrane and which occurred after anaphase and constriction of the contractile ring but prior to abscission. This calcium transient was confirmed by mobile calcium probes. Further, the Ca 2+ -sensitive protein kinase Cβ C2 domain transiently translocated to the contractile ring-associated membrane simultaneously with the calcium transient. Collectively, these results demonstrate that a calcium transient, apparently originating at the contractile ring-associated plasma membrane, promotes polar body abscission.

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics., (© 2022 Michaud et al.)

Interest in cortical excitability-the ability of the cell cortex to generate traveling waves of protein activity-has grown considerably over the past 20 years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in Xenopus laevis eggs and embryos and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties-such as wave frequency and amplitude-and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to nonfurrow cortical excitability and showed that these wave regimes are mutually exclusive.

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division. 1 , 2 During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed "cortical excitability." 3-7 In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho. 7 , 8 These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation. 9 In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics. 10 This reconstituted system spontaneously develops two distinct types of self-organized cortical dynamics: singular excitable Rho and F-actin waves, and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the excitable dynamics previously characterized in vivo. 7 These findings directly support the long-standing speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

As the interface between the cell and its environment, the cell cortex must be able to respond to a variety of external stimuli. This is made possible in part by cortical excitability, a behavior driven by coupled positive and negative feedback loops that generate propagating waves of actin assembly in the cell cortex. Cortical excitability is best known for promoting cell protrusion and allowing the interpretation of and response to chemoattractant gradients in migrating cells. It has recently become apparent, however, that cortical excitability is involved in the response of the cortex to internal signals from the cell-cycle regulatory machinery and the spindle during cell division. Two overlapping functions have been ascribed to cortical excitability in cell division: control of cell division plane placement, and amplification of the activity of the small GTPase Rho at the equatorial cortex during cytokinesis. Here, we propose that cortical excitability explains several important yet poorly understood features of signaling during cell division. We also consider the potential advantages that arise from the use of cortical excitability as a signaling mechanism to regulate cortical dynamics in cell division., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Plasma membrane integrity is essential for cellular homeostasis. In vivo, cells experience plasma membrane damage from a multitude of stressors in the extra- and intra-cellular environment. To avoid lethal consequences, cells are equipped with repair pathways to restore membrane integrity. Here, we assess plasma membrane damage and repair from a whole-body perspective. We highlight the role of tissue-specific stressors in health and disease and examine membrane repair pathways across diverse cell types. Furthermore, we outline the impact of genetic and environmental factors on plasma membrane integrity and how these contribute to disease pathogenesis in different tissues.

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these methods, we identified pools of active and inactive RhoGTPase associated with the membrane, found that RhoGDI can extract both inactive and active RhoGTPases, and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds. These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane., Competing Interests: AG, IV, PB, WB No competing interests declared, (© 2019, Golding et al.)

Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, whether actin filaments (F-actin) are required for or are even present in mitotic spindles has long been controversial. Here we have developed improved methods for simultaneously preserving F-actin and microtubules in fixed samples and exploited them to demonstrate that F-actin is indeed associated with mitotic spindles in intact Xenopus laevis embryonic epithelia. We also find that there is an "F-actin cycle," in which the distribution and organization of spindle F-actin changes over the course of the cell cycle. Live imaging using a probe for F-actin reveals that at least two pools of F-actin are associated with mitotic spindles: a relatively stable internal network of cables that moves in concert with and appears to be linked to spindles, and F-actin "fingers" that rapidly extend from the cell cortex toward the spindle and make transient contact with the spindle poles. We conclude that there is a robust endoplasmic F-actin network in normal vertebrate epithelial cells and that this network is also a component of mitotic spindles. More broadly, we conclude that there is far more internal F-actin in epithelial cells than is commonly believed.

Colocalization analysis aims to study complex spatial associations between bio-molecules via optical imaging techniques. However, existing colocalization analysis workflows only assess an average degree of colocalization within a certain region of interest and ignore the unique and valuable spatial information offered by microscopy. In the current work, we introduce a new framework for colocalization analysis that allows us to quantify colocalization levels at each individual location and automatically identify pixels or regions where colocalization occurs. The framework, referred to as spatially adaptive colocalization analysis (SACA), integrates a pixel-wise local kernel model for colocalization quantification and a multi-scale adaptive propagation-separation strategy for utilizing spatial information to detect colocalization in a spatially adaptive fashion. Applications to simulated and real biological datasets demonstrate the practical merits of SACA in what we hope to be an easily applicable and robust colocalization analysis method. In addition, theoretical properties of SACA are investigated to provide rigorous statistical justification.

Anaphase in epithelia typically does not ensue until after spindles have achieved a characteristic position and orientation, but how or even if cells link spindle position to anaphase onset is unknown. Here, we show that myosin-10 (Myo10), a motor protein involved in epithelial spindle dynamics, binds to Wee1, a conserved regulator of cyclin-dependent kinase 1 (Cdk1). Wee1 inhibition accelerates progression through metaphase and disrupts normal spindle dynamics, whereas perturbing Myo10 function delays anaphase onset in a Wee1-dependent manner. Moreover, Myo10 perturbation increases Wee1-mediated inhibitory phosphorylation on Cdk1, which, unexpectedly, concentrates at cell-cell junctions. Based on these and other results, we propose a model in which the Myo10-Wee1 interaction coordinates attainment of spindle position and orientation with anaphase onset., (© 2018 Sandquist et al.)

Proper spindle positioning at anaphase onset is essential for normal tissue organization and function. Here we develop automated spindle-tracking software and apply it to characterize mitotic spindle dynamics in the Xenopus laevis embryonic epithelium. We find that metaphase spindles first undergo a sustained rotation that brings them on-axis with their final orientation. This sustained rotation is followed by a set of striking stereotyped rotational oscillations that bring the spindle into near contact with the cortex and then move it rapidly away from the cortex. These oscillations begin to subside soon before anaphase onset. Metrics extracted from the automatically tracked spindles indicate that final spindle position is determined largely by cell morphology and that spindles consistently center themselves in the XY -plane before anaphase onset. Finally, analysis of the relationship between spindle oscillations and spindle position relative to the cortex reveals an association between cortical contact and anaphase onset. We conclude that metaphase spindles in epithelia engage in a stereotyped "dance," that this dance culminates in proper spindle positioning and orientation, and that completion of the dance is linked to anaphase onset., (© 2017 Larson and Bement. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Plasma membrane damage elicits a complex and dynamic cellular response. A vital component of this response, membrane resealing, is thought to arise from fusion of intracellular membranous compartments to form a temporary, impermeant patch at the site of damage; however, this hypothesis has been difficult to confirm visually. By utilizing advanced microscopy technologies with high spatiotemporal resolution in wounded Xenopus laevis oocytes, we provide the first direct visualization of the membrane fusion events predicted by the patch hypothesis; we show the barrier formed by patching is capable of abating exchange of material across the plasma membrane within seconds. Profound changes also occur to the plasma membrane surrounding wounds; lipid remodeling is accompanied by membrane fusion events, both conventional (e.g., exocytosis) and novel (e.g., "explodosis"). Further, we reveal additional complexity in wound-induced subcellular patterning, supporting existing evidence that extensive interactions between lipid, protein, and ionic signaling pathways shape the cellular wound response.

Rho GTPases are regulatory proteins whose patterns on the surface of a cell affect cell polarization, cell motility and repair of single-cell wounds. The stereotypical patterns formed by two such proteins, Rho and Cdc42, around laser-injured frog oocytes permit experimental analysis of GTPase activation, inactivation, segregation and crosstalk. Here, we review the development and analysis of a spatial model of GTPase dynamics that describe the formation of concentric zones of Rho and Cdc42 activity around wounds, and describe how this model has provided insights into the roles of the GTPase effector molecules protein kinase C (PKCβ and PKCη) and guanosine nucleotide dissociation inhibitor (GDI) in the wound response. We further demonstrate how the use of a 'sharp switch' model approximation in combination with bifurcation analysis can aid mapping the model behaviour in parameter space (approximate results confirmed with numerical simulation methods). Using these methods in combination with experimental manipulation of PKC activity (PKC overexpression (OE) and dominant negative conditions), we have shown that: (i) PKCβ most probably acts by enhancing existing positive feedbacks (from Rho to itself via the guanosine nucleotide exchange factor domain of Abr, and from Cdc42 to itself), (ii) PKCη most probably increases basal rates of inactivation (or possibly decreases basal rates of activation) of Rho and Cdc42, and (iii) the graded distribution of PKCη and its effect on initial Rho activity accounts for inversion of zones in a fraction (20%) of PKCη OE cells. Finally, we speculate that GDIs (which sequester GTPases) may have a critical role in defining the spatial domain, where the wound response may occur. This paper provides a more thorough exposition of the methods of analysis used in the investigation, whereas previous work on this topic was addressed to biologists and abbreviated such discussion.

Cells rapidly reseal after damage, but how they do so is unknown. It has been hypothesized that resealing occurs due to formation of a patch derived from rapid fusion of intracellular compartments at the wound site. However, patching has never been directly visualized. Here we study membrane dynamics in wounded Xenopus laevis oocytes at high spatiotemporal resolution. Consistent with the patch hypothesis, we find that damage triggers rampant fusion of intracellular compartments, generating a barrier that limits influx of extracellular dextrans. Patch formation is accompanied by compound exocytosis, local accumulation and aggregation of vesicles, and rupture of compartments facing the external environment. Subcellular patterning is evident as annexin A1, dysferlin, diacylglycerol, active Rho, and active Cdc42 are recruited to compartments confined to different regions around the wound. We also find that a ring of elevated intracellular calcium overlaps the region where membrane dynamics are most evident and persists for several minutes. The results provide the first direct visualization of membrane patching during membrane repair, reveal novel features of the repair process, and show that a remarkable degree of spatial patterning accompanies damage-induced membrane dynamics., (© 2016 Davenport et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Emergence of the cytokinetic Rho zone that orchestrates formation and ingression of the cleavage furrow had been explained previously via microtubule-dependent cortical concentration of Ect2, a guanine nucleotide exchange factor for Rho. The results of a recent publication now demonstrate that, en route from resting cortex to fully established furrow, there lies a regime of cortical excitability in which Rho activity and F-actin play the roles of the prototypical activator and inhibitor, respectively. This cortical excitability is manifest as dramatic traveling waves on the cortex of oocytes and embryos of frogs and starfish. These waves are initiated by autocatalytic activation of Rho at the wave front and extinguished by F-actin-dependent inhibition at their back. It is still unclear how propagating excitable Rho-actin waves give rise to the stable co-existence of Rho activity and F-actin density in the static cleavage furrow during cytokinesis. It is possible that some central spindle-associated signaling molecule simply turns off the inhibition of Rho activity by F-actin. However, mathematical modeling suggests a distinct scenario in which local "re-wiring" of the Rho-actin coupling in the furrow is no longer necessary. Instead, the model predicts that the continuously rising level of Ect2 produces in the furrow a qualitatively new stable steady state that replaces excitability and brings about the stable co-existence of high Rho activity and dense F-actin despite the continuing inhibition of Rho by F-actin.

Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, although Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modelling results show that waves represent excitable dynamics of a reaction-diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation.

Cell repair is attracting increasing attention due to its conservation, its importance to health, and its utility as a model for cell signaling and cell polarization. However, some of the most fundamental questions concerning cell repair have yet to be answered. Here we consider three such questions: (1) How are wound holes stopped? (2) How is cell regeneration achieved after wounding? (3) How is calcium inrush linked to wound stoppage and cell regeneration?, (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US.

Cytokinesis in animal cells depends on spindle-derived spatial cues that culminate in Rho activation, and thereby actomyosin assembly, in a narrow equatorial band. Although the nature, origin, and variety of such cues have long been obscure, one component is certainly the Rho activator Ect2. Here we describe the behavior and function of Ect2 in echinoderm embryos, showing that Ect2 migrates from spindle midzone to astral microtubules in anaphase and that Ect2 shapes the pattern of Rho activation in incipient furrows. Our key finding is that Ect2 and its binding partner Cyk4 accumulate not only at normal furrows, but also at furrows that form in the absence of associated spindle, midzone, or chromosomes. In all these cases, the cell assembles essentially the same cytokinetic signaling ensemble—opposed astral microtubules decorated with Ect2 and Cyk4. We conclude that if multiple signals contribute to furrow induction in echinoderm embryos, they likely converge on the same signaling ensemble on an analogous cytoskeletal scaffold., (© 2014 Su et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

After damage, cells reseal their plasma membrane and repair the underlying cortical cytoskeleton. Although many different proteins have been implicated in cell repair, the potential role of specific lipids has not been explored. Here we report that cell damage elicits rapid formation of spatially organized lipid domains around the damage site, with different lipids concentrated in different domains as a result of both de novo synthesis and transport. One of these lipids-diacylglycerol (DAG)-rapidly accumulates in a broad domain that overlaps the zones of active Rho and Cdc42, GTPases that regulate repair of the cortical cytoskeleton. Formation of the DAG domain is required for Cdc42 and Rho activation and healing. Two DAG targets, protein kinase C (PKC) β and η, are recruited to cell wounds and play mutually antagonistic roles in the healing process: PKCβ participates in Rho and Cdc42 activation, whereas PKCη inhibits Rho and Cdc42 activation. The results reveal an unexpected diversity in subcellular lipid domains and the importance of such domains for a basic cellular process., (© 2014 Vaughan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

A major goal of developmental biology is to explain the emergence of pattern in cell layers, tissues and organs. Developmental biologists now accept that reaction diffusion-based mechanisms are broadly employed in developing organisms to direct pattern formation. Here we briefly consider these mechanisms and then apply some of the concepts derived from them to several processes that occur in single cells: wound repair, yeast budding, and cytokinesis. Two conclusions emerge from this analysis: first, there is considerable overlap at the level of general mechanisms between developmental and single cell pattern formation; second, dynamic structures based on the actin cytoskeleton may be far more ordered than is generally recognized., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Rho GTPases-Rho, Rac, and Cdc42-control an enormous variety of processes, many of which reflect activation of these GTPases in spatially confined and mutually exclusive zones. By using mathematical models and experimental results to establish model parameters, we analyze the formation and segregation of Rho and Cdc42 zones during Xenopus oocyte wound repair and the role played by Abr, a dual guanine nucleotide exchange factor-GTPase-activating protein, in this process. The Rho and Cdc42 zones are found to be best represented as manifestations of spatially modulated bistability, and local positive feedback between Abr and Rho can account for the maintenance and dynamic properties of the Rho zone. In contrast, the invocation of an Abr-independent positive feedback loop is required to account for Cdc42 spatial bistability. In addition, the model replicates the results of previous in vivo experiments in which Abr activity is manipulated. Further, simulating the model with two closely spaced wounds made nonintuitive predictions about the Rho and Cdc42 patterns; these predictions were confirmed by experiment. We conclude that the model is a useful tool for analysis of Rho GTPase signaling and that the Rho GTPases can be fruitfully considered as components of intracellular pattern formation systems.

Microtubules control cell motility by positively regulating polarization in many cell types. However, how microtubules regulate leukocyte migration is not well understood, particularly in living organisms. Here we exploited the zebrafish system to study the role of microtubules in neutrophil migration in vivo. The localization of microtubules was visualized in motile neutrophils using various bioprobes, revealing that, in contrast to what has been seen in studies in vitro, the microtubule organizing center is positioned in front of the nucleus (relative to the direction of migration) in motile neutrophils. Microtubule disassembly impaired attraction of neutrophils to wounds but enhanced the polarity of F-actin dynamics as measured by the distribution of stable and dynamic F-actin. Microtubule depolymerization inhibited polarized phosphoinositol 3-kinase (PI(3)K) activation at the leading edge and induced rapid PI(3)K independent motility. Finally, we show that microtubules exert their effects on neutrophil polarity and motility at least in part by the negative regulation of both Rho and Rac activity. These results provide new insight into the role of microtubules in neutrophil migration in a living vertebrate and show that the motility of these professional migratory cells are subject to distinctly different rules from those established for other cell types.

Screening of small molecule libraries offers the potential to identify compounds that inhibit specific biological processes and, ultimately, to identify macromolecules that are important players in such processes. To date, however, most screens of small molecule libraries have focused on identification of compounds that inhibit known proteins or particular steps in a given process, and have emphasized automated primary screens. Here we have used "low tech" in vivo primary screens to identify small molecules that inhibit both cytokinesis and single cell wound repair, two complex cellular processes that possess many common features. The "diversity set", an ordered array of 1990 compounds available from the National Cancer Institute, was screened in parallel to identify compounds that inhibit cytokinesis in Dendraster excentricus (sand dollar) embryos and single cell wound repair in Xenopus laevis (frog) oocytes. Two small molecules were thus identified: Sph1 and Sph2. Sph1 reduces Rho activation in wound repair and suppresses formation of the spindle midzone during cytokinesis. Sph2 also reduces Rho activation in wound repair and may inhibit cytokinesis by blocking membrane fusion. The results identify two small molecules of interest for analysis of wound repair and cytokinesis, reveal that these processes are more similar than often realized and reveal the potential power of low tech screens of small molecule libraries for analysis of complex cellular processe., (Copyright © 2012 Wiley Periodicals, Inc.)

Video Abstract: Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying "signal treadmill" in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2-powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays, and proper functioning of these gradients is dependent on both contraction and F-actin turnover., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

The spindle directs chromosome partitioning in eukaryotes and, for the last three decades, has been considered primarily a structure based on microtubules, microtubule motors, and other microtubule binding proteins. However, a surprisingly large body of both old and new studies suggests roles for actin filaments (F-actin) and myosins (F-actin-based motor proteins) in spindle assembly and function. Here we review these data and conclude that in several cases the evidence for the participation of F-actin and myosins in spindle function is very strong, and in the situations where it is less strong, there is nevertheless enough evidence to warrant further investigation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The Rho GTPases-Rho, Rac, and Cdc42-regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single-cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown., Results: Using a candidate screen approach for Rho GTPase activators (guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase-activating proteins; GAPs), we find that Abr, a protein with both GEF and GAP activity, regulates Rho and Cdc42 during single-cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone, Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair., Conclusions: Abr is the first identified Rho GTPase regulator of single-cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The importance of wound healing to medicine and biology has long been evident, and consequently, wound healing has been the subject of intense investigation for many years. However, several relatively recent developments have added new impetus to wound repair research: the increasing application of model systems; the growing recognition that single cells have a robust, complex, and medically relevant wound healing response; and the emerging recognition that different modes of wound repair bear an uncanny resemblance to other basic biological processes such as morphogenesis and cytokinesis. In this review, each of these developments is described, and their significance for wound healing research is considered. In addition, overlapping mechanisms of single-cell and multicellular wound healing are highlighted, and it is argued that they are more similar than is often recognized. Based on this and other information, a simple model to explain the evolutionary relationships of cytokinesis, single-cell wound repair, multicellular wound repair, and developmental morphogenesis is proposed. Finally, a series of important, but as yet unanswered, questions is posed.

A bundle of microtubules known as the spindle midzone is rapidly assembled after anaphase onset, recruiting multiple signaling proteins that regulate cytokinesis. A new study reveals that positive feedback driven by clustering of a kinesin-6 motor underlies the explosive assembly of the spindle midzone.

Animal cells decide where to build the cytokinetic apparatus by sensing the position of the mitotic spindle. Reflecting a long-standing presumption that a furrow-inducing stimulus travels from spindle to cortex via microtubules, debate continues about which microtubules, and in what geometry, are essential for accurate cytokinesis. We used live imaging in urchin and frog embryos to evaluate the relationship between microtubule organization and cytokinetic furrow position. In normal cells, the cytokinetic apparatus forms in a region of lower cortical microtubule density. Remarkably, cells depleted of astral microtubules conduct accurate, complete cytokinesis. Conversely, in anucleate cells, asters alone can support furrow induction without a spindle, but only when sufficiently separated. Ablation of a single centrosome displaces furrows away from the remaining centrosome; ablation of both centrosomes causes broad, inefficient furrowing. We conclude that the asters confer accuracy and precision to a primary furrow-inducing signal that can reach the cell surface from the spindle without transport on microtubules.

Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing--Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds--occurs in single cells and in epithelia. Here, we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions and around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single-cell wound response, also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.

Unconventional myosins are proteins that bind actin filaments in an ATP-regulated manner. Because of their association with membranes, they have traditionally been viewed as motors that function primarily to transport membranous organelles along actin filaments. Recently, however, a wealth of roles for myosins that are not obviously related to organelle transport have been uncovered, including organization of F-actin, mitotic spindle regulation and gene transcription. Furthermore, it has also become apparent that the motor domains of different myosins vary strikingly in their biophysical attributes. We suggest that the assumption that most unconventional myosins function primarily as organelle transporters might be misguided.

In animal cells, cytokinesis is powered by a contractile ring of actin filaments (F-actin) and myosin-2. Formation of the contractile ring is dependent on the small GTPase RhoA, which is activated in a precise zone at the cell equator. It has long been assumed that cytokinesis and other Rho-dependent processes are controlled in a sequential manner, whereby Rho activation by guanine nucleotide exchange factors (GEFs) initiates a particular event, and Rho inactivation by GTPase activating proteins (GAPs) terminates that event. MgcRacGAP is a conserved cytokinesis regulator thought to be required only at the end of cytokinesis. Here we show that GAP activity of MgcRacGAP is necessary early during cytokinesis for the formation and maintenance of the Rho activity zone. Disruption of GAP activity by point mutation results in poorly focused Rho activity zones, whereas complete removal of the GAP domain results in unfocused zones that show lateral instability and/or rapid side-to-side oscillations. We propose that the GAP domain of MgcRacGAP has two unexpected roles throughout cytokinesis: first, it transiently anchors active Rho, and second, it promotes local Rho inactivation, resulting in the constant flux of Rho through the GTPase cycle.

Historically, much of our understanding of actin filaments, microtubules and intermediate filaments has come from the study of fixed cells and tissues. But the cytoskeleton is inherently dynamic, and so developing the means to image it in living cells has proved crucial. Advances in confocal microscopy and fluorescent protein technologies have allowed us to dynamically image the cytoskeleton at high resolution and so learn much more about its cellular functions. However, most of this work has been performed in cultured cells, and a critical next step is to understand how the cytoskeleton functions in the context of an intact organism. We, and others, have developed methods to image the cytoskeleton in living vertebrate embryos. Here, we describe an approach to image the cytoskeleton in embryos of the frog, Xenopus laevis, using mRNA to express fluorescently tagged cytoskeletal probes and confocal microscopy to visualize their dynamic behavior.

Vertebrate oocyte maturation is an extreme form of asymmetric cell division, producing a mature egg alongside a diminutive polar body. Critical to this process is the attachment of one spindle pole to the oocyte cortex prior to anaphase. We report here that asymmetric spindle pole attachment and anaphase initiation are required for localized cortical activation of Cdc42, which in turn defines the surface of the impending polar body. The Cdc42 activity zone overlaps with dynamic F-actin and is circumscribed by a RhoA-based actomyosin contractile ring. During cytokinesis, constriction of the RhoA contractile ring is accompanied by Cdc42-mediated membrane outpocketing such that one spindle pole and one set of chromosomes are pulled into the Cdc42 enclosure. Unexpectedly, the guanine nucleotide exchange factor Ect2, which is necessary for contractile ring formation, does not colocalize with active RhoA. Polar body emission thus requires a classical RhoA contractile ring and Cdc42-mediated membrane protrusion.

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.

Cytokinesis in animal cells is powered by the cytokinetic apparatus, a ring of filamentous actin and myosin-2 that underlies the plasma membrane and closes between the separating chromosomes. Formation of the cytokinetic apparatus is at least partially dependent on the small GTPase, Rho. Similar to other small GTPases, Rho cycles between the active (GTP-bound) and inactive (GDP-bound) states. Because of this switch-like behaviour, Rho and other members of the Rho GTPase family, such as Rac and Cdc42, have long been thought to work in a manner such that their activation and inactivation are not tightly coupled. That is, a given Rho-dependent event, such as cytokinesis, has been thought to be initiated by activation of Rho, and then, many minutes later, terminated by inactivation of Rho. Here we discuss evidence suggesting that in fact Rho undergoes rapid movement through the GTPase cycle throughout the entire process of cytokinesis, and that this cycling is necessary for proper cytokinetic apparatus function.