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A new analysis of cytokinetic furrow ingression in the Caenorhabditis elegans zygote at high spatiotemporal resolution demonstrates that, rather than being a process of steady, spatially uniform constriction, furrow ingression is modulated by complex contractile oscillations that move around the furrow, possibly in the form of propagating waves.
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Actomiosina , Caenorhabditis elegans , Animales , Caenorhabditis elegans/fisiología , Actomiosina/metabolismo , Citocinesis/fisiología , Cigoto/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
The Rho GTPases pattern the cell cortex in a variety of fundamental cell-morphogenetic processes including division, wound repair, and locomotion. It has recently become apparent that this patterning arises from the ability of the Rho GTPases to self-organize into static and migrating spots, contractile pulses, and propagating waves in cells from yeasts to mammals 1 . These self-organizing Rho GTPase patterns have been explained by a variety of theoretical models which require multiple interacting positive and negative feedback loops. However, it is often difficult, if not impossible, to discriminate between different models simply because the available experimental data do not simultaneously capture the dynamics of multiple molecular concentrations and biomechanical variables at fine spatial and temporal resolution. Specifically, most studies typically provide either the total Rho GTPase signal or the Rho GTPase activity as reported by various sensors, but not both. Therefore, it remains largely unknown how membrane accumulation of Rho GTPases (i.e., Rho membrane enrichment) is related to Rho activity. Here we dissect the dynamics of RhoA by simultaneously imaging both total RhoA and active RhoA in the regime of acute cortical excitability 2 , characterized by pronounced waves of Rho activity and F-actin polymerization 3-5 . We find that within nascent waves, accumulation of active RhoA precedes that of total RhoA, and we exploit this finding to distinguish between two popular theoretical models previously used to explain propagating cortical Rho waves.
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To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.
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Cinesinas , Microtúbulos , Cinesinas/metabolismo , Microtúbulos/metabolismo , Movimiento Celular , Citoesqueleto/metabolismoRESUMEN
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.
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Actinas , Citoesqueleto , Proteínas Activadoras de GTPasa , Proteínas Proto-Oncogénicas , Proteínas de Unión al GTP rho , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Citocinesis , Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Oocitos , Proteínas Proto-Oncogénicas/metabolismo , Xenopus , Proteínas de Unión al GTP rho/metabolismoRESUMEN
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.
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Excitabilidad Cortical , Animales , Ciclo Celular , División Celular , Citoplasma , Xenopus laevisRESUMEN
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.
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Actinas , Células Artificiales , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Citocinesis , Huso Acromático/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
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.
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Actinas , Citocinesis , Actinas/metabolismo , División Celular , Citoplasma/metabolismo , Transducción de SeñalRESUMEN
The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.
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Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Hongos/metabolismo , Hongos/fisiología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación/genética , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Unión Proteica/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Secretoras/metabolismo , Vesículas Secretoras/fisiologíaRESUMEN
Cellular morphogenesis is governed by the prepattern based on the symmetry-breaking emergence of dense protein clusters. Thus, a cluster of active GTPase Cdc42 marks the site of nascent bud in the baker's yeast. An important biological question is which mechanisms control the number of pattern maxima (spots) and, thus, the number of nascent cellular structures. Distinct flavors of theoretical models seem to suggest different predictions. While the classical Turing scenario leads to an array of stably coexisting multiple structures, mass-conserved models predict formation of a single spot that emerges via the greedy competition between the pattern maxima for the common molecular resources. Both the outcome and the kinetics of this competition are of significant biological importance but remained poorly explored. Recent theoretical analyses largely addressed these questions, but their results have not yet been fully appreciated by the broad biological community. Keeping mathematical apparatus and jargon to the minimum, we review the main conclusions of these analyses with their biological implications in mind. Focusing on the specific example of pattern formation by small GTPases, we speculate on the features of the patterning mechanisms that bypass competition and favor formation of multiple coexisting structures and contrast them with those of the mechanisms that harness competition to form unique cellular structures.
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Tipificación del Cuerpo/fisiología , Polaridad Celular/fisiología , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/metabolismo , Saccharomyces cerevisiae/metabolismo , Retroalimentación FisiológicaRESUMEN
Small GTPases are organizers of a plethora of cellular processes. The time and place of their activation are tightly controlled by the localization and activation of their regulators, guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Remarkably, in some systems, the upstream regulators of GTPases are also found downstream of their activity. Resulting feedback loops can generate complex spatiotemporal dynamics of GTPases with important functional consequences. Here we discuss the concept of positive autoregulation of small GTPases by the GEF-effector feedback modules and survey recent developments in this exciting area of cell biology.
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Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Retroalimentación , HumanosRESUMEN
Tight junctions contribute to epithelial barrier function by selectively regulating the quantity and type of molecules that cross the paracellular barrier. Experimental approaches to evaluate the effectiveness of tight junctions are typically global, tissue-scale measures. Here, we introduce Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which we used in Xenopus laevis embryos to visualize short-lived, local breaches in epithelial barrier function. These breaches, or leaks, occur as cell boundaries elongate, correspond to visible breaks in the tight junction, and are followed by transient localized Rho activation, or Rho flares. We discovered that Rho flares restore barrier function by driving concentration of tight junction proteins through actin polymerization and ROCK-mediated localized contraction of the cell boundary. We conclude that Rho flares constitute a damage control mechanism that reinstates barrier function when tight junctions become locally compromised because of normally occurring changes in cell shape and tissue tension.
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Uniones Adherentes/metabolismo , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Quinasas Asociadas a rho/metabolismo , Actinas/metabolismo , Animales , Células CACO-2/citología , Humanos , Fosfoproteínas/metabolismo , Uniones Estrechas/patología , Xenopus laevis/metabolismoRESUMEN
NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.
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Proteínas de Ciclo Celular/genética , Exocitosis/genética , Regulación Fúngica de la Expresión Génica , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Citocinesis/genética , Fosfoproteínas/clasificación , Fosfoproteínas/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure their faithful inheritance. How only a single procentriole is produced on each mother centriole remains enigmatic. We propose the first mechanistic biophysical model for procentriole initiation which posits that interactions between kinase PLK4 and its activator-substrate STIL are central for procentriole initiation. The model recapitulates the transition from a uniform "ring" of PLK4 surrounding the mother centriole to a single PLK4 "spot" that initiates procentriole assembly. This symmetry breaking requires autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4 by phosphorylated STIL. We find that in situ degradation of active PLK4 cannot break symmetry. The model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes destabilizes the PLK4 ring and produces instead a single PLK4 spot. Weakening of competition by overexpression of PLK4 and STIL causes progressive addition of supernumerary procentrioles, as observed experimentally.
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The conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeast Schizosaccharomyces pombe has two Cdc42 guanine nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, we show here that when scd1 function is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4 or Pom1 leads to disruption of polarized growth. Instead, cells adopt an isotropic-like pattern of growth, which we term PORTLI growth. Surprisingly, PORTLI growth is caused by spatially inappropriate activity of Gef1. Although most Cdc42 GEFs are membrane associated, we find that Gef1 is a broadly distributed cytosolic protein rather than a membrane-associated protein at cell tips like Scd1. Microtubules and the Tea1-Tea4-Pom1 axis counteract inappropriate Gef1 activity by regulating the localization of the Cdc42 GTPase-activating protein Rga4. Our results suggest a new model of fission yeast cell polarity regulation, involving coordination of 'local' (Scd1) and 'global' (Gef1) Cdc42 GEFs via microtubules and microtubule-dependent polarity landmarks.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Polaridad Celular , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Proteínas Quinasas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
A new study deploys optogenetics to induce the yeast bud on demand, at a site determined by a laser spot. The authors definitively prove that the initiation of cell polarization is driven by the Bem1-mediated positive feedback loop and reveal novel features of its regulation by the cell cycle.
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Proteínas de Saccharomyces cerevisiae , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales , Ciclo Celular , Polaridad Celular , Saccharomyces cerevisiae/citologíaRESUMEN
Mathematical modeling has been instrumental in identifying common principles of cell polarity across diverse systems. These principles include positive feedback loops that are required to destabilize a spatially uniform state of the cell. The conserved small G-protein Cdc42 is a master regulator of eukaryotic cellular polarization. Here we discuss recent developments in studies of Cdc42 polarization in budding and fission yeasts and demonstrate that models describing symmetry-breaking polarization can be classified into six minimal classes based on the structure of positive feedback loops that activate and localize Cdc42. Owing to their generic system-independent nature, these model classes are also likely to be relevant for the G-protein-based symmetry-breaking systems of higher eukaryotes. We review experimental evidence pro et contra different theoretically plausible models and conclude that several parallel and non-mutually exclusive mechanisms are likely involved in cellular polarization of yeasts. This potential redundancy needs to be taken into consideration when interpreting the results of recent cell-rewiring studies.
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Polaridad Celular/genética , Polaridad Celular/fisiología , Proteína de Unión al GTP cdc42/metabolismo , División Celular , Retroalimentación Fisiológica , Modelos Biológicos , Modelos Teóricos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteína de Unión al GTP cdc42/fisiologíaRESUMEN
The Rho family GTPase Cdc42 is a key regulator of eukaryotic cellular organization and cell polarity [1]. In the fission yeast Schizosaccharomyces pombe, active Cdc42 and associated effectors and regulators (the "Cdc42 polarity module") coordinate polarized growth at cell tips by controlling the actin cytoskeleton and exocytosis [2-4]. Localization of the Cdc42 polarity module to cell tips is thus critical for its function. Here we show that the fission yeast stress-activated protein kinase Sty1, a homolog of mammalian p38 MAP kinase, regulates localization of the Cdc42 polarity module. In wild-type cells, treatment with latrunculin A, a drug that leads to actin depolymerization, induces dispersal of the Cdc42 module from cell tips and cessation of polarized growth [5, 6]. We show that latrunculin A treatment also activates the Sty1 MAP kinase pathway and, strikingly, we find that loss of Sty1 MAP kinase signaling prevents latrunculin A-induced dispersal of the Cdc42 module, allowing polarized growth even in complete absence of the actin cytoskeleton. Regulation of the Cdc42 module by Sty1 is independent of Sty1's role in stress-induced gene expression. We also describe a system for activation of Sty1 kinase "on demand" in the absence of any external stress, and use this to show that Sty1 activation alone is sufficient to disperse the Cdc42 module from cell tips in otherwise unperturbed cells. During nitrogen-starvation-induced quiescence, inhibition of Sty1 converts non-growing, depolarized cells into growing, polarized cells. Our results place MAP kinase Sty1 as an important physiological regulator of the Cdc42 polarity module.
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Polaridad Celular , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/fisiología , Transducción de Señal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
A new study in fission yeasts promotes the notion that transient polarity patches that wander the cell surface at the onset of mating are discrete agents of gradient sensing. This concept unexpectedly bridges the modes of gradient sensing in eukaryotes and prokaryotes.
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Membrana Celular/fisiología , Saccharomyces cerevisiae , SchizosaccharomycesRESUMEN
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.
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Citocinesis , Actinas/metabolismo , Animales , Humanos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
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.