ABSTRACT
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.
Subject(s)
Cytoskeleton , rho GTP-Binding Proteins , rho GTP-Binding Proteins/metabolism , Cytoskeleton/metabolism , Actins/metabolism , Signal Transduction , Cell Movement , rac1 GTP-Binding Protein/metabolismABSTRACT
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.
Subject(s)
Extracellular Matrix/metabolism , Models, Biological , Wound Healing/physiology , Animals , Cell Membrane/metabolism , Cell Movement , Cell Proliferation , Cytoskeleton/metabolism , Embryo, Mammalian/physiology , Endocytosis/physiology , Hemostasis/physiology , Humans , Inflammation/metabolism , Membrane Fusion/physiology , Signal Transduction/physiologyABSTRACT
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.
Subject(s)
Cell Membrane , HomeostasisABSTRACT
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?
Subject(s)
Calcium Signaling , Wound Healing , Animals , Calcium/metabolism , Cell Membrane/physiology , Exocytosis , HumansABSTRACT
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.
Subject(s)
Microtubules/metabolism , Neutrophils/cytology , Neutrophils/metabolism , Zebrafish/metabolism , Animals , Cell Movement/physiology , Cell Polarity/physiology , Chemotaxis/physiologyABSTRACT
Cellular damage triggers rapid resealing of the plasma membrane and repair of the cortical cytoskeleton. Plasma membrane resealing results from calcium-dependent fusion of membranous organelles and the plasma membrane at the site of the damage. Cortical cytoskeletal repair results from local assembly of actin filaments (F-actin), myosin-2 and microtubules into an array that closes around the original wound site. Control of the cytoskeletal response is exerted by local activation of the small GTPases, Rho and Cdc42. Recent work has given insight into both the membrane fusion and cytoskeletal responses to plasma membrane damage and we propose that Rho GTPase activation results at least in part from the events that drive membrane repair.
Subject(s)
Actins/physiology , Cell Membrane/physiology , Cytoskeleton/physiology , Membrane Fusion/physiology , Myosins/physiology , Animals , Humans , Microtubules/physiology , Wound HealingABSTRACT
Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.
Subject(s)
Actins/metabolism , Cell Compartmentation/physiology , Cell Membrane/metabolism , Cytoplasmic Granules/metabolism , Membrane Fusion , Animals , Diglycerides/metabolism , Exocytosis/physiology , Female , Oocytes/metabolism , Protein Kinase C/metabolism , Protein Kinase C beta , Xenopus/metabolismABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Cytokinesis, the final step in cell division, is dependent on formation and closure of a ring of actin filaments (F-actin) and myosin-2 which is, in turn, dependent on activation of the small GTPase, RhoA, at the cell equator. Four new papers, including two in this issue of Developmental Cell (Petronczki et al., 2007; Birkenfeld et al., 2007), provide new insights into how RhoA activation at the equator is initiated and maintained.
Subject(s)
Cytokinesis , Cell Cycle Proteins/metabolism , Enzyme Activation , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Rho Guanine Nucleotide Exchange Factors , Spindle Apparatus/metabolism , rhoA GTP-Binding Protein/metabolism , Polo-Like Kinase 1ABSTRACT
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.
Subject(s)
Cortical Excitability , Animals , Cell Cycle , Cell Division , Cytoplasm , Xenopus laevisABSTRACT
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 (IP3R), an endoplasmic reticulum (ER) calcium channel, in spindle assembly using Trim21-mediated depletion of IP3R. Oocytes depleted of IP3R 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 IP3R 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 IP3R. We conclude that intracellular calcium signaling involving IP3R-mediated calcium release is required for meiotic spindle assembly in Xenopus oocytes.
Subject(s)
Calcium , Inositol , Animals , Xenopus laevis/metabolism , Calcium/metabolism , Inositol/metabolism , Calcium Chelating Agents/metabolism , Oocytes/metabolism , Meiosis , Spindle Apparatus/metabolism , Microtubules/metabolismABSTRACT
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 Ca2+ signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca2+ have been difficult to document in oocyte meiosis except for the global Ca2+ 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 Ca2+-sensitive protein kinase Cß 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.
Subject(s)
Calcium , Polar Bodies , Animals , Calcium/metabolism , Calcium Chelating Agents/metabolism , Male , Meiosis , Oocytes/metabolism , Polar Bodies/metabolism , Protein Kinases/metabolism , Semen/metabolismABSTRACT
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.
Subject(s)
Actins , Cytoskeleton , GTPase-Activating Proteins , Proto-Oncogene Proteins , rho GTP-Binding Proteins , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cytokinesis , Cytoskeleton/metabolism , GTPase-Activating Proteins/metabolism , Oocytes , Proto-Oncogene Proteins/metabolism , Xenopus , rho GTP-Binding Proteins/metabolismABSTRACT
Compensatory endocytosis follows regulated exocytosis in cells ranging from eggs to neurons, but the means by which it is accomplished are unclear. In Xenopus eggs, compensatory endocytosis is driven by dynamic coats of assembling actin that surround and compress exocytosing cortical granules (CGs). We have identified Xenopus laevis myosin-1c (XlMyo1c) as a myosin that is upregulated by polyadenylation during meiotic maturation, the developmental interval that prepares eggs for fertilization and regulated CG exocytosis. Upon calcium-induced exocytosis, XlMyo1c is recruited to exocytosing CG membranes where actin coats then assemble. When XlMyo1c function is disrupted, actin coats assemble, but dynamic actin filaments are uncoupled from the exocytosing CG membranes such that coats do not compress, and compensatory endocytosis fails. Remarkably, there is also an increase in polymerized actin at membranes throughout the cell. We conclude that XlMyo1c couples polymerizing actin to membranes and so mediates force production during compensatory endocytosis.
Subject(s)
Actins/physiology , Cell Membrane/metabolism , Endocytosis , Myosin Type I/physiology , Myosins/physiology , Xenopus Proteins/physiology , Xenopus laevis/physiology , Amino Acid Sequence , Animals , Calcium/physiology , Cytoskeleton/metabolism , Exocytosis , Female , In Vitro Techniques , Meiosis , Molecular Sequence Data , Myosins/metabolism , Oocytes/metabolism , Polyadenylation , Protein Binding , Secretory Vesicles/metabolism , Up-Regulation , Xenopus Proteins/metabolism , Xenopus laevis/metabolismABSTRACT
Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.
Subject(s)
Actin Cytoskeleton/metabolism , Cell Differentiation/physiology , Cell Movement/physiology , Eukaryotic Cells/metabolism , Microtubules/metabolism , Animals , Cell Division/physiology , Contractile Proteins/metabolism , Eukaryotic Cells/ultrastructure , Growth Cones/metabolism , Growth Cones/ultrastructure , Humans , Wound Healing/physiologyABSTRACT
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.
Subject(s)
Actins , Cytokinesis , Actins/metabolism , Cell Division , Cytoplasm/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Actins , Artificial Cells , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cytokinesis , Spindle Apparatus/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
In the October 7th issue of Cell, it is shown that a ring-like structure containing the centrosomal protein centriolin acts as a local recruitment site for the membrane fusion machinery that controls abscission.