Testing models of cell cortex wave generation by Rho GTPases.

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.

Patterning of the cell cortex by Rho GTPases.

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.

Bring the pain: wounding reveals a transition from cortical excitability to epithelial excitability in Xenopus embryos.

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.

Inositol 1, 4, 5-trisphosphate receptor is required for spindle assembly in Xenopus oocytes.

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.

A localized calcium transient and polar body abscission.

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.

A versatile cortical pattern-forming circuit based on Rho, F-actin, Ect2, and RGA-3/4.

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.

Cell cycle and developmental control of cortical excitability in Xenopus laevis.

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.

Rho and F-actin self-organize within an artificial cell cortex.

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.

Cortical excitability and cell division.

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.

Plasma membrane integrity: implications for health and disease.

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.

Extraction of active RhoGTPases by RhoGDI regulates spatiotemporal patterning of RhoGTPases.

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.

Spindle-F-actin interactions in mitotic spindles in an intact vertebrate epithelium.

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.

Spatially Adaptive Colocalization Analysis in Dual-Color Fluorescence Microscopy.

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.

An interaction between myosin-10 and the cell cycle regulator Wee1 links spindle dynamics to mitotic progression in epithelia.

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.

Automated mitotic spindle tracking suggests a link between spindle dynamics, spindle orientation, and anaphase onset in epithelial cells.

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.

A mathematical model of GTPase pattern formation during single-cell wound repair.

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 (PKCß 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) PKCß 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.

Membrane dynamics during cellular wound repair.

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.

How to make a static cytokinetic furrow out of traveling excitable waves.

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.