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 . These self-organizing Rho GTPase patterns have been explained by a variety of theoretical models which require multiple interacting positive and negative feedback loops.

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

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.

Inositol 1, 4, 5-trisphosphate receptor is required for spindle assembly in 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 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 (IPR), an endoplasmic reticulum (ER) calcium channel, in spindle assembly using Trim21-mediated depletion of IPR.

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 Ca signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca have been difficult to document in oocyte meiosis except for the global Ca wave induced by sperm at fertilization.

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.

Cell cycle and developmental control of cortical excitability in .

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 eggs and embryos and the changes in cortical excitability throughout early development.

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. 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." 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.

Cross-talk-dependent cortical patterning of Rho GTPases during cell repair.

Rho GTPases such as Rho, Rac, and Cdc42 are important regulators of the cortical cytoskeleton in processes including cell division, locomotion, and repair. In these processes, Rho GTPases assume characteristic patterns wherein the active GTPases occupy mutually exclusive "zones" in the cell cortex. During cell wound repair, for example, a Rho zone encircles the wound edge and is in turn encircled by a Cdc42 zone.

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.

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.

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.

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 embryonic epithelia.

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.

Living Xenopus oocytes, eggs, and embryos as models for cell division.

Xenopus laevis has long been a popular model for studies of development and, based on the use of cell-free extracts derived from its eggs, as a model for reconstitution of cell cycle regulation and other basic cellular processes. However, work over the last several years has shown that intact Xenopus eggs and embryos are also powerful models for visualization and characterization of cell cycle-regulated cytoskeletal dynamics. These findings were something of a surprise, given that the relatively low opacity of Xenopus eggs and embryos was assumed to make them poor subjects for live-cell imaging.

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.

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 embryonic epithelium. We find that metaphase spindles first undergo a sustained rotation that brings them on-axis with their final orientation.

Cell repair: Revisiting the patch hypothesis.

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 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.

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 (PKCβ and PKCη) and guanosine nucleotide dissociation inhibitor (GDI) in the wound response.

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.

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.

Cell healing: Calcium, repair and regeneration.

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?

Activator-inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium.

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.