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.

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.

Pericentrin interacts with Kinesin-1 to drive centriole motility.

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.

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.

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. Here we evaluated the contributions of cross-talk between Rho and Cdc42 to the patterning of their respective zones in wounded Xenopus oocytes using experimental manipulations in combination with mathematical modeling. The results show that the position of the Cdc42 zone relative to the Rho zone and relative to the wound edge is controlled by the level of Rho activity. In contrast, the outer boundary of the Rho zone is limited by the level of Cdc42 activity. Models based on positive feedback within zones and negative feedback from Rho to the GEF-GAP Abr to Cdc42 capture some, but not all, of the observed behaviors. We conclude that GTPase zone positioning is controlled at the level of Rho activity and we speculate that the Cdc42 zone or something associated with it limits the spread of Rho activity.

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.

Fascetto interacting protein ensures proper cytokinesis and ploidy.

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is the protein regulator of cytokinesis 1, or Fascetto (Feo) in Drosophila, which forms a homodimer to cross-link interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Feo interacting protein (FIP). Loss of FIP results in a reduction in Feo localization, rapid disassembly of interzonal MTs, and several defects related to cytokinesis failure, including polyploidization of neural stem cells. Simultaneous reduction in Feo and FIP results in very large, tumorlike DNA-filled masses in the brain that contain hundreds of centrosomes. In aggregate, our data show that FIP acts directly on Feo to ensure fully accurate cell division.

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.