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

In Vivo Tracking and 1H/19F Magnetic Resonance Imaging of Biodegradable Polyhydroxyalkanoate / Polycaprolactone Blend Scaffolds Seeded with Labeled Cardiac Stem Cells

Medium-chain length Polyhydroxyalkanoates (MCL-PHAs) have demonstrated exceptional properties for cardiac tissue engineering (CTE) applications. Despite prior work on MCL-PHA/Polycaprolactone (PCL) blends, optimal scaffold production and use as an alternative delivery route for controlled release of seeded cardiac progenitor cells (CPCs) in CTE applications in vivo has been lacking, We present herein applicability of MCL-PHA/PCL (95/5 wt%) blends fabricated as thin films with an improved performance compared to the neat MCL-PHA aiming to a) benefit from the material properties of natural and synthetic polymers, b) achieve controlled delivery and increase retention of delivered cells to the murine myocardium, c) extend the temporal window over which the release of labeled CPCs occurs compared to traditional direct injection techniques, and d) use 19F MRI/MRS to noninvasively detect, and longitudinally monitor the seeded scaffolds. Polymer characterization confirmed the chemical structure and composition of the synthesized scaffolds, while thermal, wettability, and mechanical properties were also investigated and compared in neat and porous counterparts. In vitro cytocompatibility studies were performed using perfluorocrown-ether (PFCE)-nanoparticle-labeled murine cardiac progenitor cells (CPC), and studied using confocal microscopy and 19F MRS/MRI. Seeded scaffolds were implanted and studied in the post-mortem murine heart in situ, and in two additional C57BL/6 mice in vivo (using single-layered and double-layered scaffolds) and imaged immediately after and at 7 days post-implantation. Superior MCL-PHA/PCL scaffold performance has been demonstrated compared to MCL-PHA through experimental comparisons of a) morphological data using scanning electron microscopy and b) contact angle measurements attesting to improved CPC adhesion, c) in vitro confocal microscopy showing increased SC proliferative capacity, d) mechanical testing that elicited good overall responses. In vitro MRI results justify the increased seeding density, increased in vitro MRI signal, and improved MRI visibility in vivo, in the double-layered compared to the single-layered scaffolds. Histological evaluations (bright-field, cytoplasmic (Atto647) and nuclear (DAPI) stains) performed in conjunction with confocal microscopy imaging attest to CPC binding within the scaffold, subsequent release and migration to the neighboring myocardium, and to increased retention in the murine myocardium in the case of the double-layered scaffold. Thus MCL-PHA/PCL blends possess tremendous potential for controlled delivery of CPCs and to maximize possible regeneration in myocardial infarction

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

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

Human Retinal Gene Therapy for Leber Congential Amaurosis Shows Advancing Retinal Degeneration Despite Enduring Visual Improvement

Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5–11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term