Investigating the mechanism of self-organized cortical patterning in an artificial cortex

细胞皮层是基本细胞功能的基础，包括促进细胞分裂的细胞形状变化。皮质由丝状肌动蛋白（F-肌动蛋白）和质膜组成，在胞质分裂过程中重塑，将细胞一分为二。最近的研究表明，在大规模重塑之前，皮质也动态地以小GTdR RhoA和F-肌动蛋白的相干亚细胞波图案化，这种现象称为“皮质兴奋性”。在发育中的胚胎中，这些波出现在细胞的整个表面上，然后随着细胞分裂的进行而进入细胞动力学沟，并且已经被提出来支持分裂平面的快速和灵活的建立。研究支持和调节皮质模式的机制目前受到缺乏技术方法的限制，这些方法可以弥合我们对生化反馈信号和皮质模式形成的理解，包括信号分子的分子调节，膜动力学和细胞骨架重塑。这种知识差距的一个突破是申请人开发了一种“人工皮质”，由支持的脂质双层（SLB）和非洲爪蟾卵提取物制成，其成功地在无细胞系统中重建了活性Rho和F-肌动蛋白动力学。与体内皮质兴奋性一样，人工皮质中的图案化取决于Rho活性和F-肌动蛋白聚合。这种新颖的，合成的方法来研究皮质图案是一个理想的系统，系统地研究的作用，个别因素（如上游GTdR调节，膜组成和流动性，细胞周期状态）在调节皮质动力学。使用人工皮质作为皮质图案化的模型，这项MOSAIC K99/R 00奖的提案旨在了解皮质图案形成是如何调节的，以及图案化如何重塑细胞皮质以执行胞质分裂等基本功能。Landino博士将研究驱动皮质波形成（Aim 1）、皮质细胞骨架重塑（Aim 2）以及皮质模式在支持成功细胞分裂中的作用（Aim 3）的因素。这项工作的结果将扩大我们的知识皮质的分子调节皮质兴奋性的出现，和动态模式在细胞分裂中的作用。Landino博士的长期职业目标是建立一个独立的研究小组，调查调节皮层模式和细胞分裂的机制。拟议的培训将为Landino博士提供额外的科学专业知识，包括电子显微镜和循环提取物制备方面的技术培训，并进一步建立人工皮层作为了解细胞皮层生化和结构调控的有用平台。Landino的专业发展，包括研究实验室管理的正式培训，领导多元化，公平和包容性的工作场所，以及支持Landino博士申请教师职位的定制计划。密歇根大学的示范性科学和专业环境非常适合支持本提案中概述的培训，并确保Landino博士成功启动独立研究计划。

The cell cortex underlies essential cellular functions, including cell shape changes that facilitate cell division. Comprised of a meshwork of filamentous actin (F-actin) and the plasma membrane, the cortex is remodeled during cytokinesis, physically dividing the cell in two. Recent work has shown that prior to large-scale remodeling, the cortex is also dynamically patterned with coherent subcellular waves of the small GTPase RhoA and F-actin, a phenomenon termed “cortical excitability”. In developing embryos, these waves appear over the entire surface of the cell and then feed into the cytokinetic furrow as cell division progresses and have been proposed to support the rapid and flexible establishment of the division plane. Investigating the mechanisms that support and regulate cortical patterning is currently limited by a lack of technical approaches that can bridge our understanding of biochemical feedback signaling and cortical pattern formation, including the molecular regulation of signaling molecules, membrane dynamics, and cytoskeletal remodeling. A breakthrough in this gap in knowledge has been the development by the applicant of an “artificial cortex”, made from supported lipid bilayers (SLBs) and Xenopus egg extract, which successfully reconstitutes active Rho and F-actin dynamics in a cell-free system. Like in vivo cortical excitability, patterning in the artificial cortex depends on Rho activity and F-actin polymerization. This novel, synthetic approach to investigating cortical patterning is an ideal system for systematically examining the role of individual factors (such as upstream GTPase regulators, membrane composition and fluidity, cell cycle state) in regulating cortical dynamics. Using the artificial cortex as a model for cortical patterning, this proposal for a MOSAIC K99/R00 Award seeks to understand how cortical pattern formation is regulated and how patterning remodels the cell cortex to perform essential functions like cytokinesis. Dr. Landino will investigate the factors that drive cortical wave formation (Aim 1), cytoskeletal remodeling at the cortex (Aim 2), and the role of cortical patterning in supporting successful cell division (Aim 3). The results of this work will expand our knowledge of the molecular regulation of the cortex underlying the emergence of cortical excitability, and the role of dynamic patterning in cell division. Dr. Landino's long-term career goal is to establish an independent research group investigating the mechanisms that regulate cortical patterning and cell division. The proposed training will provide Dr. Landino with additional scientific expertise, including technical training in electron microscopy and preparation of cycling extract, and further establish the artificial cortex as a useful platform for understanding the biochemical and structural regulation of the cell cortex. This award will further Dr. Landino's professional development including formal training in research laboratory management, leading a diverse, equitable, and inclusive workplace, and a tailored plan to support Dr. Landino's application to faculty positions. The exemplary scientific and professional environment at the University of Michigan is ideally suited to support the training outlined in this proposal and ensure Dr. Landino's success in launching an independent research program.