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。皮层由丝状肌动蛋白（F-actin）和质膜组成，在细胞质分裂过程中被重塑，将细胞物理地分成两部分。最近的研究表明，在大规模重塑之前，皮质也会动态地形成小GTPase RhoA和F-actin的连贯亚细胞波，这种现象被称为“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，然后随着细胞分裂的进行进入细胞动力学沟，并被认为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，这些技术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。这一知识缺口的突破是由支持脂质双分子层（slb）和爪蟾卵提取物制成的“人工皮质”的开发，它成功地在无细胞系统中重建了活跃的Rho和f -肌动蛋白动力学。与体内皮质兴奋性一样，人工皮质的模式也取决于Rho活性和f -肌动蛋白聚合。这种新颖的、综合的研究皮质模式的方法是系统地检查个体因素（如上游GTPase调节剂、膜组成和流动性、细胞周期状态）在调节皮质动力学中的作用的理想系统。这个MOSAIC K99/R00奖的提案使用人工皮层作为皮质模式的模型，试图理解皮质模式的形成是如何被调节的，以及模式如何重塑细胞皮层以执行细胞分裂等基本功能。Landino博士将研究驱动皮层波形成的因素（目标1），皮层细胞骨架重塑（目标2），以及皮层模式在支持细胞成功分裂中的作用（目标3）。这项工作的结果将扩大我们对皮层兴奋性出现的分子调控的认识，以及动态模式在细胞分裂中的作用。兰迪诺博士的长期职业目标是建立一个独立的研究小组，研究调节皮质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的技术培训，并进一步建立人工皮层作为理解细胞皮层生化和结构调节的有用平台。该奖项将进一步促进Landino博士的专业发展，包括在研究实验室管理方面的正式培训，领导多元化，公平和包容的工作场所，以及为Landino博士申请教师职位提供量身定制的计划。密歇根大学堪称典范的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博士成功启动一个独立的研究项目。

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.