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

细胞皮质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。由丝状肌动蛋白(F-肌动蛋白)和质膜组成的网络，皮质在胞质分裂时被重塑，物理上将细胞一分为二。最近的研究表明，在大规模重塑之前，皮质也会动态地被小GTP酶RhoA和F-肌动蛋白的相干亚细胞波所构筑，这种现象被称为“皮质兴奋性”。在发育中的胚胎中，这些波出现在整个细胞表面，然后随着细胞分裂的进行进入细胞动态沟，并被认为支持快速而灵活地建立分裂平面。目前，对支持和调节皮质模式的机制的研究受到限制，因为缺乏技术手段来沟通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控、膜动力学和细胞骨架重塑。这一知识空白的一个突破性进展是申请者开发了一种“人工皮质”，它由支持的脂质双层(SLB)和非洲爪哇卵提取液制成，它成功地在无细胞系统中重建了活性Rho和F-肌动蛋白的动力学。就像活体皮质的兴奋性一样，人工皮质中的图案依赖于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一个理想的系统，可以系统地研究单个因素(如上游GTP酶调节因子、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。使用人工皮质作为皮质图案的模型，这项镶嵌K99/R00奖的提案试图了解皮质图案的形成是如何调节的，以及图案是如何重塑细胞皮质以执行细胞质分裂等基本功能的。兰迪诺博士将研究驱动皮质波形成的因素(目标1)、皮质细胞骨架重塑的因素(目标2)，以及皮质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背后的皮层分子调控的了解，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，研究调节大脑皮层模式和细胞分裂的机制。拟议中的培训将为兰迪诺博士提供更多的科学专业知识，包括电子显微镜的技术培训和循环提取物的制备，并进一步将人工皮质确立为了解细胞皮质的生化和结构调控的有用平台。该奖项将进一步促进兰迪诺博士的专业发展，包括在研究实验室管理方面的正式培训，领导一个多样化、公平和包容的工作场所，以及支持兰迪诺博士申请教职的量身定做的计划。密歇根大学模范的科学和专业环境非常适合支持本提案中概述的培训，并确保兰迪诺博士成功启动独立研究项目。

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.