The role of biomolecular condensates in regulating the cytoskeleton.

项目摘要。 肌动蛋白细胞骨架的动态重组和精确定位是复杂细胞的关键 形态、细胞运动和细胞信号等过程。虽然有大量的肌动蛋白库存 调控蛋白及其生化活性、这些生化活性的空间调控 贯穿整个细胞仍然是理解细胞内组织的一个关键鸿沟。 生物分子凝聚物已成为控制生物化学各个领域的中心机制。 来自进化分化系统的几项研究指出，肌动蛋白组装可能是 由凝析油控制。具体地说，一些肌动蛋白调节器具有本质上紊乱的特征 区域(IDR)，以及F-肌动蛋白形成的一些部位具有归因于凝析油的生物物理性质。在……里面 在某些情况下，这些组件可能是通过相分离形成的，但在另一些情况下，凝析油似乎会出现 通过不同的机制。目前尚不清楚的是，生物分子凝聚体是如何对局部化的 肌动蛋白细胞骨架的组装以及这种调控模式如何控制细胞的形态发生。 在这项提案中，我将确定核糖核蛋白(RNP)凝结的机制， 同时含有RNA和蛋白质，在时间和空间上形成肌动蛋白细胞骨架的组装模式。这就做 使用在合胞真菌Ashbya Cotsypii(“Ashbya”)中看到的菌丝分枝作为模型系统 破译缩合物和肌动蛋白调控之间的联系。众所周知，肌动蛋白的集中浓缩- 相互作用的蛋白质导致菌丝顶端局部极化的细胞骨架网络，以及 阿什比亚。格拉德费尔特实验室的研究表明，RNA结合蛋白WHI3是促进形成所必需的 在阿什比亚的新的极地。值得注意的是，Whi3与Forin Bni1和极性的mRNA转录本缩合 蛋白Spa2在现有的和初始的分支位置。Ashbya提供了一个强大的系统来研究 凝聚体在肌动蛋白调节中的作用，因为凝聚体的基本和生理作用可能是遗传的 在活细胞中解剖。我的初步数据显示，Whi3包裹的珠子能够使极化肌动蛋白成核 阿什比亚无细胞提取物中的网络，开启了将遗传学的力量与无细胞相结合的能力 萃取物，细胞骨架发现的主力。有了这个新的分析，我将区分两个模型 缩合物如何通过(I)肌动蛋白的局部翻译或(Ii)通过改变 AIM 1中缩合控制的肌动蛋白调节器的活性。然后我将确定多个Whi3缩合是如何在 在Aim 2中，一个共同的细胞质对Ashbya的复杂形态有贡献。这项工作将揭示 生物分子凝聚物如何控制肌动蛋白细胞骨架的空间组织的机制 这些组件驱动复杂的细胞形态，这是许多细胞的基本特征。

Project Summary. The dynamic restructuring and precise positioning the actin cytoskeleton is essential for complex cell morphologies, cell motility, and cell signaling among other processes. While there is a large inventory of actin regulatory proteins and their biochemical activities, the spatial regulation of these biochemical activities throughout the cell still represents a key gap in understanding intracellular organization. Biomolecular condensates have emerged as a central mechanism for controlling diverse areas of biochemistry. Several studies from evolutionarily divergent systems point to the possibility that actin assembly may be controlled by condensates. Specifically, some actin regulators have hallmark features of intrinsically disordered regions (IDRs), and some sites where F-actin forms have biophysical properties ascribed to condensates. In some cases, these assemblies likely form by phase separation, but in others the condensates appear to emerge by different mechanisms. What isn’t clear is how biomolecular condensates specifically contribute to the localized assembly of the actin cytoskeleton and how this mode of regulation controls cell morphogenesis. In this proposal, I will identify the mechanisms by which ribonucleoprotein (RNP) condensates, containing both RNA and protein, pattern the assembly of the actin cytoskeleton in time and space. I will use the mycelial branching seen in the syncytial fungus Ashbya gossypii (“Ashbya”) as a model system for deciphering the links between condensates and actin regulation. It is known that focused enrichment of actin- interacting proteins leads to a local polarized cytoskeletal network at hyphal tips, and incipient branch sites in Ashbya. studies in the Gladfelter lab have shown the RNA-binding protein, Whi3, is required to promote formation of new polarity sites in Ashbya. Notably, Whi3 condenses with mRNA transcripts for the formin Bni1 and polarity protein Spa2 at existing and incipient branch sites. Ashbya provides a powerful system to study the role of condensates in actin regulation because the essential and physiological role of condensates can be genetically dissected in live cells. My preliminary data show Whi3-coated beads are capable of nucleating polarized actin networks in Ashbya cell-free extracts, opening up the ability to combine the power of genetics with cell-free extracts, a workhorse of cytoskeletal discovery. With this new assay, I will distinguish between two models for how condensates may regulate actin assembly through either (i) the local translation of or (ii) by changing the activity of condensate-controlled actin regulators in Aim 1. I will then identify how multiple Whi3 condensates in a common cytoplasm contribute to the complex morphology of Ashbya in Aim 2. This work will reveal mechanisms for how biomolecular condensates control spatial organization of the actin cytoskeleton, and how these assemblies drive complex cell morphology, an essential feature of many cells.