The role of biomolecular condensates in regulating the cytoskeleton.

生物分子缩合物在调节细胞骨架中的作用。

• 批准号: 10751631
• 负责人: Zach Geisterfer
• 金额: $ 6.91万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751631
• 关键词: Actins; Area; Biochemical; Biochemistry; Biological Assay; Biological Models; Cells; Cellular Morphology; Cellular biology; Complex; Cytoplasm; Cytoskeletal Modeling; Cytoskeleton; Data; Development; Disease; Equipment and supply inventories; F-Actin; Fluorescence Microscopy; Frequencies; Genetic; Growth; Link; Malignant Neoplasms; Measures; Messenger RNA; Mission; Modeling; Molecular Genetics; Morphogenesis; Morphology; Muscle; Neurons; Pattern; Phase; Phenotype; Physical condensation; Physiological; Polymers; Positioning Attribute; Post-Translational Regulation; Probability; Process; Proteins; Proteomics; Public Health; RNA; RNA-Binding Proteins; Regulation; Research; Ribonucleoproteins; Role; Signal Transduction; Site; System; Testing; Time; Transcript; Translations; United States National Institutes of Health; Visualization; Work; biophysical properties; cell motility; fungus; genetic regulatory protein; mathematical model; mutant; polymerization; simulation

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.

项目摘要。 肌动蛋白细胞骨架的动态重组和精确定位对于复杂细胞至关重要 形态、细胞运动和细胞信号传导等过程。 调节蛋白及其生化活动，这些生化活动的空间调节 整个细胞仍然是理解细胞内组织的一个关键差距。 生物分子凝聚体已成为控制生物化学不同领域的中心机制。 来自进化分歧系统的几项研究指出，肌动蛋白组装可能是 具体来说，一些肌动蛋白调节因子具有本质上无序的标志性特征 区域（IDR）以及 F-肌动蛋白形成的一些位点具有归因于凝聚物的生物物理特性。 在某些情况下，这些组件可能是通过相分离形成的，但在其他情况下，似乎会出现冷凝物 目前尚不清楚生物分子凝聚物如何具体促进局部化。 肌动蛋白细胞骨架的组装以及这种调节模式如何控制细胞形态发生。 在这个提案中，我将确定核糖核蛋白（RNP）缩合的机制， 含有RNA和蛋白质的肌动蛋白细胞骨架在时间和空间上的组装模式。 使用合胞真菌 Ashbya gossypii（“Ashbya”）中看到的菌丝分支作为模型系统 破译凝结物和肌动蛋白调节之间的联系众所周知，肌动蛋白的集中富集。 蛋白质相互作用导致菌丝尖端局部极化的细胞骨架网络和初始分支位点 Gladfelter 实验室的研究表明，RNA 结合蛋白 Whi3 是促进形成所必需的。 Ashbya 中新的极性位点值得注意的是，Whi3 与 formin Bni1 和极性的 mRNA 转录物缩合。 Ashbya 中现有和初期分支位点的 Spa2 蛋白提供了一个强大的系统来研究其作用。 凝结物在肌动蛋白调节中的作用，因为凝结物的基本和生理作用可以通过遗传来确定。 我的初步数据显示 Whi3 包被的珠子能够使极化肌动蛋白成核。 Ashbya 无细胞提取物中的网络，开启了将遗传学与无细胞结合的能力 提取物，细胞骨架发现的主力，通过这个新的，我将区分两种模型分析。 凝结物如何通过（i）局部翻译或（ii）改变肌动蛋白组装来调节肌动蛋白组装 目标 1 中凝结物控制的肌动蛋白调节剂的活性。然后我将确定多个 Whi3 凝结物如何在 在目标 2 中，共同的细胞质有助于 Ashbya 的复杂形态。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，以及如何 这些组件驱动复杂的细胞形态，这是许多细胞的基本特征。