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。 动态恢复和精确定位肌动蛋白细胞骨架对于复杂细胞至关重要 形态，细胞运动和细胞信号传导和其他过程。虽然有大量肌动蛋白库存 调节蛋白及其生化活性，这些生化活性的空间调节 整个细胞仍然代表了理解细胞内组织的关键差距。 生物分子冷凝物已成为控制生物化学潜水区域的中心机制。 来自进化不同系统的几项研究表明，肌动蛋白组装可能是 由冷凝水控制。具体而言，一些肌动蛋白调节剂具有本质上无序的标志性特征 区域（IDR）以及一些F-肌动蛋白形式具有生物物理特性的位点。 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，冷凝水似乎出现了 通过不同的机制。尚不清楚的是生物分子冷凝物如何针对局部贡献 肌动蛋白细胞骨架的组装以及这种调节方式如何控制细胞形态发生。 在此提案中，我将确定核糖核蛋白（RNP）凝结的机制， 同时包含RNA和蛋白质，将肌动蛋白细胞骨架的组装在时空和空间中进行模式。我会 在合成真菌Ashbya Gossypii（“ Ashbya”）中使用的菌丝分支作为模型系统 解解冷凝水与肌动蛋白调节之间的联系。众所周知，集中富集肌动蛋白 - 相互作用的蛋白质导致菌丝尖端的局部两极化细胞骨架网络，并在 阿什比亚。在Gladfelter Lab中的研究表明，RNA结合蛋白WHI3需要促进形成 Ashbya的新极性网站值得注意的是，Whi3用mRNA转录本的formin bni1和极性凝结 现有和初期分支部位的蛋白SPA2。 Ashbya提供了一个强大的系统来研究 肌动蛋白调节中的冷凝水，因为冷凝水的基本和物理作用可以是一般的 在活细胞中解剖。我的初步数据表明，含有含有的珠子的珠子能够对极化肌动蛋白进行成核 无ashbya细胞提取物中的网络，开放了将遗传学功能与无细胞相结合的能力 提取物，是细胞骨架发现的主力。通过此新测定，我将区分两个模型 冷凝水如何通过（i）通过更改或（ii）的局部翻译来调节肌动蛋白组件。 AIM 1中的冷凝物控制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 一个常见的细胞质在AIM 2中有助于Ashbya的复杂形态。这项工作将揭示 生物分子凝结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，这是许多细胞的重要特征。