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是复杂细胞的关键 形态学、细胞运动性和细胞信号传导等过程。虽然有大量肌动蛋白 调节蛋白及其生化活性，这些生化活性的空间调节 仍然代表着理解细胞内组织的一个关键差距。 生物分子凝聚物已经成为控制生物化学不同领域的中心机制。 一些来自进化分歧系统的研究指出，肌动蛋白组装可能是 由冷凝物控制。具体来说，一些肌动蛋白调节剂具有内在无序的标志性特征， 区域（IDR），以及一些F-肌动蛋白形式具有归因于缩合物的生物物理性质的位点。在 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，冷凝物似乎出现了 通过不同的机制。目前尚不清楚的是，生物分子凝聚物如何特别有助于局部化 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。 在这个建议中，我将确定核糖核蛋白（RNP）缩合的机制， 包含RNA和蛋白质，在时间和空间上模拟肌动蛋白细胞骨架的组装。我会 使用在合胞体真菌棉阿舒囊霉（Ashbya gossypii）（“阿舒囊霉”）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节之间的联系。据了解，集中富集肌动蛋白- 相互作用的蛋白质导致在菌丝顶端的局部极化的细胞骨架网络，以及在菌丝顶端的初始分支位点。 阿什比亚Gladfelter实验室的研究表明，RNA结合蛋白Whi 3是促进形成 阿什比亚新的极性点值得注意的是，Whi 3与mRNA转录物缩合，用于表达Bni 1和极性。 蛋白Spa 2在现有的和初始的分支网站。Ashbya提供了一个强大的系统来研究 在肌动蛋白调节中的缩合物，因为缩合物的基本和生理作用可以在遗传上是 在活细胞中解剖。我的初步数据显示，Whi 3包被的珠子能够使极化的肌动蛋白成核， 网络阿舒囊菌无细胞提取物，开辟了联合收割机的能力，结合遗传学的力量与无细胞 提取物，细胞骨架发现的主力。通过这种新的分析，我将区分两种模型， 缩合物如何通过（i）局部翻译或（ii）改变肌动蛋白的结构来调节肌动蛋白的组装， Aim 1中缩合物控制的肌动蛋白调节剂的活性。然后，我将确定多个Whi 3如何在 一个共同的细胞质有助于复杂的形态阿舒囊菌在目的2。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，以及如何 这些组装体驱动复杂的细胞形态，这是许多细胞的基本特征。