The Biochemical Basis for the Mechanics of Cytokinesis

细胞分裂机制的生化基础

• 批准号: 10824516
• 负责人: DOUGLAS N ROBINSON
• 金额: $ 0.62万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10824516
• 关键词: Actins; Binding; Biochemical; Biomechanics; Cell Adhesion; Cell Proliferation; Cell Shape; Cell division; Cells; Chemicals; Complex; Crosslinker; Cytokinesis; Cytoplasm; Defect; Development; Disease; Dynamin; Event; Feedback; Fluorescence; Gene Expression; Genetic; Grant; Lectin; Length; Life; Maintenance; Mechanics; Messenger RNA; Microtubules; Modeling; Molecular; Myosin Type II; Nocodazole; Organism; Pathway interactions; Process; Production; Property; Protein Secretion; Proteins; Proteomics; RNA; RNA Recognition Motif; Race; Research; Ribonucleoproteins; Signal Transduction; Spectrum Analysis; Surface; System; Techniques; Tissues; cortexillin I; crosslinking and immunoprecipitation sequencing; interest; knock-down; migration; overexpression; parent grant; single molecule; transcriptome sequencing; viscoelasticity

PROJECT SUMMARY GM66817-18 PARENT GRANT Cell shape change is fundamental for cell division, migration, and tissue formation, and defects in cell shape change are one of the early hallmarks of disease. In our research, we study cell shape change, focusing on the biomechanical systems, and we utilize cytokinesis as an elegant model process. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive cell shape change, including cytokinesis furrow ingression. We identified key molecular pathways that control these properties and found that the circuitry is wired like a control system complete with feedback loops that allows mechanical and chemical signals to tune the accumulation of the contractile machinery. In this proposal, we will build upon our understanding of cell shape change and the mechano- responsive contractility network. We use a suite of techniques, including genetics, proteomics, Single Molecule Pulldown (SiMPull), and Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS/FCCS) to study this network. We discovered that many of the proteins in the mechano-responsive contractility network are organized into complexes in the cytoplasm, forming Contractility Kits (CKs). Several CK components have unknown functions in the context of cell contractility and are the subject of this proposal. Among these, the lectin discoidin 1A, traditionally viewed as a secreted protein, assembles with the CKs in the cytoplasm and is necessary for a key protein, the actin crosslinker cortexillin I, to localize fully to the cortex. Moreover, discoidin 1A has a complex genetic relationship with cortexillin I and its binding partners, IQGAP1 and IQGAP2. We will determine how discoidin 1 operates in the CKs and promotes cortical assembly. Next, we are studying two ribonucleoproteins, RNP1A and RNP1B. Both proteins contain predicted RNA-recognition motifs. RNP1A is also required for normal cortexillin I mRNA levels. We originally identified RNP1A over-expression as a genetic suppressor of the microtubule-destabilizer nocodazole (same study that gave rise to the RacE-14-3-3-myosin II pathway that we discovered). We have now found that RNP1A is required for normal microtubule length, cell adhesion, and cortical mechanics. We conducted RNAseq analysis and found that several CK proteins have altered gene expression in rnp1A knockdown cells. To identify RNAs that the RNP1s might bind, we have used CLIP-Seq and identified several RNAs as interactors of RNP1A and RNP1B. One of particular interest is the dynamin-like protein 1, which localizes to the cleavage furrow cortex where it assists in actin and myosin II organization. Others are involved in macropinocytosis, another essential cell shape change event that also draws upon much of the CK machinery. Here, we will flesh out how the RNP1s impact CK assembly, expression, and function. Overall, these studies will decipher how the CK network operates and integrates with other cellular systems, leading to a deeper understanding of cell shape change processes more generally.

项目概要 GM66817-18 家长补助金 细胞形状的变化是细胞分裂、迁移和组织形成的基础，细胞形状的缺陷 变化是疾病的早期标志之一。在我们的研究中，我们研究细胞形状的变化，重点关注 生物力学系统，我们利用胞质分裂作为一个优雅的模型过程。在这笔赠款的有效期内，我们 已经证明了主动力产生、皮质张力、表面曲率和 粘弹性驱动细胞形状变化，包括胞质分裂沟进入。我们确定了关键分子 控制这些属性的途径，并发现电路像控制系统一样接线，并配有 反馈回路允许机械和化学信号调节收缩的积累 机械。在这个提案中，我们将建立在我们对细胞形状变化和机械作用的理解的基础上 响应性收缩网络。我们使用一套技术，包括遗传学、蛋白质组学、单分子 Pulldown (SiMPull) 以及荧光相关和互相关光谱 (FCS/FCCS) 进行研究 这个网络。我们发现机械响应收缩网络中的许多蛋白质都是 在细胞质中组织成复合物，形成收缩性试剂盒（CK）。多个 CK 组件具有 细胞收缩性中的未知功能是本提案的主题。其中，凝集素 盘状蛋白 1A 传统上被视为分泌蛋白，与细胞质中的 CK 组装， 关键蛋白肌动蛋白交联剂皮质西林 I 是完全定位到皮质所必需的。此外，盘状蛋白 1A 与皮质西林 I 及其结合伴侣 IQGAP1 和 IQGAP2 具有复杂的遗传关系。我们将 确定 discoidin 1 如何在 CK 中发挥作用并促进皮质组装。接下来我们要学习两个 核糖核蛋白 RNP1A 和 RNP1B。两种蛋白质都含有预测的 RNA 识别基序。 RNP1A 也是 正常皮质西林 I mRNA 水平所需的。我们最初将 RNP1A 过度表达确定为遗传因素 微管不稳定剂诺考达唑的抑制剂（同一研究产生了 RacE-14-3-3-肌球蛋白 II 我们发现的途径）。我们现在发现 RNP1A 是正常微管长度、细胞 粘附力和皮质力学。我们进行了 RNAseq 分析，发现一些 CK 蛋白具有 rnp1A 敲低细胞中基因表达发生改变。为了识别 RNP1 可能结合的 RNA，我们使用了 CLIP-Seq 并鉴定了几种 RNA 作为 RNP1A 和 RNP1B 的相互作用子。特别令人感兴趣的一项是 动力蛋白样蛋白 1，定位于裂沟皮层，协助肌动蛋白和肌球蛋白 II 组织。其他参与巨胞饮作用，这是另一个重要的细胞形状变化事件，也吸引了 大部分CK机械。在这里，我们将详细阐述 RNP1 如何影响 CK 组装、表达和 功能。总的来说，这些研究将破译 CK 网络如何运作以及如何与其他细胞集成 系统，从而更深入地了解细胞形状变化过程。