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.

项目摘要 GM66817-18父母赠款 细胞形状变化对于细胞分裂，迁移和组织形成至关重要，细胞形状缺陷 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，我们利用细胞因子作为优雅的模型过程。在这笔赠款的一生中，我们 已经证明了活跃力产生，皮质张力，表面曲率和 粘弹性驱动细胞形状的变化，包括细胞因子沟插入。我们确定了钥匙分子 控制这些属性并发现电路像控制系统一样连接的途径 反馈回路允许机械和化学信号调整收缩的积累 机械。在此提案中，我们将基于对细胞形状变化和机械的理解 - 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 下拉（Simpull）以及荧光相关性和互相关光谱（FCS/FCC） 这个网络。我们发现机械响应性收缩性网络中的许多蛋白质是 组织成细胞质中的复合物，形成收缩力试剂盒（CKS）。几个CK组件有 在细胞收缩性的背景下，未知功能是该提案的主题。其中，凝集素 传统上被视为分泌蛋白的盘状1a与细胞质中的CK组装在一起，IS 对于关键蛋白质，肌动蛋白交叉链接蛋白I的蛋白质I完全定位于皮质。而且，盘状 1A与Cortexillin I及其结合伙伴IQGAP1和IQGAP2具有复杂的遗传关系。我们将 确定盘中1在CK中的运行方式并促进皮质组件。接下来，我们正在研究两个 核糖核蛋白，RNP1A和RNP1B。两种蛋白质都包含预测的RNA识别基序。 RNP1A也是 正常皮质脱甲基蛋白I mRNA水平所需。我们最初将RNP1A过表达确定为遗传 微管destabilizer nocodazole的抑制剂（同一研究引起了Race-14-3-3-肌球蛋白II 我们发现的途径）。现在，我们发现正常微管长度需要RNP1A 粘附和皮质力学。我们进行了RNASEQ分析，发现几种CK蛋白具有 RNP1A敲低细胞中基因表达改变。为了识别RNP1可能绑定的RNA，我们使用了 夹子seq并将几个RNA确定为RNP1A和RNP1B的交互作用。特别感兴趣的之一是 Dynamin样蛋白1，该蛋白1位于裂解沟皮皮质中，该皮质有助于肌动蛋白和肌球蛋白II 组织。其他人则参与大型细胞增多症，这是另一个必不可少的细胞形状变化事件，也绘制 在大部分CK机械上。在这里，我们将充实RNP1如何影响CK组件，表达和 功能。总体而言，这些研究将破译CK网络如何与其他细胞集成 系统，更深入地了解细胞形状变化过程。