The Biochemical Basis for the Mechanics of Cytokinesis

项目摘要 GM 66817 -18 PAYNET GRANT 细胞形状的改变是细胞分裂、迁移和组织形成的基础， 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，重点是 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。在这段时间里，我们 已经证明了主动力的产生、皮质张力、表面曲率和 粘弹性驱动细胞形状变化，包括胞质分裂沟内移。我们发现了关键分子 控制这些特性的通路，并发现电路像一个控制系统一样连接， 反馈回路，允许机械和化学信号调节收缩的积累， 机械.在这项提案中，我们将建立在我们对细胞形状变化和机械的理解基础上， 反应性收缩网络。我们使用一套技术，包括遗传学，蛋白质组学，单分子 下拉（SiMPull）和荧光相关和互相关光谱（FCS/FCCS）来研究 这个网络。我们发现，机械反应收缩网络中的许多蛋白质是 在细胞质中组织成复合物，形成收缩性试剂盒（CK）。几个CK组件具有 在细胞收缩性的背景下未知的功能，是这个建议的主题。其中，凝集素 discoidin 1A，传统上被认为是一种分泌蛋白，在细胞质中与CK组装， 这是一个关键蛋白质，肌动蛋白交联剂皮质素I，完全定位于皮质所必需的。此外，discoidin 1A与cortexillin I及其结合伴侣IQGAP 1和IQGAP 2具有复杂的遗传关系。我们将 确定盘状蛋白1如何在CKs中起作用并促进皮质组装。接下来，我们学习两个 核糖核蛋白，RNP 1A和RNP 1B。这两种蛋白质都含有预测的RNA识别基序。RNP 1A也是 这是正常皮质素I mRNA水平所必需的。我们最初将RNP 1A过度表达确定为一种遗传性的 微管去稳定剂诺考达唑的抑制剂（与产生RacE-14-3-3-肌球蛋白II的研究相同 我们发现的路径）。我们现在已经发现RNP 1A是正常微管长度、细胞生长和细胞增殖所必需的。 粘附和皮质力学。我们进行了RNAseq分析，发现几种CK蛋白具有 在rnp 1A敲低细胞中改变基因表达。为了鉴定RNP 1可能结合的RNA，我们使用了 CLIP-Seq和鉴定了几种RNA作为RNP 1A和RNP 1B的相互作用物。其中一个特别感兴趣的是 动力蛋白样蛋白1，定位于卵裂沟皮质，在那里协助肌动蛋白和肌球蛋白II organization.其他参与巨胞饮，另一个重要的细胞形状变化事件，也提请 大部分的CK机器。在这里，我们将充实RNP 1如何影响CK组装，表达， 功能总的来说，这些研究将破译CK网络如何运作，并与其他蜂窝网络集成。 系统，从而更深入地了解细胞形状变化过程更普遍。

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.