The Biochemical Basis for the Mechanics of Cytokinesis

项目摘要 胞质分裂，一个细胞分裂成两个子细胞，作为一个优雅的细胞行为，突出了细胞的分裂。 许多细胞形状变化过程所需的生物力学系统。在这段时间里，我们 证明了如何相互作用的积极力量的生产，皮层张力，表面曲率，和粘弹性， 驱动胞质分裂沟内移。我们确定了控制这些特性的关键分子途径， 电路就像一个控制系统，带有反馈回路， 调节收缩机制的信号。在这一建议中，我们将建立在我们的 理解胞质分裂和这种机械反应性收缩网络。我们使用一套技术， 包括遗传学，蛋白质组学，单分子下拉（SiMPull），荧光相关和交叉， 相关光谱（FCS/FCCS）来研究这个网络。我们发现，许多蛋白质中， 机械响应收缩网络在细胞质中被组织成复合物，形成收缩网络。 试剂盒（CK）。几种CK组分在细胞收缩性的背景下具有未知的功能， 这个提议。其中，传统上被视为分泌蛋白的盘状凝集素1A与 细胞质中的CK，是一个关键蛋白，肌动蛋白交联剂皮质素I，完全定位于 大脑皮层此外，discoidin 1A与cortexillin I及其结合配偶体具有复杂的遗传关系 和调节器IQGAP 2。我们将确定discoidin 1如何在CKs中起作用并促进皮质组装。 接下来，我们研究两种核糖核蛋白，RNP1A和RNP1B。两种蛋白质都含有预测的RNA- 识别基序RNP1A也是正常皮质素I mRNA水平所必需的。我们最初发现RNP1A 作为微管去稳定剂诺考达唑的遗传抑制剂的过表达（同样的研究引起了 我们发现的RacE-14 - 3 - 3-肌球蛋白II途径）。我们现在已经发现RNP1A是必需的， 正常微管长度。考虑到皮质素I mRNA水平的变化，我们进行了RNAseq分析， 并发现几种CK蛋白在rnp1A敲低细胞中改变了基因表达。为了识别RNA RNP1B可能会结合，我们正在使用CLIP-Seq，并在初步研究中发现RNP1B 可能与14 - 3 - 3 mRNA结合。在这里，我们将充实RNP1如何影响CK组装和表达。最后， 我们发现腺嘌呤核苷酸移位酶（ANT，由网骨藻中的ancA编码）与 与肌球蛋白II和racE基因。有趣的是，我们发现CK蛋白无效（例如，Cortexillin I）具有 代谢活动减少，导致ATP产生减少和能量状态降低。我们将探讨如何 通过ANT增加能量产生可以绕过CK蛋白的一些细胞功能。总的来说， 这些研究将试图破译CK网络是如何运作的，以及如何与其他过程相结合，例如 mRNA水平和代谢，从而更深入地了解细胞形状的变化过程 一般来说。

PROJECT SUMMARY Cytokinesis, the division of a cell into two daughter cells, serves as an elegant cell behavior that highlights the biomechanical systems required for many cell shape change processes. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive 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 cytokinesis and this 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 partner and regulator 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. Given the changes in mRNA levels of cortexillin I, 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 are using CLIP-Seq and have already found in a preliminary study that RNP1B may bind to 14-3-3 mRNA. Here, we will flesh out how the RNP1s impact CK assembly and expression. Finally, we discovered that the adenine nucleotide translocase (ANT, encoded by ancA in Dictyostelium) interacts genetically with myosin II and racE. Intriguingly, we are finding that CK protein nulls (e.g., cortexillin I) have reduced metabolic activity, leading to reduced ATP production and a lower energetic state. We will probe how increasing energy production through ANT can bypass some of the cellular functions of the CK proteins. Overall, these studies will seek to decipher how the CK network operates and integrates with other processes, such as mRNA levels and metabolism, leading to a deeper understanding of the cell shape change process more generally.