The Biochemical Basis for the Mechanics of Cytokinesis

细胞分裂机制的生化基础

基本信息

批准号：
10685956
负责人：
DOUGLAS N ROBINSON
金额：
$ 33.73万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
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/10685956
关键词：
Actins Actomyosin Adenine Nucleotide Translocase Affinity Area Binding Biochemical Biological Assay Biology Biomechanics Bypass Cell Proliferation Cell Shape Cell model Cell physiology Cells Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Collection Colorectal Cancer Complex Crosslinker Cytokinesis Cytoplasm Cytoprotection Cytoskeletal Proteins Cytoskeleton Data Development Dictyostelium Dictyostelium discoideum Diffusion Disease Endowment Engineering Environmental Hazards Exposure to Feedback Filament Fluorescence Gene Expression Genetic Grant Hepatocyte Human In Vitro KIT gene Lectin Length Life Link Liquid substance Lung Maintenance Malignant neoplasm of pancreas Mammalian Cell Maps Measurement Mechanical Stress Mechanics Messenger RNA Metabolic Metabolism Microtubules Molecular Myoblasts Myosin Type II Nocodazole Organism Outcome Oxidative Phosphorylation Pathway interactions Phenotype Process Production Property Protein Secretion Proteins Proteomics RNA RNA Recognition Motif RNA-Binding Proteins Race Ribonucleoproteins Role Signal Transduction Site Spectrum Analysis Surface Surveys System Techniques Transcript Work cell behavior cell cortex cellular imaging cortexillin I crosslinking and immunoprecipitation sequencing daughter cell follow-up in vivo individualized medicine knock-down mRNA Stability mutant overexpression physical process reconstitution single molecule small molecule stoichiometry transcriptome sequencing viscoelasticity

项目摘要

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.

项目摘要细胞因子分为两个子细胞的细胞因子，是一种优雅的细胞行为，突出显示许多细胞形状变化过程所需的生物力学系统。在这笔赠款的一生中，我们有展示了活性力产生，皮质张力，表面曲率和粘弹性的相互作用驱动细胞因子沟插入。我们确定了控制这些特性并发现的关键分子途径电路像控制系统一样连接，并配有反馈回路，可以机械和化学信号调节收缩机械的积累。在此提案中，我们将建立在我们的基础上了解细胞因子和该机械响应性收缩性网络。我们使用一套技术，包括遗传学，蛋白质组学，单分子下拉（Simpull）以及荧光相关性和交叉相关光谱（FCS/FCC）研究该网络。我们发现机械响应性收缩性网络被组织成细胞质中的复合物，形成了收缩力套件（CKS）。几个CK组件在细胞收缩性的背景下具有未知功能，并且是主题该提议。其中，传统上将凝集素盘状1a视为分泌的蛋白质，与细胞质中的CKS是关键蛋白肌动蛋白交叉链链甲基甲基蛋白I的必要条件，才能完全定位到皮质。此外，Discoidin 1a与Cortexillin I及其结合伴侣具有复杂的遗传关系和调节器IQGAP2。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，我们正在研究两种核糖核蛋白RNP1A和RNP1B。两种蛋白质都包含预测的RNA- 识别图案。正常的皮质辛I mRNA水平也需要RNP1A。我们最初确定RNP1A 过表达作为微管destabilizer nocodazole的遗传抑制剂（同一研究产生到我们发现的Race-14-3-3-肌球蛋白II途径）。我们现在发现RNP1A是必需的正常的微管长度。鉴于Cortexillin I的mRNA水平的变化，我们进行了RNASEQ分析并发现几种CK蛋白改变了RNP1A敲低细胞中的基因表达。识别RNA RNP1可能会结合，我们正在使用夹子序列，并且在初步研究中已经发现RNP1B 可以与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易位酶（ANT，由dictyostelium中的ANCA编码）相互作用与肌球蛋白II和种族一起遗传。有趣的是，我们发现CK蛋白质无效（例如，Cortexillin I）具有代谢活性降低，导致ATP产生降低和降低能量状态。我们将调查如何通过蚂蚁增加能量产生可以绕过CK蛋白的某些细胞功能。全面的，这些研究将寻求破译CK网络如何运行并与其他流程集成，例如 mRNA水平和代谢，导致对细胞形状变化的更深入了解更多一般来说。