A SH3 domain-based protein interaction network drives cytokinesis from contraction into abscission.

基于SH3结构域的蛋白相互作用网络驱动细胞质分裂从收缩到脱落。细胞周期中的过渡是由胞质溶胶及其底层蛋白质网络的组成和组织的变化引起的。这些差异通常受到相对较弱和短暂的蛋白质相互作用变化的影响。通过它们的合作和协同性质，这些网络仍然能够精确地确定酶和蛋白质机器的行动时间和地点。因此，深入表征这些动态蛋白质基质是理解细胞过程的确切时间和空间激活的先决条件。细胞分离过程中的脱落是在出芽酵母中通过合成次级隔膜实现的，次级隔膜紧随AMR收缩和PS合成。作为本提案的中心假设，我假设从AMR收缩到脱落的转变伴随着中央细胞动力学复合物的溶解和nba1复合物的同时形成。这两种复合物都是由小的sh3结构域相互作用网络构建的，并与Hof1p和Cyk3p共享两个共同的亚基。该建议旨在描述这两种复合物在时间和空间分辨率上的细胞形成和溶解，并将这些事件与细胞质分裂期间同时发生的其他活动联系起来。其亚基的特性让我们假设nba1复合物在细胞分裂过程中支持胞外分泌，并微调GDP和gtp负载的rho - gtpase之间的平衡。我们将建立在细胞分裂过程中跟踪单个分泌囊泡的方法，并监测rho - gtpase的活性。使用我们的基于Split-Ubiquitin的酵母阵列，我们将额外搜索RhoGAPS， gef和关键的rho依赖性效应物的结合伙伴。在细胞分裂过程中，将测试获得的hit对这些蛋白的定位和活性的影响。对CCC-和nba1复合物及其个体相互作用的功能注释所提出的分析的核心是创建只有一个相互作用被选择性消除的等位基因。我们将利用我们最近建立的基于分裂泛素的生/死策略，通过非结合选择丰富这些等位基因。所需的突变将由NGS识别，随后将其设计到酵母的基因组中，以测试它们对AMR收缩到脱落过渡的影响。该建议的目的是理解和可视化细胞质分裂作为协作蛋白相互作用状态的顺序进展。我们的分析将确定该网络中作为监管点的节点。这些干预节点对于协调染色体分离和脱落是重要的。我们相信这些节点及其调控机制是通过进化而保存下来的。

A SH3 domain-based protein interaction network drives cytokinesis from contraction into abscission.The transitions during the cell cylce are caused by changes in the composition and organization of the cytosol and its underlying protein networks. These differences are often affected by changes in comparatively weak and temporal protein interactions. Through their cooperative and synergistic nature these networks are still able to precisely determine the when and where of the enzymes and protein machines actions. The in-depth characterization of these dynamic protein matrices is thus a prerequisite for understanding the exact timing and spatial activation of cellular processes.Abscission during cell separation is achieved in the budding yeast by the synthesis of the secondary septum that immediately follows AMR constriction and PS synthesis. As central hypothesis of this proposal I postulate that the transition from AMR contraction to abscission is accompanied by the dissolution of the central cytocinetic complex and the simultaneous formation of the Nba1-complex. Both complexes are built by small SH3-domain interactions networks and share with Hof1p and Cyk3p two common subunits. The proposal aims at describing the cellular formation and dissolution of both complexes in time and spatial resolution, and to correlate these events with the other simultaneously occurring activities during cytokinesis.The identities of its subunits let us assume that the Nba1-complex supports exocytosis during cytokinesis and fine-tunes the balance between GDP- and GTP-loaded Rho-GTPases. We will establish methods to follow single secretory vesicles during cytokinesis and monitor the activities of the Rho-GTPases. Using our Split-Ubiquitin based yeast arrays we will additionally search for binding partners of the RhoGAPS, GEFs and the critical Rho-dependent effectors. The obtained hits will be tested for their impact on localization and activities of theses proteins during cytokinesis.Central for the proposed analysis of the CCC- and the Nba1-complex and the functional annotations of their individual interactions are the creation of alleles where only one interaction is selectively eliminated. We will harness our recently established Split-ubiquitin based life/death strategy that enriches these alleles through a selection for non-binding. The desired mutations will be identified by NGS and subsequently engineered into the genome of the yeast to test their impact onto the transition from AMR contraction to abscission.The aim of the proposal is to understand and visualize cytokinesis as sequential progression of collaborating protein interaction states. Our analysis will identify the nodes within this network that serve as points of regulation. These nodes of intervention are important to coordinate chromosome separation with abscission. We are convinced that these nodes and the mechanism of their regulation are conserved through evolution.