The Biochemical Basis for the Mechanics of Cytokinesis

描述(申请人提供)：细胞质分裂是细胞形状变化的戏剧性例子，在此过程中，收缩环的机械收缩导致有丝分裂结束时细胞分离。机械力主要由基于肌动蛋白的肌球蛋白-马达蛋白产生。胞质分裂对于正常的细胞增殖是必不可少的，由于它在癌症等增殖性疾病中的作用而具有医学价值。 在盘状网柄藻中，两个具有互补细胞分布的肌动蛋白束蛋白之间的遗传相互作用已被确定。皮质素I定位于收缩环，而强皮质素则在皮质上丰富，但不在收缩环之外。Dyacortin是一种新型的二聚体肌动蛋白结合蛋白。一个dynacortin片段(C181)破坏了天然的dynacortin复合体，并抑制了皮质菌素I突变株的胞质分裂缺陷。这意味着，细胞已经进化出独特的肌动蛋白交联蛋白，具有互补的细胞分布，协调细胞形状的变化。 在这项研究中，我们将利用各种平衡和动力学技术研究重组表达和纯化的全长dynacortin、C181和异源二聚体与肌动蛋白结合/结合的机制。由于dynacortin在体内是磷酸化的，因此将从Dictybone fium中提纯该蛋白，并测试其结合和捆绑肌动蛋白细丝的能力。磷酸化dynacortin的激酶将通过在体外磷酸化dynacortin的能力而纯化到均一组分。这些酶将通过质谱学进行鉴定，并使用基因敲除技术和GFP融合蛋白的荧光显微镜进行研究。纯化的酶将被用来磷酸化重组动力皮质素，以便可以充分定量特定磷酸化状态的影响。另一种蛋白质，DdERM，也是在皮质素I的抑制屏幕中分离出来的，将被研究。DdERM与Ezrin-Radixinmoesin蛋白家族有关，该家族将皮质肌动蛋白细胞骨架连接到质膜上。我们假设DdERM将共享这一财产。通过对该蛋白的研究，可以评估肌动蛋白皮层网络与质膜连接在胞质分裂中的作用。最后，涉及皮质形状控制的新基因将通过饱和皮质素I抑制因子的遗传选择实验和筛选肌球蛋白II缺失细胞的增强子来识别。

DESCRIPTION (provided by applicant): Cytokinesis is a dramatic example of a cell shape change during which the mechanical constriction of the contractile ring leads to cell separation at the end of mitosis. The mechanical forces are largely generated by actin-based myosin-motor proteins. Cytokinesis is essential for normal cell proliferation and is of medical interest for its role in hyperproliferative diseases such as cancer. In Dictyostefium discoideum, genetic interactions have been identified between two actin bundling proteins that have complementary cellular distributions. Cortexillin I is localized to the contractile ring while dynacortin is cortically enriched but excluded from the contractile ring. Dynacortin is a novel dimeric actin bundling protein. A fragment of dynacortin (C181) disrupts the native dynacortin complex and suppresses the cytokinesis defect of cortexillin I mutant strains. The implication is that cells have evolved distinct actin cross-linking proteins with complementary cellular distributions that orchestrate cell shape changes. In this proposal, the mechanisms of actin binding/bundling by recombinantly expressed and purified full-length dynacortin, C181, and the heterodimer will be studied using a variety of equilibrium and kinetic techniques. Since dynacortin is phosphorylated in vivo, the protein will be purified from Dictyostefium and tested for its ability to bind and bundle actin filaments. Kinases that phosphorylate dynacortin will be purified to homogeneity by following their ability to phosphorylate dynacortin in vitro. These enzymes will be identified by mass spectroscopy and studied using gene knockout technology and fluorescence microscopy of GFP-fusion proteins. Purified enzymes will be used to phosphorylate recombinant dynacortin so that the effect of a particular phosphorylation state may be fully quantitated. Another protein, DdERM, which was also isolated in a suppressor screen of cortexillin I, will be studied. DdERM is related to the ezrin-radixinmoesin family of proteins, which tether the cortical actin cytoskeleton to the plasma membrane. We hypothesize that the DdERM will share this property. By studying this protein, the role of tethering of the actin cortical meshwork to the plasma membrane in cytokinesis may be evaluated. Finally, new genes involved in cortical shape control will be identified by saturating the genetic selection experiment for suppressors of cortexillin I and by screening for enhancers of myosin-II null cells.