Molecular Genetics of the Cytoskeleton

细胞的形状、极性和运动由肌动蛋白细胞骨架控制。F-肌动蛋白细丝如何组装成功能阵列仍然知之甚少。有许多F-肌动蛋白结合蛋白能够使肌动蛋白细丝交联，这些蛋白在产生的肌动蛋白细丝的排列和结合活性的调节上有所不同。它们可以产生平行的、反平行的或正交的F-肌动蛋白阵列，但这些蛋白质之间复杂的相互作用尚不清楚。此外，许多肌动蛋白结合蛋白受钙离子等因素的调节，这一点的重要性尚不清楚。这项提议寻求使用分子遗传学和成像方法来继续研究细胞用来调节肌动蛋白细丝阵列组装的机制。肌球蛋白II已被发现是网柄蕨类细胞细胞骨架完整性的主要贡献者。肌球蛋白II对施加在表面的力的贡献将被调查，并使用弹性底物分析与其他肌动蛋白结合蛋白的贡献进行比较。肌球蛋白II对哺乳动物细胞完整性的贡献将通过RNAi在组织培养细胞中抑制肌球蛋白LLB的表达来研究。对ABP120和α-肌动蛋白的研究表明，这些蛋白的肌动蛋白结合域调节它们与F-肌动蛋白细丝的结合。这导致了一种假设，即并不是所有细胞中的肌动蛋白细丝都是相同的，因为这些蛋白质可以区分细胞不同部分的细丝。这一重要思想将通过研究肌动蛋白结合域在体内和体外的性质来进一步研究。钙离子在调节α-肌动蛋白定位中的作用也将被研究，以确定在什么情况下钙离子调节变得重要。Fimbrin是这类肌动蛋白交联蛋白的另一个成员。Dictyostelials细胞中有两种Fimbrin同源物，FimA和FIMB。FIMA基因类似于哺乳动物的flmbrin，具有EF-Hand和两个肌动蛋白结合域。FIMB是一种新近发现的同源基因，缺乏EF手，但具有PH结构域和Talin同源结构域。每种蛋白质的功能将通过基因破坏、突变和定位技术进行研究。这些蛋白质的肌动蛋白结合域的结构也将被确定，以研究它们识别肌动蛋白细丝的不同机制。

Cell shape, polarity and motility are controlled by the actin cytoskeleton. How F-actin filaments are assembled into functional arrays is still poorly understood. There are numerous F-actin binding proteins that are capable of cross-linking actin filaments and these proteins vary in the arrangement of filaments produced and the regulation of the binding activity. They can produce parallel, anti-parallel or orthogonal arrays of F-actin, but the complex interplay of these proteins is poorly understood. IN addition, a number of actin-binding proteins are regulated by factors such as Ca++ and the circumstance where this is important is not known. This proposal seeks to use molecular genetic and imaging approaches to continue to investigate the mechanisms used by cells to regulate the assembly of actin filament arrays. Myosin II has been found to be a major contributor to cytoskeletal integrity of Dictyostelium cells. The contribution of myosin II to forces applied to the surface will be investigated and compared to the contributions of other actin binding proteins using a flexible substratum assay. The contribution of myosin II to mammalian cell integrity will be investigated by using RNAi to inhibit the expression of myosin llB in tissue culture cells. Studies of ABP120 and alpha-actinin have shown that the actin binding domains of these proteins regulate their association with F-actin filaments. This has led to the hypothesis that not all actin filaments in cells are the same, since these proteins can distinguish between filaments in different parts of the cell. This important idea will be further investigated by studying the properties of the actin binding domains in vivo and in vitro. The role of Ca++ in regulating alpha-actinin localization will also be investigated in order to determine under what circumstances Ca++ regulation becomes important. Fimbrin is another member of this class of actin crosslinking proteins. There are two homologs of fimbrin in Dictyostelium cells, fimA and fimB. The fimA gene is similar to mammalian flmbrin having EF-hands and two actin-binding domains. FimB is a recently discovered homolog that lacks EF hands, but has PH domains and a talin homology domain. The function of each protein will be investigated by gene disruption, mutagenesis and localization techniques. The structure of the actin binding domains of each of these proteins will also be determined to investigate the mechanism by which they differentially recognize actin filaments.