Regulation and Function of Low Molecular Weight Tropomyosins

这项工作的目的是了解原肌球蛋白（TM）异构体的多样性在肌动蛋白丝的组织和功能的作用。在非肌肉细胞中，肌动蛋白丝在细胞运动、膜皱褶、丝状伪足形成、细胞分裂、细胞内运输、细胞形状调节、粘附和粘附介导的信号传导中起重要作用。TM是肌动蛋白结合蛋白家族，其结合到丝状肌动蛋白的两个沟。尽管它们在所有真核细胞中表达，但不同的形式是特定细胞类型的特征。在成纤维细胞中，TM以含有284个酸的高分子量同种型（HMW）（TM-1、TM-2和TM-3）或含有248个酸的低分子量同种型（LMW）（TM-4、TM-5（NM-1）、TM-5a和TM-5 b）存在。在成纤维细胞中发现的多个TM是微丝系统的组成部分，尽管不同的同种型如何有助于肌动蛋白丝组装和功能知之甚少。这些卷曲螺旋相互作用如何影响TM与肌动蛋白丝的相互作用及其随后的细胞功能将被确定。使用纯化的组件，异构体功能的基本属性将被研究，如卷曲螺旋结构（同源二聚体与异源二聚体）如何影响它们对F-肌动蛋白的亲和力和与其他TM的协同性。此外，TM的F-肌动蛋白的结合是如何影响钙调素将被研究。这些研究还将为进一步的实验提供基础，以确定TM沿着肌动蛋白丝的组成如何影响肌球蛋白I和肌球蛋白II的功能。将用GFP-TM融合蛋白分析活成纤维细胞中每种同种型的动态定位，以确定特定的TM是否结合到肌动蛋白丝的空间上不同的子集，包括收缩环、丝状伪足、板状伪足、将通过表达突变的TM和引入抑制性反义寡脱氧核苷酸或RNA来破坏它们的功能来研究特定TM在运动过程中的参与。这些实验将确定特定亚型的突变或减少是否会导致细胞功能的变化，如微丝束和丝状伪足的丢失或胞质分裂的缺陷。总的来说，这些研究将为特定TM亚型的关联如何有助于不同肌动蛋白丝结构的组装和调节提供重要的新见解，并将为未来的研究提供基础。此外，这些研究将提供具体的TM如何与不同的肌动蛋白组件，包括应力纤维，收缩环和丝状伪足的机制的见解。

The goal of this work is to understand the role of tropomyosin (TM) isoform diversity in actin filament organization and function. In nonmuscle cells, actin filaments play important roles in cell movement, membrane ruffling, filopodium formation, cell division, intracellular transport, regulation of cell shape, adhesion and adhesion-mediated signaling. TMs are a family of actin-binding proteins that bind to both grooves of filamentous actin. Although they are expressed in all eukaryotic cells, different forms are characteristic of specific cell types. In fibroblasts TMs exist as high molecular weight isoforms (HMW) containing 284 acids (TM-1, TM-2 and TM-3) or low molecular weight isoforms (LMW), containing 248 acids (TM-4, TM-5(NM-1), TM-5a, and TM-5b). The multiple TMs found in fibroblasts are an integral part of the microfilament system, although how the different isoforms contribute to actin filament assembly and function is poorly understood.In fibroblasts, HMW TMs exist as homodimers whereas LMW TMs can exist as heterodimers. How these coiled-coil interactions affect the interaction of TMs with actin filaments and their subsequent cellular function will be determined. Using purified components, fundamental properties of isoform function will be studied, such as how the coiled-coil structure (homodimers versus heterodimers) affects their affinity for F-actin and cooperativity with other TMs. In addition, how the binding of TMs to F-actin is affected by caldesmon will be studied. These studies will also provide the foundation for further experiments to determine how the composition of TMs along actin filaments affect the function of myosin I and myosin II.The dynamic localization of each isoform in living fibroblasts will be analyzed with GFP-TM fusion proteins, to determine if specific TMs bind to spatially distinct subsets of actin filaments including the contractile ring, filopodia, lamellipodia, and stress fibers.The involvement of specific TMs in motile processes will be studied by disrupting their function by expression of mutant TM and introduction of inhibitory antisense oligodeoxynucleotides or RNAs. These experiments will determine whether a mutation or decrease in a specific isoform will lead to changes in cell function, such as loss of microfilament bundles and filopodia or defects in cytokinesis.Collectively, these studies will provide important new insights into how the association of specific TM isoforms contribute to the assembly and regulation of distinct actin filament structures, and will provide the foundation for future studies. Furthermore, these studies will provide mechanistic insights into how specific TMs associate with different actin assemblies including stress fibers, contractile ring and filopodia.