Regulation and Function of Low Molecular Weight Tropomyosins

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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.