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Deciphering the tropomyosin code: Uncovering how different tropomyosin isoforms bind and assemble on actin filaments

Abstract

Actin is a highly conserved protein that assembles into many three-dimensional structures, each with a unique subset of actin-binding proteins that produces a unique three-dimensional architecture capable of performing a specific function. These actin networks are central to cellular processes such as motility, adhesion, endocytosis, and division. Yet how actin filaments can recruit the correct subset of actin-binding proteins while avoiding the incorrect ones remains unclear. Recent literature demonstrated that in non-muscle cells, a different tropomyosin isoform(s) is present at every single actin network. Tropomyosins are coiled-coil proteins that regulate access to actin filament binding sites and now widely believed to determine which actin structure will be built. Despite this, we know very little about the assembly and function of tropomyosins on actin filaments. This is partly due to the fact that mammalian cells express over 20 different tropomyosin isoforms. In the Mullins lab, we have chosen Drosophila melanogaster S2 cells as the model system for the study of tropomyosins because S2 cells are easy to grow, highly susceptible to gene inhibition using RNAi, have well-characterized actin networks, and most importantly, only express three tropomyosin isoforms. In this thesis, we investigate how these Drosophila tropomyosins assemble on actin filaments. In Chapter 1, we designed a microfluidic system that allows us to manipulate microscopy experiments at a level never before achieved in the Mullins lab. In Chapter 2, we characterize binding and dynamic properties of the classic Drosophila tropomyosin Tm1A. In Chapter 3, we discovered how to get Drosophila tropomyosins Tm1J and Tm2A to bind actin filaments and then characterize these tropomyosins.

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