Monitoring the Real-Time Binding of Tropomyosin to Actin using Total Internal Reflection Fluorescence Microscopy

Abstract

Tropomyosin, an elongated actin binding protein, regulates the access of numerous other proteins onto the thin filament in virtually all cell types. This association, in turn, regulates various functions. Tropomyosin monomers assemble end-to-end to form a continuous polymeric chain that lies along the length of the actin filament. A number of different tropomyosin isoforms are present in all three muscle types: viz. smooth, cardiac, and skeletal. In cardiac and skeletal muscle, tropomyosin and troponin are the primary regulators of contraction. A plethora of tropomyosin isoforms are also present in non-muscle cells where they associate with cytoskeletal actin and play a role in numerous cellular processes including lamellipodia formation and actin-based cell motility. Moreover, various mutations in tropomyosin are associated with diseases such as congenital fiber type distortion (CFTD), ulcerative colitis, nemaline myopathy, and various cardiomyopathies. Tropomyosin is a modular protein, and individually charged residues within its repeating domains are thought to be critical for actin binding. However, the mechanism of tropomyosin binding to actin is still uncertain. In the present study, we have acquired real-time videos of skeletal and smooth tropomyosin isoforms binding to actin filaments using total internal reflection fluorescence microscopy (TIRF). The videos suggest that weak monomer binding facilitates the gradual formation of small “nuclei” of tropomyosin monomers and/or oligomers. Once the nucleus is formed, the affinity of tropomyosin for actin increases exponentially and additional monomers are able to rapidly add to either end of the growing tropomyosin chain lying on F-actin.