Direct Visualization of Tropomyosin Isoform Binding to F-Actin

Abstract

There are more than forty different isoforms in mammalian cells produced via alternative splicing from four separate genes. These isoforms have varying expression levels in different cell types and are targeted to specific regions of cells. In turn, the proper regulation of actin-binding protein interactions relies on the ability of different isoforms, each with its own unique actin-binding properties, to target a particular subset of actin filaments and define filament functions. Despite the large number of studies that have investigated the various cellular roles played by tropomyosin, the mechanism of differential binding to actin filaments remains uncertain.In this work, we used TIRF microscopy to monitor Alexa-532-labeled binding to actin. We found that random, weak monomer binding leads to the formation of sites with enhanced affinity for the actin filament-a process most likely dependent on the end-to-end interactions. Stepwise changes in fluorescence intensity observed indicate that three molecules are required to form a stable nucleation site. From these sites, subsequent chain elongation is rapid and appears to depend on the strength of such end-to-end tropomyosin linkages. This mechanism, which is strongly dependent on polymer formation rather than initial monomer binding onto the actin filament, suggests that isoform sorting may be intrinsic to the end-to-end linkage sites. A mixture of Tm2-Cy3 and Tm5NM1-Cy5.5 labeled tropomyosins was shown to segregate on F-actin and form distinct patches along actin filaments. We hypothesize that the C-terminal region of tropomyosin, which is highly variable among isoforms, is the primary determinant in distinguishing among different isoforms.