Single Molecule Imaging of Actin-Tropomyosin Complexes

The actin cytoskeleton is fundamental for cellular function and highly organized in space and time. The numerous functions of actin filaments ranging from determining cell shape to cell motility are regulated by a variety of actin-binding proteins. Tropomyosins are highly conserved critical constituents of actin filaments and differentially regulate filament stability and function. Tropomyosins are dimeric coiled-coil proteins that form head-to-tail polymers along the major groove of the actin filament. However, the mechanism of tropomyosin polymerisation and how it regulates actin functional diversity in combination with other actin-binding proteins is still uncertain. To understand these assembly mechanisms we have developed an imaging approach combining a microfluidic device and total internal reflection fluorescence (TIRF) microscopy to investigate the architecture of actin-tropomyosin filaments at different stages of the assembly process.