Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin.

Abstract

Actin filaments are dynamic polymers whose length depends on regulated monomer association and dissociation at their ends. Actin barbed-end dynamics are relatively better understood, primarily due to the approximately tenfold faster subunit on/off rates at barbed versus pointed ends. We present experimental approaches to selectively assay actin pointed-end regulation using bulk biochemistry, single filament imaging, and live cell microscopy with an emphasis on tropomodulins (Tmods), a conserved family of eukaryotic proteins that specifically cap pointed ends. Average pointed-end assembly/disassembly rates are measured in bulk solution using pyrene-labeled actin and barbed end-capping protein CapZ. Direct rate measurements of individual pointed ends are performed via microfluidic-assisted total internal reflection fluorescence microscopy (mf-TIRF). Actin pointed-end dynamics in living cells are examined in striated muscle cells expressing fluorescent actin, where the regular arrays of 1- to 2-μm-long actin filaments in sarcomeres enable visualization of filament pointed and barbed ends. These assays will also help advance our understanding of other pointed end regulators, including cyclase-associated protein and leiomodins, which have been implicated in filament stabilization, disassembly, and elongation. This work is relevant to the musculoskeletal field, where precise regulation of filament lengths is particularly critical for sarcomere organization and striated muscle contraction.