Reconstitution of Dynamic Actin Cables with Tunable Lengths

Abstract

The sizes and functions of many actin-based cellular structures, such as microvilli, stereocilia, filopodia and polarized actin cables, depend critically on the precise control of actin bundle length and turnover dynamics. Yeast cells offer an ideal system in which to study principles of cytoskeletal length control, because they contain discrete linear actin cables that extend to well-defined lengths (3-7 μm), matching the cell compartment size, despite undergoing highly rapid turnover. Yeast is also simpler compositionally and more genetically amenable compared to most animal cells. Over the past 30 years, the factors involved in yeast actin cable formation have been identified, and many of these individual parts have been studied for their biochemical activities. Yet it has remained unclear how this complex group of proteins works in concert to build a single actin-based structure of a specified length and architecture. Here, we used purified components to reconstitute the formation of yeast actin cables that dynamically turnover and have steady-state lengths similar to those observed in vivo. Cables are rapidly polymerized at one end by formins immobilized on beads, in a profilin-dependent manner, and disassembled at the other end by the combined actions of Cofilin, Coronin, and AIP1. Cable length is tuned by Tropomyosin, which decorates the sides of actin cables and antagonizes the disassembly-promoting factors. Capping protein restricts actin polymerization to the formin-coated beads. Bundling proteins with different properties alter the geometry and dynamics of the cable networks. Together, our results offer new mechanistic insights into the sculpting of actin cables through orchestrated assembly, turnover, and crosslinking.