Biased localization of actin binding proteins by actin filament conformation

Andrew R Harris,Andreas R Bausch,Daniel A Fletcher,Pamela Jreij,Aaron M Joffe,Brian Belardi

doi:10.1038/s41467-020-19768-9

Abstract

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1–CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

Highlights

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear
We investigated whether filament conformational changes could be sensed by a common class of actin-binding domain, tandem calponin homology domains (CH1–CH2), and if differences in affinity for F-actin conformations could potentially influence the localization of CH1–CH2-containing proteins to different actin structures in cells
Using a combination of live cell imaging and in vitro single molecule binding kinetics measurements, we find that mutants of the actin-binding domain of utrophin (CH1–CH2) localize to different actin structures and exhibit different binding kinetics on actin filaments whose conformational state has been altered biophysically and biochemically

Summary

Introduction

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop. In addition to branched actin networks in the lamellipodium, and stress fibers in the cell body, advances in both optical and electron microscopy continue to reveal more details about the organization and assembly of a broader range of actin structures, including filopodia[3], asters and stars[4], podosomes[5] and patches[6] In each of these structures, the interaction of actin filaments with a vast set of accessory proteins promotes the formation of distinct cytoskeletal architectures. We show that this mechanism potentially extends to native actin-binding domains, suggesting that sensitivity to actin filament conformational states could be playing an important role in the organization and regulation of actin-binding proteins in actin filament structures

Methods

Results

Conclusion