The actin filament bundling protein α-actinin-4 actually suppresses actin stress fibers by permitting actin turnover

Abstract

Cells organize actin filaments into contractile bundles known as stress fibers that resist mechanical stress, increase cell adhesion, remodel the extracellular matrix, and maintain tissue integrity. α-actinin is an actin filament bundling protein that is thought to be essential for stress fiber formation and stability. However, previous studies have also suggested that α-actinin might disrupt fibers, making the true function of this biomolecule unclear. Here we use fluorescence imaging to show that kidney epithelial cells depleted of α-actinin-4 via shRNA or CRISPR/Cas9, or expressing a disruptive mutant make more massive stress fibers that are less dynamic than those in WT cells, leading to defects in cell motility and wound healing. The increase in stress fiber mass and stability can be explained, in part, by increased loading of the filament component tropomyosin onto stress fibers in the absence of α-actinin, as monitored via immunofluorescence. We show using imaging and cosedimentation that α-actinin and tropomyosin compete for binding to F-actin and that tropomyosin shields actin filaments from cofilin-mediated disassembly and in cells. Perturbing tropomyosin in cells lacking α-actinin-4 results in a complete loss of stress fibers. Our results with α-actinin-4 on stress fiber organization are the opposite of what might have been predicted from previous biochemistry and further highlight how the complex interactions of multiple proteins competing for filament binding lead to unexpected functions for actin-binding proteins in cells.

Spatial integration of mechanical forces by alpha-actinin establishes actin network symmetry

“…For instance α-actinin competes with tropomyosins, which protects actin filaments from cofilin induced severing and activate myosinII (Gateva et al, 2017). This competition determines the rate of actin turn-over and thereby the assembly of stress fiber formation and the regulation of cell motility (Kemp and Brieher, 2018). We also found that alpha-actinin knockdown perturbs the orientation of contractile forces.…”

Post-mitotic expansion of cell nuclei requires ACTN4-mediated nuclear actin filament bundling

“…The underlying mechanism appears to be based on the ability of ACTN4 to bundle actin filaments (Honda et al, 1998;Winkelman et al, 2016), however, we also observed a role for actin assembly by nuclear ACTN4. Interestingly, ACTN4 has been previously shown to affect actin assembly or filament turnover (Kemp and Brieher, 2018). Hence, it is conceivable that a coordinated mechanism between actin assembly and bundling might be at work, further emphasizing the highly complex and dynamic nature of the actin cytoskeleton.…”

Functional Characterization and Proteomic Analysis of Porcine Deltacoronavirus Accessory Protein NS7

“…ACTN4 is a conserved, abundant, and ubiquitous actin-binding protein belonging to the spectrin superfamily, which modulates a wide range of processes [51][52][53][54][55][56][57][58]. As a component of the cytoskeleton, ACTN4 plays crucial roles in remodeling of the actin cytoskeleton, and in stress fiber formation and stability [59,60]. Stress fibers are contractile bundles of actin filaments that span the entire length of the cell and are anchored at both ends at focal adhesions, so actin presents a challenging hurdle to virus entry [61,62].…”