Journal Article

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

Weitz, David A., Bidan, Cécile M., Krishnan, Ramaswamy, Pollak, Martin R., Ehrlicher, Allen J., Guo, Ming

2015

Overview

Significance In this study, we examine how proteins that cross-link actin filaments control certain biophysical aspects of living cells. We studied α-actinin-4 (ACTN4) a dimeric rod-shaped homodimer, of particular interest because mutations in its actin binding domain cause a human disease characterized by dysfunction of the kidney’s glomeruli; however, the mechanical impact of such mutations are unknown. We find that the human disease-causing K255E mutation in ACTN4 leads to a change in cellular biophysical properties, increasing the affinity for actin increases cellular forces and work, while decreasing cell movement. These observations describe the effects of variable cross-linking on cellular forces and dynamics, and reveal how pathology may arise mechanically from disruptive point mutations in cytoskeletal proteins. The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: actin / Actinin - chemistry / Actinin - genetics / Actinin - physiology / Actins - metabolism / Amino Acid Substitution / Binding Sites - genetics / Biological Sciences / Biomechanical Phenomena / Cell Line / cell movement / Cell Movement - genetics / Cell Movement - physiology / Cells / Cross-Linking Reagents / crosslinking / Cytoplasm / Cytoskeleton / Fluorescence / Fluorescence Recovery After Photobleaching / HeLa Cells / human diseases / Humans / kidneys / Kinetics / microfilaments / Microscopy, Confocal / Models, Biological / Mutagenesis, Site-Directed / Mutation / Photobleaching / point mutation / Protein Binding / Proteins / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / Traction