Alpha-actinin binding kinetics modulate cellular dynamics and force generation

Abstract

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 crosslinker 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.Alpha-actinin | actin | kidney disease | cell mechanics | traction force M ovement, morphology, and force production are essential aspects of animal life. At the cellular level, these mechanics are largely determined by the actin cytoskeleton-a network of actin filaments connected by cross-linkers to create a 3D biopolymer frame. These cross-linkers are not permanent, but bind transiently. Studies using reconstituted proteins show that when these cross-linkers are attached to and connect multiple filaments, they create a network that behaves like a weak elastic solid. When cross-links unbind, actin filaments are free to slide past one another, producing a network that behaves more like a viscous fluid (1, 2). This dynamic cross-linking makes the actin network a viscoelastic material that is solid-like on short timescales such as seconds, yet fluid-like on longer timescales such as minutes (3, 4).The timescale of the transition from solid to fluid-like behavior in reconstituted actin networks is set by the duration of cross-linking, which is in turn set by cross-linker dissociation rate, K off (1, 5, 6). The ability for an actin network to move between solid and fluid-like states may be an essential mechanism to balance mechanical and structural integrity of an elastic solid with adaptability and movement (1, 7-9). Despite this clear physical role of actin cross-linking in reconstituted network mechan...

Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton

“…3E). Similar to a previous report [ 31 ], the higher affinity K255E mutant showed much slower recovery than wild-type (Fig 3F,G). Interestingly, while wild-type α-actinin 4 showed slower recovery under agarose overlay, the K255E mutant showed no significant change in recovery time (τ) or immobile fraction (Fig.…”

Dynamic cross-links tune the solid–fluid behavior of living cells

“…Alpha-actinin is a cross-linker that forms loose bundles of actin filaments, but a mutated version known as K255E, leads to kidney dysfunction. Using fluorescence recovery after photobleaching, Ehrlicher et al (3) show that the mutated alpha-actinin (K255E) binds actin filaments together for nearly three times as long as the normal alpha-actinin in WT cells. This suggests that stress relaxation in the cytoskeleton of K255E cells will occur more slowly, allowing these cells to resist larger forces than normal.…”

“…By using traction force microscopy, Ehrlicher et al (3) show that K255E cells generate higher traction forces resulting in increased work done on the environment. These solid-like Overall, the study by Ehrlicher et al shows that, by only changing the dynamic of actin cross-linkers, it is possible to tune the solid and fluid-like behavior of living cells.…”

“…Overall, the study by Ehrlicher et al (3) shows that, by only changing the dynamics of actin cross-linkers, it is possible to tune the solid and fluid-like behavior of living cells. On one hand, the implication is that a molecular-scale defect can dramatically change the ability of a cell to function under the pressure and force typically experienced in physiological tissues (e.g., kidney, heart, lungs, etc.).…”

“…A fundamental understanding of actin network physics was developed using in vitro systems (4-7), setting the stage for recent discoveries of active diffusion driven processes in vivo (8)(9)(10). The current study by Ehrlicher et al (3) relates fundamental actin network behavior to a known human kidney disease by manipulating dynamic cross-linking to tie it all together.…”