Dynamic actin crosslinking governs the cytoplasm’s transition to fluid-like behavior

Abstract

Cells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments changes in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is clear in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequencydependent viscoelastic properties of the cytoplasm in living cells using multiharmonic excitation and in situ optical trap calibration. At previously unexplored long timescales, we observe that the cytoskeleton fluidizes. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255E+/-cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255E+/-cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

“…Heterogeneity may originate from multiple non-exclusive sources. At the micron-scale, obstruction by organelles (Gu et al, 2007;Parkinson et al, 2008) and other cytoplasmic structures such as condensates, as well as localized active mixing, could contribute to cytoplasmic variability (Chaubet et al, 2020). At the nanometer-scale there are some enticing sources of heterogeneity that remain unexplored, notably those intrinsic to the macromolecular milieu: crowder density, size, charge, and hydrophobicity.…”

Subtle changes in crosslinking drive diverse anomalous transport characteristics in actin–microtubule networks

“…The cytoskeleton is a complex network of filamentous proteins, including semiflexible actin filaments and rigid microtubules [1][2][3] . Numerous crosslinking proteins that can link actin to actin [4][5][6][7] , microtubules to microtubules [8][9][10] , and actin to microtubules [11][12][13][14][15] enable the cytoskeleton to adopt diverse architectures and stiffnesses to drive key processes such as cell motility, meiosis and apoptosis 10,[16][17][18] . These varying structural and rheological properties, in turn, directly impact the intracellular transport of vesicles and macromolecules traversing the cytoplasm 14,15,19,20 .…”