Liquid behavior of cross-linked actin bundles

Abstract

The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.actin | phase separation | liquid crystal | cytoskeleton T he cellular cytoplasm is a hierarchical array of diverse, soft materials assembled from biological molecules that work in concert to support cell physiology (1). The actin cytoskeleton constitutes a spectrum of materials constructed from the semiflexible polymer actin (F-actin) that are crucial in diverse physical processes ranging from cell division and migration to tissue morphogenesis (2, 3). Cross-linking and regulatory proteins assemble actin filaments into bundles and networks with varied composition, mechanics, and physiological function (4). The mechanical properties of actin assemblies regulate force generation and transmission to dynamically control morphogenic processes from the subcellular to tissue length scales (5, 6).A mechanistic understanding of cytoplasmic mechanics is obscured by the rich complexity of in vivo cytoskeletal assemblies (7) and has been investigated via in vitro model systems (8, 9). Vastly different material properties have been accessed through varying filament length, concentration, and cross-linking. For semidilute concentrations of long actin filaments (>1 μm), the mean spacing between actin filaments, or mesh size, is much smaller than the filament length. In this case, cross-linking proteins mechanically constrain actin filaments to result in space-spanning networks that are viscoelastic gels (10). The structure of cross-linked actin networks is kinetically determined, reflecting a metastable state (11, 12) that requires motor-driven stresses for significant shape changes (13). In contrast, highly concentrated solutions of short actin filaments (<1 μm) align due to entropic effects and form equilibrium liquid crystal phases (14). Liquid crystal theory has been introduced as a framework to understand actin cortex mechanics and mitotic spindle shape (5, 15), but the existence of liquid crystal-like phases at physiological conditions is uncertain.Liquid-like phases of proteins and nucleic acids have been found within the cytoplasm and are thought to be important in subcellular...

Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin

“…2b), indicating its properties as a nematic liquid crystal. It has already been shown that F-actin forms a nematic phase at high concentrations 15,[27][28][29] . From observations in the bulk solution, it has been reported that the nematic liquid crystallization of F-actin depends on its concentration and length like many other rod-like molecules, and the liquid crystal phase tends to appear at 50 μM or higher concentrations of F-actin 29,30 .…”

“…Recently, it was suggested that the high concentration and crowded environment of the cytoplasm plays an important role in the construction of cell morphology via the regulation of cytoskeletons, especially F-actin networks 14,15 . Interestingly, it has become more clear that these cytoskeletons lack specific regulatory factors, suggesting a plausible scenario whereby cell morphology is generated in a self-organizing manner depending on the highly concentrated and crowded conditions in the cytoplasm.…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[58] Cytoskeletonr econstitution has also been investigated in dropletsf ormed by associative phase separation. [154] In another notable study,t he dissipative self-assembly of FtsZ filaments in RNA/polypeptide complex coacervates in the presence of GTP was demonstrated [155] (Figure 4E). [152] On the basis of the low energy cost for interfaciala rea increase associated witht he low surfacet ensions of all-aqueous droplets, af ew studies startedt oi nvestigate how the local accumulationo fp olymerisable proteins could produce dropletd eformations and, ultimately,d ivision.…”

“…[153] Shape instabilities and self-division were also reported in anisotropic liquid droplets formed by actin filaments. [154] In another notable study,t he dissipative self-assembly of FtsZ filaments in RNA/polypeptide complex coacervates in the presence of GTP was demonstrated [155] (Figure 4E). Significantly,d irectional elongation of the coacervate droplets along the filaments, followed by their fission,a ssociated with the division of the FtsZ fibrils into two, was reporteda nd explained in terms of diffusion reaction kinetics coupled to capillaryf orces.…”

Self-organizing motors divide active liquid droplets

“…We recently reported another type of macromolecular droplet, where the actin cross-linker, filamin, condenses short actin filaments into liquid droplets (17). While actin filament-based liquids have characteristics of conventional liquids, they are anisotropic due to internal structure from densely packed filaments, so instead form liquid crystals (17)(18)(19). The internal liquid structure of molecular liquid crystals can be harnessed to drive processes such as the spatial localization of colloidal particles (20,21).…”