Actin-microtubule dynamic composite forms responsive active matter with memory

Abstract

Active cytoskeletal materials in vitro demonstrate self-organizing properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possess adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite where self-assembling microtubules and actin filaments collectively self-organize due to the activity of microtubule-percolating molecular motors. We demonstrate that microtubules spatially organize actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli, suggesting possible autoregulatory behavior in changing mechanochemical environments. We thus establish an artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.

Keywords: active materials; cytoskeleton; structural memory.

Fig. 1.

Motile dynamic microtubule system self-organizes into quasi-parallel microtubule streams. (A) Schematic representation of the system. (B) Intensity inverted fluorescence micrographs of dynamic microtubule motility assay (Top) and the corresponding color-coded orientation analysis (Bottom). (C) Polar histogram of the orientation of microtubules is narrowing with time. (D) Time trace of the global order, S, and fluorescence intensity, I, of dynamic microtubules in the motility assay. (E) Time traces of equidistant vertical slices of Pearson correlation coefficient matrix of fluorescence micrographs of dynamic microtubule motility assay. (F) The correlation slices in E can be approximated by an exponential decay with the parameters of the correlation decay time (time constant) and asymptotic correlation amplitude and represented by corresponding temporal profiles (Inset). (G) Intensity inverted fluorescence micrographs of photobleaching of microtubule stream (Left; green arrowheads indicate the limits of photobleached area) formed during 30 min of ordering and the corresponding kymograph (Right; orange lines indicate the photobleached area, green dashed lines indicate the front of receding photobleached areas, and asterisks denote the time of photobleaching). (H) Time traces of the global order for various initial concentrations of microtubule seeds and free tubulin. All experiments were repeated at least four times with similar results. Data in H are represented as mean value (solid blue line) ± SD (shaded area) (n = 4 per condition).

Fig. 2.

Assembling actin filaments self-organize in a locally nematic fashion with low global order. (A) Schematic representation of the system. (B) Intensity inverted fluorescence micrographs of dense elongating actin filaments (Top) and the corresponding color-coded orientation analysis (Bottom) show local nematic ordering. (C) Polar histogram of the orientation of actin filaments is narrowing only a little with time, indicating low global order. (D) Time trace of the global order, S, and fluorescence intensity, I, of growing actin system. (E) Time traces of equidistant vertical slices of Pearson correlation coefficient matrix of fluorescence micrographs of growing actin system. (F) Time traces of the global order for two initial concentrations of free actin monomers. All experiments were repeated at least four times with similar results. Data in F are represented as mean value (solid blue line) ± SD (shaded area) (n = 4 per condition).

Fig. 3.

Interactions between microtubules and actin filaments mutually promote the self-organization of the cytoskeletal composite. (A) Schematic representation of the system. (B) Color-coded maximum fluorescence intensity projections of gliding microtubule (Top) pushing actin filaments (Bottom), effectively organizing them. (C) Gliding microtubule steered and guided by actin filaments. Image representation as in B. (D) Schematic representation of the interactions forming a feedback loop between actin and microtubules. (E) Multichannel fluorescence micrographs of self-assembling and self-organizing actin-microtubule composite. Individual fluorescence channels are displayed as intensity inverted images. The overlay image is noninverted for clarity. (F) Time traces of the global order, S, and fluorescence intensity, I, of microtubules (Top) and actin filaments (Center), and their mutual alignment, S_c, (Bottom) in the composite system. (G) Time traces of the global order of microtubules (Left) and the corresponding steady-state order (Right) for three indicated initial biochemical conditions. All experiments were repeated at least four times with similar results. Data in G are represented as mean value ± SD (Left) and median ± 75th percentile (Right), where notches display the variability of the median between samples (n = 4 per condition).

Fig. 4.

Response of the cytoskeletal composite to chemical and physical perturbations reveal the structural memory of the system. (A) Color-coded orientation analysis of microtubules (Top) and actin filaments (Center) before (Left) and after (Right) the addition of CaCl₂. Corresponding time trace of the global actin order (Bottom). (B) Orientation color-coded maximum fluorescence intensity projection (200 s) of microtubules (Top) and orientation color-coded micrographs of actin filaments before (Left) and after (Right) the addition of gelsolin. Corresponding polar histograms of microtubule orientation (Bottom) show microtubule dispersion. (C) Color-coded orientation analysis of microtubules (Top) after the first (Left) and second (Right) cycle of microtubule polymerization in the absence of actin. Corresponding polar histograms of microtubule orientation (Bottom) show reorientation in different directions. (D) Color-coded orientation analysis of microtubules (Top) and actin filaments (Center) after the first (Left) and second (Right) cycle of microtubule polymerization. Corresponding polar histograms of microtubule orientation (Bottom) show the retention of the orientation. Time traces of the fluorescence intensities (Top Right) and global order (Bottom Right) of microtubules and actin filaments. The shaded area corresponds to the cooling cycle. (E) Diversion of repolymerized microtubules (second cycle) from the dominant direction before depolymerization (first cycle) in the absence and presence of F-actin memory as in examples shown in C and D, respectively.