Interplay of self-organization of microtubule asters and crosslinking protein condensates

Abstract

The cytoskeleton is a major focus of physical studies to understand organization inside cells given its primary role in cell motility, cell division, and cell mechanics. Recently, protein condensation has been shown to be another major intracellular organizational strategy. Here, we report that the microtubule crosslinking proteins, MAP65-1 and PRC1, can form phase separated condensates at physiological salt and temperature without additional crowding agents in vitro. The size of the droplets depends on the concentration of protein. MAP65 condensates are liquid at first and can gelate over time. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. The droplet size directly controls the number of projections in the microtubule asters, demonstrating that the MAP65 concentration can control the organization of microtubules. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate, returning the MAP65 molecules to solution. This work implies that there is an interplay between condensate formation from microtubule-associated proteins, microtubule organization, and condensate dissolution that could be important for the dynamics of intracellular organization.

Protein condensate fusion mapping using Hippopede curve

“…The sample chambers were coated with a triblock copolymer (Pluronic F127) so that the droplets do not wet the surface with a high contact angle, as previously described (3). Given the axial symmetry of the fusion process, the 3D droplets that sit on the chamber bottom surface can be projected down to a plane around the axis.…”

“…When the droplets coalesce, the volume of two condensates remain constant (3), and the surface area decreases to a minimum value when there is a single sphere (Fig. 3(b)).…”

“…Thus, the estimate of the inverse capillary velocity is ∼ 1.75 − 2 times higher for the hippopede method compared to the ellipse fit method. Using the viscosity calculated from Stokes-Einstein equation (3), we estimate that the surface tension of liquid-like droplets is γ e ∼ 5.2 μN/m from ellipse method and 1.75-fold lower, γ h ∼ 2.9 μN/m for hippopede curve estimate.…”

“…The protein purification and imaging chamber design were the same here as previously reported (3). Briefly, MAP65 and MPA65-GFP plasmids were gifts from am Dixit (Washington University, St. Louis) and purified as described (21,22).…”

Fundamental Aspects of Phase-Separated Biomolecular Condensates

“…18,103,114,135,146,268,300,385−387 Over time many liquid-like condensates turn into solid-like gels, a process referred as aging or maturation. 10,[62][63][64]72,82,260,261,326,332,365,376,379,388 Gelation and aging have been studied by coarse-grained simulations 147,225,226,336,389 Theoretical models will be very useful for providing a conceptual framework. Lastly we take note of growing efforts in engineering biomolecular condensates.…”