Physical effects of crowdant size and concentration on collective microtubule polymerization.

Abstract

The polymerization of cytoskeletal filaments is regulated by both biochemical pathways, as well as physical factors such as crowding. The effect of crowding in vivo emerges from the density of intracellular components. Due to the complexity of the intracellular environment, most studies are based on either in vitro reconstitution or theory. Crowding agent (crowdants) size has been shown to influence polymerization of both actin and microtubules (MTs). Previously, the elongation rates of MT dynamics observed at single filament scale were reported to decrease with increasing concentrations of small but not large crowdants, and this correlated with in vivo viscosity increases. However, the exact nature of the connection between viscosity, crowdant size, nucleation, and MT elongation has remained unclear. Here, we use in vitro reconstitution of bulk MT polymerization kinetics and microscopy to examine the collective effect of crowdant molecular weight, volume occupancy, and viscosity on elongation and spontaneous polymerization. We find MT elongation rates obtained from bulk polymerization decrease in the presence of multiple low-molecular weight (LMW) crowdants, while increasing with high-molecular weight (HMW) crowdants. Lattice Monte Carlo simulations of an effective model of collective polymerization demonstrate reduced polymerization rates arise due to decrease in monomer diffusion due to small-sized crowdants. However, MT polymerization in the absence of nucleators, de novo, shows a crowdant size independence of polymerization rate and critical concentration, depending solely on concentration of the crowdant. In microscopy, we find LMW crowdants result in short but many filaments, while HMW crowdants increase filament density, but have little effect on lengths. The effect of crowdant volume fraction ϕ_C and size in de novo polymerization match simulations, demonstrating crowdants affect elongation independent of nucleation. Thus, the effect of viscosity on collective MT dynamics, i.e., filament numbers and lengths, shows crowdant size dependence for elongation, but independence for de novo polymerization.