Microtubule polymerization tuned by macromolecular crowdant size and density

Abstract

Microtubule (MT) polymerization is regulated by biochemical as well as physical factors such as macromolecular crowding. Crowding agents or crowdants affect MT elongation rates differently depending on crowdant size due to opposing ef-fects on polymerization: microviscosity reduces polymer elongation, while volume exclusion increases reaction rates by local concentration. In order to address how crowdant size and concentration collectively affect MT populations, we combine in vitro MT polymerization experiments with kinetic Monte Carlo simulations. Our experiments in bulk with nucleators validate decreasing MT elongation rates with increasing concentrations of small molecular weight crowdants in bulk assays and a corresponding increase for large crowdants. Kinetic Monte Carlo simulations can explain the result with packing fractions dependence of small as compared to large crowdants increasing microviscosity more dramatically. In contrast MT bulk polymerization rates in absence of nucleators increased with crowdant con-centration, irrespective of their size, with a corresponding decrease in the critical concentration. Microscopy of filament growth dynamics demonstrates that small crowdants result in shorter filaments in a concentration dependent manner, consis-tent with their role in reducing elongation rates, but this decrease is compensated by increased number of filaments. Large crowdants increase the filament numbers while elongation is slightly decreased. Our results provide evidence for MT nucle-ation being rate-limited and elongation diffusion limited, resulting in differences in the effect of crowdant sizes on nucleation and elongation. These results are of gen-eral relevance to understand physical effects of crowding on collective cytoskeletal polymerization dynamics.