Microtubules are dynamic intracellular polymers whose ends stochastically switch between periods of polymerization and depolymerization. Regulation of microtubule dynamics is key to the function of microtubule cytoskeleton in intracellular transport, cell division and cell motility. One of the microtubule behaviors that has long been observed in cells is ‘treadmilling’, a phenomenon characterized by simultaneous polymerization at one polymer end, and depolymerization at the other end. Treadmilling is an essential feature of active cytoskeletal polymers underlying actin-based cell motility, bacterial cell division and transport, and reorganization of microtubule arrays in plants. Nevertheless, how cells coordinate growth at microtubule plus ends and shrinkage at microtubule minus ends to achieve treadmilling was not known.
Figure: Microtubule polymers undergoing treadmilling in silico. The colors represent time, from blue to gold. Image courtesy of EJ Lawrence and G Arpag, produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).
In cells, microtubule dynamics are regulated by a myriad of microtubule-associated proteins, which can act on either or both microtubule ends to modulate the rates of polymerization, depolymerization and switching frequencies between the two states. Given the complexity of the microtubule regulatory network in cells, the conditions that lead to treadmilling are non-trivial. To address this problem, we employed computational modeling of the effects of multi-protein ensembles on microtubule dynamics, which allowed us to determine a regime conducive to microtubule treadmilling. Our simulations predicted that a combination of four microtubule regulatory proteins – XMAP215, EB1, CLASP2 and MCAK – constitutes a minimal-component system for microtubule treadmilling. We then used in vitro experiments with purified recombinant protein components and total-internal-reflection-fluorescence microscopy to confirm our modeling predictions and, for the first time, reconstitute cellular-like microtubule treadmilling outside of cells.
By combining computational and in vitro reconstitution approaches, our work provides a deeper understanding of a fundamental cellular phenomenon, demonstrating how active polymer systems can be tuned to give rise to complex polymer behavior. Given the essential roles of microtubules in many cellular processes, and the particular relevance of microtubule network regulation in the context of neurodegenerative disorders and cancer, our ability to control microtubule behavior has important relevance for human health.
Funding by the HFSP Career Development Award was instrumental in getting this project off the ground, as it provided the initial support for the computational aspects of this work.