In vitro reconstitution of the Collective Effects in Microtubule Transport and Dynamics

Abstract

The transport of dynamic microtubules (MTs) driven by dyneins is essential for spindle assembly, asymmetric cell division, axonal growth and organelle positioning. While the single-molecule mechano-chemistry of dyneins has advanced extensively, their role in collective MT transport, remains unclear. Additionally, MTs in animal somatic cells are typically nucleated by centrosomes forming radial arrays or asters. MT aster transport in oocytes and single-cell embryos involves a tug-of-war of dyneins that is resolved by a combination of MT polymerization dynamics regulation, motor localization and self-organized clustering. This work encompasses (a) the length and motor number dependence of microtubule transport by yeast dyneins, (b) aster-based transport of nuclei in Saccharomyces cerevisiae and (c) compares polymerization dynamics of MTs from plant and animal sources. The work shows that microtubules transported by teams of immobilized S. cerevisiae dynein transits from random to directed motion with increasing MT lengths and motor densities, both in vitro and in silico. Velocity, diffusivity and directionality, all reflect a coordination of transport above a threshold motor numbers. Our analysis of in vivo data of astral MTs transported by dynein during S. cerevisiae mitosis supports a functional role for such a transition. Based on these findings, a general ‘search and orient’ mechanism is proposed for organelle positioning by MT asters. The comparative study on the role of MT polymerization dynamics from multiple sources: goat, porcine and plant (mung beans) demonstrates nucleation limited polymerization in all these diverse species, suggesting a central role of nucleators such as centrosomes in determining MT filament distributions. The model proposed highlights the importance of critical concentration in determining filament length distributions. Optimizations with ex vivo reconstitutions of nucleators such centrosomes and spindle pole body will advance our understanding of the collective effects of motor numbers, MT length dynamics and transport of complex geometry in eukaryotic cells.