Crowding and evolutionary divergence in microtubule assembly and combinatorial effects in kinesin-1 transport.

Abstract

Microtubule (MT) polymerization and motor driven transport drive cellular processes like intracellular transport and cell division. In this thesis, we study how physical and chemical factors collectively affect microtubule polymerization and transport focussing mainly on- (a) effect of crowding on MT polymerization, (b) evolutionary divergence of polymerization kinetics comparing plant and animal brain tubulin and (c) effect of microtubule binding tail domain on full length kinesin-1 driven collective MT transport. Macromolecular crowding influences actin polymerization. However, its effect on microtubule polymerization is less well known. Previous work showed that single MT growth exhibits a crowdant size-dependence, with increase in viscosity by small crowdants attributed to such effects. However, the physical effect of crowdant size, concentration and viscosity on collective MTpolymerization remains unclear. Brain tubulin when polymerized in bulk, in the presence of stabilized MT ‘seeds’, shows a crowdant size and concentration dependence: small crowdants decrease elongation rates and large crowdants increase. However, bulk viscosity measured using micron sized beads increased irrespective of crowdant size. Interestingly, de novo tubulin polymerization in the absence of seeds shows a crowdant size independent increase, with a corresponding decrease in critical concentration. A size-independent increase in MT density but reduced lengths only for small crowdants observed in label free imaging confirms a differential effect of crowdant size on MT nucleation and elongation. While tubulin sequence is highly conserved, the kinetics across species are reported to diverge. Previously, activity purified mung bean tubulin showed transient kinetics, lower critical concentration, and shorter microtubule filaments compared to brain tubulin. Simulations attributed this to a higher GTP hydrolysis. However low yield prevented us from testing this experimentally. In order to address this, we used a TOG-based affinity chromatography approach described earlier. We observed the kcat of GTP hydrolysis of mung tubulin 11X higher than goat brain tubulin. The activity cycling based purification was further optimized to achieve higher yields of assembly competent mung bean tubulin. We further proceeded to study the co-polymerization of kinetically distinct mung and brain tubulin. While mung tubulin polymerizes poorly with brain MT seeds, brain dimers successfully elongate mung MT filaments. This kinetic asymmetry is likely driven by high GTPase activity of mung tubulin, which we address partially by forming copolymers of mung and brain tubulin in the presence of GTP analogues de novo. Kinesin-1 motor mediated collective microtubule transport is well studied. However, its tail with ATP independent MT binding and IAK domains, remain less explored. Engineering bacterial constructs of the tail domain and IAK-deleted kinesin mutants, I show a coin tossing of kinesin motile head and immotile tail induces spatio-temporal MT patterns. Furthermore, we observe the tail to form higher ordered structures both in the presence and absence of microtubules. Our FRAP based measurements confirm these structures to be non-mobile droplet formation by the tail domain. In conclusion, we show how diverse factors like crowding, evolutionary divergence and structural components collectively shape MTpolymerization and motor-driven transport. These provide insights into physical processes determining cytoskeleton-motor dynamics inside cells.