Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

Abstract

The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.

Lattice defects induce microtubule self-renewal

“…On the other hand, the frequency of lattice defects in vivo may be reduced by other proteins that support tubulin assembly at the microtubule end (Akhmanova and Steinmetz, 2008), which could eliminate defects and enforce a homogeneous lattice. Nevertheless, lattice defects occur in vivo and have been shown to impact the motion of microtubule motors (Gramlich et al, 2017; Liang et al, 2016), microtubule-severing enzymes (Diaz-Valencia et al, 2011; Davis et al, 2002), and the recruitment of microtubule-associated proteins (de Forges et al, 2016). Our results suggest that dimer turnover promotes the remodeling of the microtubule's conformation around sites of lattice defects and thereby confers on it an unexpected degree of structural plasticity.…”

Single-molecule fluorescence microscopy review: shedding new light on old problems

“…Significant mechanistic insights into kinesin-based transport, for example, were obtained by single-molecule TIRF imaging of single GFP-labelled kinesins [122]. …”

Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots

“…Further, recent work has shown that microtubule defects can inhibit the transport of motor proteins needed to traffic intracellular cargos. 34, 35 Another recent study demonstrated that intracellular transport can be enhanced when microtubules are stretched in live neurons. 36 These studies imply that mechanical stresses and even damage to microtubules can affect the biological functions and activities of microtubule filaments.…”