Microtubule assembly dynamics: new insights at the nanoscale

Abstract

Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.

Figure 1

Microtubule structure at the nanoscale. Microtubules in vivo are typically composed of 13 individual protofilaments organized into a tube configuration. The individual protofilaments consist of stacked αβ tubulin heterodimers with an exchangeable GDP/GTP binding site on the β subunit (GDP-tubulin αβ subunits are shown in white and green; GTP-tubulin αβ subunits shown in white and magenta). A GTP-tubulin “cap” stabilizes MT growth (left, magenta), possibly by keeping individual protofilament subunits in close proximity to each other. In contrast, depolymerizing MT tips tend to have curled protofilaments due to the preferential outward kinking of exposed GDP-tubulin subunits (right). Cartoons depict output from the mechanochemical model of VanBuren et al [25].

Figure 2

Depiction of experimental assays for measuring various aspects of nanoscale MT dynamics. (A) The experimental assay designed by Kerssemakers et al [12] for measuring MT assembly dynamics at the nanoscale. A microtubule-attached bead (orange, bead not to scale) is centered via a two-component “keyhole” optical trap (dark blue/ light blue). The dark blue “point” trap serves to keep the MT plus-end (magenta) in contact with a microfabricated barrier (hatched vertical section, direction of force shown by orange arrow), and the light blue “line” trap serves to orient the MT perpendicular to the barrier wall. Deflection of the bead away from the center of the “point” trap (dark blue) reflects leading protofilament length fluctuations at the MT plus-end (magenta). (B) An assay designed by Schek et al [18] similarly measured MT plus-end leading protofilament length fluctuations (magenta) via deflection of a bead (orange, bead not to scale) away from the center point of a laser trap (dark blue). In contrast to the Kerrsemakers et al assay, the position of the laser trap itself was updated at 10 Hz frequency to maintain a constant force at the MT tip as the MT increased in length during polymerization. (C) In an assay designed by Grishchuk et al [26], brief deflections in the position of a bead (orange, bead not to scale) conjugated to the microtubule lattice (green/magenta) are recorded as the MT depolymerizes past the position of the bead. Since the GDP-tubulin subunits in the depolymerizing protofilaments prefer to curl radially outward, they should displace the bead as the depolymerization proceeds past the site of bead attachment. Thus, forces produced by the depolymerizing plus-end are inferred from the magnitude of bead deflections from the center of the trap (laser trap – dark blue; resisting force provided by trapped bead shown as orange arrow). (D) In an assay designed by Franck et al [30] tension is applied at MT plus-ends by pulling a dynamic MT plus-end (magenta) away from a laser-trapped bead (laser trap – blue, bead – orange, not to scale), while maintaining the bead at a constant position relative to the center of the trap. Mechanochemical coupling is achieved between the MT plus-end (magenta) and the bead (orange) via the Dam1p protein complex (red), which forms a key kinetochore component in yeast. MT length fluctuations are recorded by measuring the distance between the MT minus-end and the bead position.