Nanoscopic Structural Fluctuations of Disassembling Microtubules Revealed by Label‐Free Super‐Resolution Microscopy

Abstract

Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, label‐free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with increased organization of protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a stochastic mechanism of switching from the disassembly to the assembly phase.

Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins

“…In that study, the authors imaged microtubules bound to standard microscope cover glass. This was later extended to gliding assays employed by Andrecka et al As microtubules exhibit anisotropic scattering profiles, their iSCAT contrast is polarization-dependent relative to their orientations, a property previously explored for GNRs in both iSCAT and darkfield modalities. ,,, Parallelized polarization-sensitive iSCAT detection recently enabled studies of not only microtubule motion but also disassembly . Differential image contrasts from the two orthogonal polarization projections (Figure A) were presented in the form of a composite kymograph (Figure B) to show the change in relative isotropic and anisotropic scattering contributions, indicating a mass change at the microtubule tip (Figure C).…”

“…75,77,99,153 Parallelized polarization-sensitive iSCAT detection recently enabled studies of not only microtubule motion but also disassembly. 154 Differential image contrasts from the two orthogonal polarization projections (Figure 9A) were presented in the form of a composite kymograph (Figure 9B) to show the change in relative isotropic and anisotropic scattering contributions, indicating a mass change at the microtubule tip (Figure 9C). Incremental images, temporally separated by 19 ms, provided quantitative information on disassembly rates and were used to reconstruct super-resolution images which enabled detection of tubulin oligomer detachment, with a theoretical detection limit of ∼3 tubulin dimers (Figure 9D).…”

“…(E), (F), (G), and (H) correspond to extension, attachment, tension, and detachment, respectively. (A–D) Adapted with permission from ref . Copyright 2021 John Wiley and Sons.…”

Approaches to visualize microtubule dynamics in vitro

“…Впечатляющие эксперименты на основе iSCAT реализованы в работах [68] и [69] для изучения структуры растущих концов микротрубочек. В первом случае iSCAT позволил напрямую наблюдать встраивание димеров тубулина в тело микротрубочки с помощью золотых наночастиц размером 20 нм, ковалентно прикреплённых к молекулам рекомбинантного тубулина.…”

Fast Leaps between Millisecond Confinements Govern Ase1 Diffusion along Microtubules

“…[22,23] It has been used to observe the motion of motor proteins, [24,25] the interactions of membrane proteins, [26] protein diffusion in lipid membranes and live cells, [27,28] label-free detection of single proteins, [29,30] and protein conformational changes. [31] Here, we explore fine details of the interaction of the Ase1 protein with the MT lattice by employing fast 3D tracking of single proteins with nanometer precision and 20 µs temporal resolution. Within the Ase1 trajectories, we reveal millisecond and sub-millisecond confinements associated with the Ase1 interaction with single tubulin dimers.…”