Luchniak, A. | bioRxiv Science

bioRxiv2022

Dynamic microtubules slow down during their shrinkage phase

Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural and chemical properties of these phases remain poorly understood. The transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but is rather a multi-step process whose frequency increases with the growth time: the microtubule ages as the older microtubule tip becomes more unstable. Aging shows that the growing phase is not a single state but comprises several substates of increasing instability. To investigate whether the shrinking phase is also multi-state, we characterized the kinetics of microtubule shrinkage following catastrophe using an in vitro reconstitution assay with purified tubulins. We found that the shrinkage speed is highly variable across microtubules and that the shrinkage speed of individual microtubules slows down over time by as much as several fold. The shrinkage slowdown was observed in both fluorescently labeled and unlabeled microtubules as well as in microtubules polymerized from tubulin purified from different species, suggesting that the shrinkage slowdown is a general property of microtubules. These results indicate that microtubule shrinkage, like catastrophe, is time-dependent and that the shrinking microtubule tip passes through a succession of states of increasing stability. We hypothesize that the shrinkage slowdown is due to destabilizing events that took place during growth which led to multi-step catastrophe. This suggests that the aging associated with growth is also manifest during shrinkage with the older, more unstable growing tip being associated with a faster depolymerizing shrinking tip. Statement of SignificanceThe dynamics of the microtubule cytoskeleton is crucial for several functions in eukaryotic cells. Microtubule dynamics is traditionally described by constant growth and shrinkage speeds with first order transitions between the growth and shrinkage phases. However, catastrophe, the transition from growth to shrinkage, increases with microtubule age and is not a first order process. In contrast to the common assumption that microtubules shrink with constant speed, here we show that shrinking microtubule tips undergo step-wise slowdown during depolymerization. Our results suggest that microtubule shrinkage, like catastrophe, is a multi-step process. This finding is important for understanding the molecular nature of microtubule dynamic instability and how microtubule shrinkage can be modulated by microtubule associated proteins.

biophysics↗

bioRxiv2018

Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

biophysics↗