The size of the EB cap determines instantaneous microtubule stability

Abstract

The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.DOI: http://dx.doi.org/10.7554/eLife.13470.001

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

“…We find that, in agreement with theory, growth fluctuations can be considered as Gaussian white noise and cap size fluctuations are well described by the mean-reverting Ornstein-Uhlenbeck (OU) process (35)(36)(37) with a typical timescale that is determined entirely by the maturation rate. This explains the timescale of previously observed stability fluctuations during microtubule growth (14). Furthermore, the expected and measured amplitude of the cap size fluctuations indicates that microtubules are far from instability during most of their growth time.…”

“…The majority of this EB cap is lost during a period of several seconds before catastrophe occurs (16,19), indicating that the EB binding region is critical for stability. In agreement with this notion, faster-growing microtubules that have larger caps were found to be more stable after sudden tubulin removal (14). During regular steady-state growth, cap size and microtubule stability seemed to fluctuate on a timescale of several seconds (14), the origin of which is unclear.…”

“…In agreement with this notion, faster-growing microtubules that have larger caps were found to be more stable after sudden tubulin removal (14). During regular steady-state growth, cap size and microtubule stability seemed to fluctuate on a timescale of several seconds (14), the origin of which is unclear.…”

“…The exact properties of these cap size fluctuations are unknown because the GTP in the growing microtubule end region cannot be directly visualized. However, end-binding proteins of the EB family have been shown recently to bind to the protective cap (13)(14)(15)(16). Fluorescent EBs can therefore be used to indirectly visualize the cap at the individual microtubule level (14).…”

Organelles – understanding noise and heterogeneity in cell biology at an intermediate scale

“…This hypothesis has been proven using live-cell imaging experiments, where stochastic fluctuations in growth rates have been observed for neurite outgrowth (Odde and Buettner, 1998) as well as endocytotic activity (Dey et al, 2014). Similar fluctuations are seen when cellular processes are reconstituted in vitro -for example, microtubules display momentary fluctuations in stability that influence microtubule growth and shrinkage rates (Duellberg et al, 2016). Many organelles also undergo fission and fusion, processes which themselves are potentially subject to stochastic variation.…”