Direct observation of individual tubulin dimers binding to growing microtubules

Abstract

The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on-and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date. SIGNIFICANCEMicrotubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on-and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.

Beyond the GTP‐cap: Elucidating the molecular mechanisms of microtubule catastrophe

“…[87] These fast rates of single-molecule tubulin dimer kinetics have recently been contested by new measurements using interferometric scattering microscopy with gold nanoparticle-labeled tubulin. [88,89] Here, the authors reported two distinct, albeit slower tubulin off-rates, corresponding to different sites of tubulin association at the microtubule end distinguished by the number of longitudinal and lateral bonds formed. Regardless of the exact rates of tubulin on/off kinetics, the effects of microtubule growth fluctuations are likely to be important for the size of the GTP-cap and overall microtubule stability.…”

“…Careful examinations using light imaging approaches and improved image analysis techniques have provided recent evidence of dramatic changes in the microtubule end structure over time, including microtubule end tapering, protofilament splitting, and repair (Figure 4), [39,41,73,98,102] as well as allowed measurements of tubulin polymerization kinetics at a single-molecule level. [88,89] Nevertheless, investigation of the microtubule end at the ultra-structural level still requires high-resolution EM approaches.…”

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

“…Microtubules with higher protofilament number have a smaller angular separation between protofilaments, and this can affect the strength of lateral tubulin bonds and the ease with which they form. The current understanding of the molecular details of microtubule growth relies on combining experiments with modeling (43)(44)(45)(46)(47)(48). For example, one recent model of microtubule polymerization assumes that growing microtubule tips have flared protofilaments, to the end of which tubulin dimers are added before the protofilaments associate with each other laterally (49).…”

XMAP215 promotes microtubule catastrophe by disrupting the growing microtubule end

“…Aside from the nucleotide composition, both kinetics of microtubule assembly and the structure of the growing microtubule end remain areas of intense interest (Kerssemakers et al, 2006) (Schek et al, 2007) (Gardner et al, 2011a) (Mickolajczyk et al, 2019) (Guesdon et al, 2016)(Estévez-Gallego et al, 2020) (Mcintosh et al, 2018) (Atherton et al, 2018) (Gudimchuk et al, 2020). The addition of inherently curved GTP-tubulin dimers to the ends of microtubule protofilaments and their subsequent straightening into closed polymer lattice have been reported to result in a variety of intermediate structures, including disconnected and splayed individual protofilaments (Mcintosh et al, 2018) (Gudimchuk et al, 2020), multi-protofilament sheets (Chrétien et al, 1995) (Guesdon et al, 2016) (Atherton et al, 2018) and overall tapered microtubule ends (Mandelkow et al, 1991) (Reid et al, 2019).…”