The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

Abstract

12Determining how microtubules (MTs) are nucleated is essential for understanding how the 13 cytoskeleton assembles. Yet, half a century after the discovery of MTs and ab-tubulin subunits 14 and decades after the identification of the γ-tubulin ring complex (γ-TuRC) as the universal MT 15 nucleator, the underlying mechanism largely remains a mystery. Using single molecule studies, 16we uncover that γ-TuRC nucleates a MT more efficiently than spontaneous assembly. The laterally 17 interacting array of γ-tubulins on γ-TuRC facilitates the lateral association of αβ-tubulins, while 18 longitudinal affinity between γ/αβ-tubulin is surprisingly weak. During nucleation, 3-4 αβ-tubulin 19 dimers bind stochastically to γ-TuRC on average until two of them create a lateral contact and 20 overcome the nucleation barrier. Although γ-TuRC defines the nucleus, XMAP215 significantly 21 increases reaction efficiency by facilitating ab-tubulin incorporation. In sum, we elucidate how 22 MT initiation occurs from γ-TuRC and determine how it is regulated. 23Results 49 50 Reconstituting and visualizing microtubule nucleation from γ-TuRC 51To study how γ-TuRC nucleates MTs (Fig. 1A), we purified endogenous γ-TuRC from Xenopus 52 egg extracts and biotinylated the complexes to immobilize them on functionalized glass ( Fig. S1A-53 C). Upon perfusing fluorescent αβ-tubulin, we visualized MT nucleation live with total internal 54 reflection fluorescence microscopy (TIRFM). Strikingly, MT nucleation events occurred 55 specifically from single γ-TuRC molecules ( Fig. 1B; Fig. S1D and Movie S1-2). Kymographs 56 revealed that attached γ-TuRC assembled ab-tubulin into a MT de novo starting from zero length 57 within the diffraction limit of light microscopy (Fig. 1C), ruling out an alternative model where 58MTs first spontaneously nucleate and then become stabilized via γ-TuRC. By observing the 59 fiduciary marks on the MT lattice ( Fig. 1C) and generating polarity-marked MTs from attached g-60 TuRC (Fig. S1E), we showed that γ-TuRC caps the MT minus-end, while only the plus-end 61 polymerizes. Altogether, our results show that γ-TuRC directly nucleates MTs. 62 63 Defining the microtubule nucleus on γ-TuRC 64To determine how γ-TuRC nucleates MTs, we measured the kinetics of MT nucleation for a 65 constant density of γ-TuRC and increasing αβ-tubulin concentration ( Fig. 1D and Movie S3). 66 Surprisingly, γ-TuRC nucleated MTs starting from 7 µM tubulin (Fig. 1D), which is higher than 67 the minimum tubulin concentration (C*) needed for growth at pre-formed MT plus-ends (C* = 1.4 68 µM, Fig. 1E). Furthermore, the number of MTs nucleated from γ-TuRC increased non-linearly 69 with tubulin concentration as opposed to the linear increase in MT's growth-speed with tubulin 70 concentration (Fig. 1E). By measuring the number of MTs nucleated over time with varying ab-71 interferometry, we compared the interaction of ab-tubulin dimers with themselves versus with g-118 tubulin. Specific interactions between probe-bound αβ-tubulin and increasing concentratio...

XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions

“…XMAP215's effect on microtubule nucleation correlates with its polymerase activity, independent of the domains present. This provides support for a model of independent action by XMAP215 during microtubule nucleation, as suggested previously [19], [27], [36].…”

“…However, increasingly higher resolution structures of purified -TuRC show that the template is not perfect-the 13-member tubulin ring presented by the complex does not perfectly match the geometry of the microtubule [14]- [16]. Further, the fraction of purified -TuRC that nucleates in vitro is consistently low [15], [17]- [19]. It is hypothesized that, in order to nucleate well, -TuRC must be activated in vivo through interactions with other proteins [20]- [22].…”

“…There is a rapidly growing body of evidence that at least one of the crucial co-factors for the -TuRC is the XMAP215 family of microtubule polymerases. The XMAP215 family promotes microtubule growth from various templates, including the -TuRC, isolated centrosomes, and GMPCPP-stabilized seeds [15], [19], [23]- [26], and it also promotes microtubule nucleation in reactions without any template [23], [27]- [30]. Across these studies, the role of XMAP215 in nucleation was generally thought to be due to its polymerase activity.…”

Assembly of the asymmetric human γ-tubulin ring complex by RUVBL1-RUVBL2 AAA ATPase

“…Thus, assuming a template nucleation mechanism, major conformational changes may be required to stimulate TuRC nucleation activity. Alternatively, these changes may be brought about passively once a microtubule has formed on TuRC (43). In this case, nucleation activity may not be stimulated by a conformational change in TuRC but at the level of the nascent microtubule, by stabilizing factors or by the local availability of --tubulin heterodimers.…”

Compartmentalized control of Cdk1 drives mitotic spindle assembly

“…Conversely, a-tubulin was substantially present in the soluble fraction and much less in the pellet fraction, as expected because of the lack of spindle assembly, as well as the MAPs Map4 and ch-Tog (Figure 2B). In metaphase cells, instead, a-tubulin and significant amounts of Map4 and ch-Tog, the latter also involved in activating centrosome-independent, intra-spindle, microtubule nucleation, were present in the pellet fraction, reflecting spindle assembly (David et al, 2019;Thawani et al, 2020;Figure 2B). Distribution of NuMA1 and g-tubulin in soluble and pellet fractions did not substantially change between prometaphase and metaphase cells (Figure 2B).…”