Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts

Abstract

Microtubules are polymers assembled from tubulin α-β-heterodimers. They typically display lateral α-α and β-β-homotypic interactions, except at one region, called the seam, where heterotypic α-β and β-α interactions occur. Here, we decorated microtubules assembled in vitro or in cytoplasmic Xenopus egg extracts with kinesin-motor domains, and analyzed their lattice organization using dual axis cryo-electron tomography followed by segmented sub-tomogram averaging. In both conditions, microtubules incorporated variable protofilament and/or tubulin subunit helix start numbers. While microtubules assembled in vitro displayed variable numbers of seams, those assembled in extracts displayed preferentially one seam. The seam location varied within individual microtubules implying the presence of lattice holes. Thus, the formation of discontinuous microtubule lattices is an intrinsic property of tubulin assembly, a process that is controlled in cells.

Microtubules self-repair in living cells

“…This was suggestive of the loss and reincorporation of tubulin dimers in the lattice of microtubules, an hypothesis further demonstrated by the visualization of this process with fluorescent tubulin dimers (Schaedel et al, 2019). These exchanges were suggested to occur at specific defect sites where the lattice structure displayed defects such as missing dimers or dislocation in the organization of protofilaments (Chrétien & Fuller, 2000;Chretien et al, 1992;Guyomar et al, 2021;Schaedel et al, 2019). This hypothesis is consistent with the analysis of microtubule response to bending cycles during which they appeared to soften in a non-elastic response, and were further seen to recover their initial mechanical state by the incorporation of new dimers in their damaged lattice (Schaedel et al, 2015).…”

Molecular motors enhance microtubule lattice plasticity

“…These structures are inherent in the lattice and are created during the polymerization process. The same holds for recently identi- * karin.john@univ-grenoble-alpes.fr fied multiseam MTs [18], which entail the existence of point defects of the size of a tubulin monomer. However, the experimentally observed increase in shaft plasticity due to severing enzymes and molecular motors [13][14][15][16] suggests the nucleation of defects in the intact lattice.…”

Dynamic microtubules slow down during their shrinkage phase

“…This model implies that the destabilization "steps" that induce catastrophe alter the microtubule lattice, perhaps in the form of structural inhomogeneity. Recent optical and electron microscopy methods that facilitate the detection of subtle structural changes of the tips or along the microtubule wall (28,(46)(47)(48) may provide novel insights into the molecular basis of these destabilization steps and how the shrinking microtubule tip structure evolves during slowdown.…”

“…A potential structural basis of multi-step catastrophe is the evolution of the tip structure, a form of memory (25, 26). For example, randomly occurring lattice defects, such as a change in protofilament number (27) or lattice-start number (28) may irreversibly alter the structural state of the growing tip making it more prone to catastrophe. Thus, length-dependent catastrophe, in addition to the growth speed fluctuations, also indicates that the growing tip changes its properties (i.e., state) over time.…”