Mechanics of the Microtubule Seam Interface Probed by Molecular Simulations and in Vitro Severing Experiments

Abstract

Microtubules (MTs) are structural components essential for cell morphology and organization. It has recently been shown that defects in the filament’s lattice structure can be healed to create stronger filaments in a local area and ultimately cause global changes in MT organization and cell mobility. The ability to break, causing a defect, and heal appears to be a physiologically relevant and important feature of the MT structure. Defects can be created by MT severing enzymes and are target sites for complete severing or for healing by newly incorporated dimers. One particular lattice defect, the MT lattice ‘‘seam” interface, is a location often speculated to be a weak site, a site of disassembly, or a target site for MT binding proteins. Despite seams existing in many MT structures, very little is known about the seam’s role in MT function and dynamics. In this study, we probed the mechanical stability of the seam interface by applying coarse-grained indenting molecular dynamics. We found that the seam interface is as structurally robust as the typical lattice structure of MTs. Our results suggest that, unlike prior results that claim the seam is a weak site, it is just as strong as any other location on the MT, corroborating recent mechanical measurements.

Factors Underlying Asymmetric Dynamics of Disaggregase and Microtubule Severing AAA+ Machines

“…This recalls our previous findings that a pushing (wedging), rather than an unfoldase, action for severing proteins leads to statistically identical distributions compared to the distributions from in-vitro katanin severing assays. 78,79 The pore constriction corresponding to the NBD2, which was characterized by monitoring the diagonal distance between PL3 loops, was found to display a similar behavior as the one for NBD1. As shown in Fig.…”

“…This recalls our previous findings that a pushing (wedging), rather than an unfoldase, action for severing proteins leads to statistically identical distributions compared to the distributions from in-vitro katanin severing assays. 78,79…”

Molecular Investigations into the Unfoldase Action of Severing Enzymes on Microtubules

“…the Regular (REG) model, which represents the GDP type lattice, and the All Homogenous model (AHM), which mimics the GMPCPP type lattice [Kononova et al, 2014, Jiang et al, 2017, Szatkowski et al, 2019, as described in the Methods section. Here we examined the unfoldase mechanism where the MT severing enzyme removes dimers through pulling on the CTT(s) of the tubulin dimer.…”

“…For the MT12 and MT16 lattices with free plus ends, we changed somewhat the procedure to determine the bending angles. Operationally, following our previous approach [Szatkowski et al, 2019], we obtained the angles between the following 3 selected points ( Fig. S22): (1) a pseudo position created by translating the coordinates of the center of mass of the fixed dimer by 50 Å towards the minus end (Fig.…”

“…1JFF)[Löwe et al, 2001] used in construction of MT lattice for our simulations, we pulled on the terminal residues with actual density in the C-terminal region of the α-and β-tubulin monomers. We applied point forces on selected residue(s) from the C-terminal regions of β-tubulin (β) and from α-and β-tubulin (both) at a speed of 2.0 µm/s, the same speed used in our indentation simulations as well as in AFM experiments[Szatkowski et al, 2019]. We selected dimers located either on the seam (the A-lattice region of the MT filament) or away from the seam (the B-lattice region of the MT filament)[ McIntosh et al, 2009] .…”