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.

Microtubule network remodeling is an essential process for cell development, maintenance, cell division, and motility. Microtubule‐severing enzymes are key players in the remodeling of the microtubule network; however, there are still open questions about their fundamental biochemical and biophysical mechanisms. Here, we explored the ability of the microtubule‐severing enzyme katanin to depolymerize stabilized microtubules. Interestingly, we found that the tubulin C‐terminal tail (CTT), which is required for severing, is not required for katanin‐catalyzed depolymerization. We also found that the depolymerization of microtubules lacking the CTT does not require ATP or katanin's ATPase activity, although the ATP turnover enhanced depolymerization. We also observed that the depolymerization rate depended on the katanin concentration and was best described by a hyperbolic function. Finally, we demonstrate that katanin can bind to filaments that lack the CTT, contrary to previous reports. The results of our work indicate that microtubule depolymerization likely involves a mechanism in which binding, but not enzymatic activity, is required for tubulin dimer removal from the filament ends.

Microtubules (MTs) and their associated proteins are essential for many cellular processes, including maintenance of cellular structure, cell motility, cell division, and intracellular transport. Kinesin superfamily (KIFs) proteins are molecular motors that directionally transport organelles and cargos along MTs. As the first-discovered kinesins, the Kinesin-1 superfamily (KIF5s) is a group of highly processive motor proteins. Despite KIF5B's importance in cellular health, atomic level insight to the structure, dynamics, and microtubule interface are lacking. Magic Angle Spinning (MAS) NMR spectroscopy is well suited for structure and dynamics characterization of KIF5B motor domain in complex with MTs. We present an investigation into the atomic-resolution structure of KIF5B motor domain in complex with polymeric MTs by MAS NMR. We applied multidimensional (2D and 3D) homo-and heteronuclear experiments in U-13 C, 15 N-kinesin/MT complex for resonance assignments. All homo-and heteronuclear correlation spectra exhibited high resolution and revealed that more than 80% of the residues are present in the spectra. Chemical shift predictions were performed by ShiftX2 using the X-ray structure of KIF5B bound to a/b tubulin dimer. Based on the 2D/3D spectra, together with SHIFTX2 predictions, characteristic spin system assignments were identified and one third of residue specific assignments have been achieved. In addition, we applied homonuclear experiments in [1,6-Glu-13 C]-[U-15 N]-kinesin/MT complex and 1 H detection experiments under fast MAS in U-2 H, 13 C, 15 N-kinesin/MT complex to reduce spectral complexity and enhance resolution.

Amyloid fibrils formed from leukocyte chemotactic factor 2 (LECT2), a secreted human cytokine, are associated with kidney failure in the disease amyloid LECT2 (ALECT2) amyloidosis. This rare disease was recognized in 2008 and has a variable prevalence worldwide. The mechanisms which lead to ALECT2 fibril deposition are not known and there are no treatments other than kidney transplant. The LECT2 gene harbors a single nucleotide polymorphism that leads to either a valine or isoleucine residue at position 40 of the mature protein. Most of the individuals diagnosed with ALECT2 amyloidosis are homozygous for valine at this position, which led us to hypothesize that the valine-containing variant of LECT2 protein is less stable and more prone to aggregation than the isoleucine-containing variant. Here, we investigate the structure, stability and aggregation of both variants of recombinant LECT2. Both variants have similar structures in solution; unfold in similar concentrations of urea; and aggregate at similar rates under native-like conditions, forming structures that bind to thioflavin T. Chelation of the structural zinc ion destabilizes both variants to a similar extent, and increases the rate at which they aggregate. We do not observe a consistent difference in stability or aggregation between the variants of LECT2, so we suggest that the presence of the valine residue at position 40 does not determine whether an individual is at increased risk of ALECT2 amyloidosis.