Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

We report the cryo-EM structure and dynamic parameters for unmodified α1B/βI+βIVb microtubules. These microtubules display markedly different dynamics compared to heterogeneous brain microtubules, and their dynamic parameters can be proportionally tuned by the addition of a recombinant neuronal tubulin isoform with different dynamic properties.

Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit “dynamic instability.” This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

on the exact molecular mechanism of MCU regulation. Our recent structural insight of the N-terminal domain revealed a b-grasp-like fold containing the MCU regulating acidic patch (MRAP) that binds Mg 2þ /Ca 2þ with mM affinity, destabilizes MCU, shifts self-association equilibrium to monomer and attenuates [Ca 2þ ] m uptake. The weak binding affinity for Mg 2þ is well suited for the regulation of MCU as mitochondrial matrix have higher Mg 2þ concentration (0.2-2 mM). Our recent identification of Mg 2þ as regulator of MCU channel, intrigued us to explore the molecular link between MCU and the unstudied Mg 2þ selective transporter, Mrs2p for mitochondrial ion homeostasis and bioenergetics. To establish the link between MCU and Mrs2p, we generated a CRISPR/Cas9-mediated Mrs2p global knockout mouse model. Mitochondrial Mg 2þ channel activity (I Mrs2p ) was measured in mitoplast obtained from cardiomyocytes isolated from WT and Mrs2p -/mice by adopting our well devised patch-clamp experiments. Knocking out Mrs2p significantly ablated I Mrs2p validating Mrs2p as an authentic mammalian mitochondrial Mg 2þ channel. Additionally, simultaneous measurement of [Ca 2þ ] m uptake and DJm was performed in cardiomyocytes isolated from WT and Mrs2p -/mice. In line with ablated I Mrs2p MCU-mediated Ca 2þ uptake was increased in Mrs2p -/myocytes, reinforcing the concept of Mg 2þ -dependent MCU regulation. Conversely, loss of MCU did not alter the I Mrs2p , suggesting a cation dependent MCU regulation that is consistent with other Ca 2þ channels including L-type, RyRs, IP 3 Rs, and CRAC. Mitochondrial ATP synthase has been shown recently to be vital not only for cellular energy production but also for energy dissipation and cell death. We identified and characterized a large non-selective uncoupling channel within the ATP synthase c-subunit ring, the persistent opening of which initiates cell death. We have continuing evidence for the crucial role of this channel in mitochondrial permeability transition (mPT). We have now purified ATP synthase from porcine heart mitochondria and performed single-channel studies. Excised proteoliposome patch-clamp recordings demonstrate that highly pure and fully assembled ATP synthase monomers form large conductance, Ca 2þ -sensitive and voltage-gated channels. We confirmed the monomeric state of ATP synthase by cryo-electron microscopy studies of ATP synthase reconstituted into proteoliposomes. We have also heterologously overexpressed and purified human ATP synthase c-subunit from E. coli plasma membranes. We show that human c-subunit purified from bacteria forms large conductance channels identical to those purified from HEK-293 cells. The channel is gated by polar amino acid residues situated at the mouth of the pore and by the hydrophilic F 1 portion of ATP synthase. We find that dissociation of ATP synthase F 1 from F O occurs when we expose primary hippocampal neurons to glutamate toxicity, suggesting that the non-reversible dissociation of F 1 from F O is pathological. We have successfully k...