Tubulins are essential for the reproduction of many eukaryotic viruses, but historically bacteriophage were assumed not to require a cytoskeleton. Here we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with a novel, extended C-terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in vitro formed filaments, indicating how interactions between the C-terminus and the following monomer drive polymerization. Finally, we show that PhuZ assembles a spindle-like array required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. This is the first example of a prokaryotic tubulin array that functions analogously to the microtubule-based spindles of eukaryotes.

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.

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

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...