Distinct conformations of the kinesin Unc104 neck regulate a monomer to dimer motor transition

Caenhorhabditis elegans Unc104 kinesin transports synaptic vesicles at rapid velocities. Unc104 is primarily monomeric in solution, but recent motility studies suggest that it may dimerize when concentrated on membranes. Using cryo-electron microscopy, we observe two conformations of microtubule-bound Unc104: a monomeric state in which the two neck helices form an intramolecular, parallel coiled coil; and a dimeric state in which the neck helices form an intermolecular coiled coil. The intramolecular folded conformation is abolished by deletion of a flexible hinge separating the neck helices, indicating that it acts as a spacer to accommodate the parallel coiled-coil configuration. The neck hinge deletion mutation does not alter motor velocity in vitro but produces a severe uncoordinated phenotype in transgenic C. elegans, suggesting that the folded conformation plays an important role in motor regulation. We suggest that the Unc104 neck regulates motility by switching from a self-folded, repressed state to a dimerized conformation that can support fast processive movement

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either α or β tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces

Orientation and structure of the Ndc80 complex on the microtubule lattice

The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential α/β-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends

An Improved Model for Dynamin Assembly Revealed by Cryo-EM

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Myosin-I nomenclature

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption

Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages