Kinesin Kar3 and Vik1 Go Head to Head

The yeast kinesin motor protein Kar3 forms a heterodimer with a nonmotor protein Vik1. A study in this issue by Allingham et al. (2007) reveals that Vik1 unexpectedly has a structure similar to a kinesin motor domain yet lacks a nucleotide-binding site and is thus catalytically inactive. However, this does not hinder movement of the heterodimer because other features of the remarkably divergent Vik1 motor domain are retained, including the ability to bind microtubules

The construction and evaluation of the International Morse Code Selection Test.

Thesis (Ed.D.)--Boston University

Surface topography of microtubule walls decorated with monomeric and dimeric kinesin constructs

The surface topography of opened-up microtubule walls (sheets) decorated with monomeric and dimeric kinesin motor domains was investigated by freeze-drying and unidirectional metal shadowing. Electron microscopy of surface-shadowed specimens produces images with a high signal/noise ratio, which enable a direct observation of surface features below 2 nm detail. Here we investigate the inner and outer surface of microtubules and tubulin sheets with and without decoration by kinesin motor domains. Tubulin sheets are flattened walls of microtubules, keeping lateral protofilament contacts intact. Surface shadowing reveals the following features: (i) when the microtubule outside is exposed the surface relief is dominated by the bound motor domains. Monomeric motor constructs generate a strong 8 nm periodicity, corresponding to the binding of one motor domain per beta -tubulin heterodimer. This surface periodicity largely disappears when dimeric kinesin motor domains are used for decoration, even though it is still visible in negatively stained or frozen hydrated specimens, This could be explained by disorder in the binding of the second (loosely tethered) kinesin head, and/or disorder in the coiled-coil tail. (ii) Both surfaces of undecorated sheets or microtubules, as well as the inner surface of decorated sheets, reveal a strong 4 nm repeat (due to the periodicity of tubulin monomers) and a weak 8 nm repeat (due to slight differences between alpha- and beta -tubulin). The differences between alpha- and beta -tubulin on the inner surface are stronger than expected from cryo-electron microscopy of unstained microtubules, indicating the existence of tubulin subdomain-specific surface properties that reflect the surface corrugation and hence metal deposition during evaporation. The 16 nm periodicity visible in some negatively stained specimens (caused by the pairing of cooperatively bound kinesin dimers) is not detected by surface shadowing

Dissection of Kinesin's Processivity

The protein family of kinesins contains processive motor proteins that move stepwise along microtubules. This mechanism requires the precise coupling of the catalytic steps in the two heads, and their precise mechanical coordination. Here we show that these functionalities can be uncoupled in chimera of processive and non-processive kinesins. A chimera with the motor domain of Kinesin-1 and the dimerization domain of a non-processive Kinesin-3 motor behaves qualitatively as conventional kinesin and moves processively in TIRF and bead motility assays, suggesting that spatial proximity of two Kinein-1 motor domains is sufficient for processive behavior. In the reverse chimera, the non-processive motor domains are unable to step along microtubules, despite the presence of the Kinesin-1 neck coiled coil. Still, ATP-binding to one head of these chimera induces ADP-release from the partner head, a characteristic feature of alternating site catalysis. These results show that processive movement of kinesin dimers requires elements in the motor head that respond to ADP-release and induce stepping, in addition to a proper spacing of the motor heads via the neck coiled coil

Microtubule Interaction Site of the Kinesin Motor

AbstractKinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface. The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule. The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/α5) that corresponds topologically to the major actin-binding domain of myosin. Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores

Report of Second Meeting for the Purpose of Obtaining the Views of the Three Affiliated Tribes of the Fort Berthold Reservation on the Lieu Lands Offered by the Secretary of War, 1946

Report of the second meeting held in the office of Assistant Secretary of the Interior C. Girard Davidson for the purpose of obtaining the views of the Three Affiliated Tribes of the Fort Berthold Reservation of the lieu lands offered by the Secretary of War. Includes a list of attendees and a transcript of the meeting discussing the Three Affiliated Tribes\u27 rejection of the offer of lieu lands made by the Secretary of Interior and Department of War to the Fort Berthold Reservation. See also: Report of Meeting for the Purpose of Obtaining the Views of the Three Affiliated Tribes of the Fort Berthold Reservation on the Lieu Lands Offered by the Secretary of War, 1946https://commons.und.edu/langer-papers/1147/thumbnail.jp

The novel KIF1A missense variant (R169T) strongly reduces microtubule stimulated ATPase activity and is associated with NESCAV syndrome

KIF1A is a microtubule-dependent motor protein responsible for fast anterograde transport of synaptic vesicle precursors in neurons. Pathogenic variants in KIF1A have been associated with a wide spectrum of neurological disorders. Here, we report a patient presenting a severe neurodevelopmental disorder carrying a novel de novo missense variant p.Arg169Thr (R169T) in the KIF1A motor domain. The clinical features present in our patient match with those reported for NESCAV syndrome including severe developmental delay, spastic paraparesis, motor sensory neuropathy, bilateral optic nerve atrophy, progressive cerebellar atrophy, epilepsy, ataxia, and hypotonia. Here, we demonstrate that the microtubule-stimulated ATPase activity of the KIF1A is strongly reduced in the motor domain of the R169T variant. Supporting this, in silico structural modeling suggests that this variant impairs the interaction of the KIF1A motor domain with microtubules. The characterization of the molecular effect of the R169T variant on the KIF1A protein together with the presence of the typical clinical features indicates its causal pathogenic effect

Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization

Engineering the Processive Run Length of the Kinesin Motor

Conventional kinesin is a highly processive molecular motor that takes several hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two heads of the kinesin dimer, but the specific factors that determine kinesin's run length (distance traveled per microtubule encounter) are not known. Here, we show that the neck coiled-coil, a structure adjacent to the motor domain, plays an important role in governing the run length. By adding positive charge to the neck coiled-coil, we have created ultra-processive kinesin mutants that have fourfold longer run lengths than the wild-type motor, but that have normal ATPase activity and motor velocity. Conversely, adding negative charge on the neck coiled-coil decreases the run length. The gain in processivity can be suppressed by either proteolytic cleavage of tubulin's negatively charged COOH terminus or by high salt concentrations. Therefore, modulation of processivity by the neck coiled-coil appears to involve an electrostatic tethering interaction with the COOH terminus of tubulin. The ability to readily increase kinesin processivity by mutation, taken together with the strong sequence conservation of the neck coiled-coil, suggests that evolutionary pressures may limit kinesin's run length to optimize its in vivo function

Microtubule-dependent Plus- and Minus End–directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus

Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end–directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end–directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1–10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end–directed vesicular transport, significantly reduced the extent and the frequency of minus end–directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end–directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end– directed cytoplasmic dynein and an unknown plus end– directed activity