To develop our understanding of myosin-1a function in vivo, we have created a mouse line null for the myosin-1a gene. Myosin-1a knockout mice demonstrate no overt phenotypes at the whole animal level but exhibit significant perturbations and signs of stress at the cellular level. Among these are defects in microvillar membrane morphology, distinct changes in brush-border organization, loss of numerous cytoskeletal and membrane components from the brush border, and redistribution of intermediate filament proteins into the brush border. We also observed significant ectopic recruitment of another short-tailed class I motor, myosin-1c, into the brush border of knockout enterocytes. This latter finding, a clear demonstration of functional redundancy among vertebrate myosins-I, may account for the lack of a whole animal phenotype. Nevertheless, these results indicate that myosin-1a is a critical multifunctional component of the enterocyte, required for maintaining the normal composition and highly ordered structure of the brush border.

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.

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Conventional kinesin is a highly processive, plus-enddirected microtubule-based motor that drives membranous organelles toward the synapse in neurons. Although recent structural, biochemical, and mechanical measurements are beginning to converge into a common view of how kinesin converts the energy from ATP turnover into motion, it remains difficult to dissect experimentally the intermolecular domain cooperativity required for kinesin processivity. We report here our presteady-state kinetic analysis of a kinesin switch I mutant at Arg 210 (NXXSSRSH, residues 205-212 in Drosophila kinesin). The results show that the R210A substitution results in a dimeric kinesin that is defective for ATP hydrolysis and a motor that cannot detach from the microtubule although ATP binding and microtubule association occur. We propose a mechanistic model in which ATP binding at head 1 leads to the plus-end-directed motion of the neck linker to position head 2 forward at the next microtubule binding site. However, ATP hydrolysis is required at head 1 to lock head 2 onto the microtubule in a tight binding state before head 1 dissociation from the microtubule. This mechanism optimizes forward movement and processivity by ensuring that one motor domain is tightly bound to the microtubule before the second can detach.Kinesin is a highly processive, dimeric mechanoenzyme that travels along microtubules toward their plus-ends in discrete 8-nm steps, each step tightly coupled to a single ATP turnover (1-3). Recent evidence from a variety of experimental approaches has focused our attention to the proposal presented by Rice et al. (4) that ATP binding induces a pronounced conformational change in the neck linker region, which docks the neck linker onto the catalytic core and propels the unattached kinesin head forward to find the next binding site on the microtubule. This model is based on a disorder-to-order transition in the neck linker region for monomeric kinesin constructs. The neck linker of the Mt⅐K 1 complex was shown to be mobile in the presence of ADP, existing in an equilibrium with two predominant conformations trapped by cryo-electron microscopy. However, upon the addition of ATP or nonhydrolyzable ATP analogs to the Mt⅐K complex, the neck mobility ceased with the neck linker element tightly associated with the catalytic core. This ordered state was reversed by the addition of ADP or loss of nucleotide. In addition, the cryo-electron microscopy of this proposed ATP state revealed a single discrete orientation of the neck linker with the carboxyl terminus of the motor domain directed toward the plus-end of the microtubule (4). Xing et al. (5) have reported for a monomeric kinesin motor domain two discrete structural transitions induced by ADP binding and another produced by ATP binding. These three conformations revealed by fluorescence resonance energy transfer were consistent with the results reported by Rice et al. (4). Furthermore, biochemical studies of dimeric kinesin have demonstrated that ATP binding (or nonhydrolyzabl...

Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other. Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s ؊1 , yet rate-limiting ADP release from the second motor domain occurs at 1.4 s ؊1 . We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule⅐Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minusend of the microtubule. The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head. ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor. Ncd1 is a microtubule-activated ATPase of the kinesin superfamily involved in spindle assembly and stabilization during female meiosis and the early divisions of the Drosophila embryo (1-4). Conventional kinesin, the founding member of the kinesin superfamily, moves membranous organelles from the neuron cell body to the synapse (reviewed in Refs. 5-7). The motor domains of Ncd and kinesin are ϳ40% identical in amino acid sequence, and their three-dimensional atomic structures are nearly superimposable (8 -10). Despite this high degree of similarity, there are striking differences in their motor behavior that are important for biological function in vivo. First, Ncd translocates to the minus-end of microtubules, while kinesin moves to the plus-end (2, 4, 11). Additionally, kinesin is a highly processive motor, taking several steps per encounter with the microtubule, while Ncd has been shown to be nonprocessive, both mechanically and chemically (12-15). Ncd and kinesin also differ in their motility and ATPase rates; Ncd is a much slower motor.Comparison of the kinesin and Ncd crystal structures suggests that despite the structural similarity of the individual catalytic motor domains, there is a different overall orientation of the heads within the Ncd dimer in comparison to those of the kinesin dimer, both in solution and when bound to the microtubule (16 -21). The neck linker, a sequence of ϳ15 amino acids directly adjacent to the catalytic core, has been implicated in specifying the direction of movement for kinesin superfamily members and to be important for force generation (17,(22)(23)(24). For Ncd, the neck linker includes 13 amino acids N-terminal to the catalytic core. There is high sequence homology among minus-end directed kinesin motors, yet this neck linker sequence is different fro...