The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.

During vertebrate cell division, chromosomes oscillate with periods of smooth motion interrupted by abrupt reversals in direction. These oscillations must be spatially constrained in order to align and segregate chromosomes with high fidelity, but the molecular mechanism for this activity is uncertain. We report here that the human kinesin-8 Kif18A has a primary role in the control of chromosome oscillations. Kif18A accumulates as a gradient on kinetochore microtubules in a manner dependent on its motor activity. Quantitative analyses of kinetochore movements reveal that Kif18A reduces the amplitude of preanaphase oscillations and slows poleward movement during anaphase. Thus, the microtubule-depolymerizing kinesin Kif18A has the unexpected function of suppressing chromosome movements. Based on these findings, we propose a molecular model in which Kif18A regulates kinetochore microtubule dynamics to control mitotic chromosome positioning.

Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1.08 ؎ 0.09 steps for each ATP hydrolyzed at 1 mM ATP. Therefore, under low loads in which the drag force < < 1 pN, coupling between the chemical and mechanical cycles of kinesin is tight, consistent with conventional power stroke models. Our results rule out models that require two or more ATPs/step, such as some thermal ratchet models, or that propose multiple steps powered by single ATPs.Conventional kinesin is a protein machine that steps along the surface of a microtubule as it carries a membrane-bound organelle toward the periphery of a cell (1-3). The size of the steps is ϳ8 nm (4,5). This is the distance between consecutive binding sites along the microtubule protofilament (6, 7), and a single kinesin molecule can take hundreds of steps without detaching (8, 9), even against opposing loads as high as ϳ6 pN (10 -12). The steps are driven by the hydrolysis of ATP; kinesin is an ATPase (13) whose speed of movement increases linearly with ATP concentration until it approaches a maximum of about 800 nm/s (8, 14), and AMP-PNP, 1 a nonhydrolyzable analog of ATP, arrests movement (15, 16). Despite intensive biochemical, biophysical, and structural investigations over the last few years, there remains considerable uncertainty as to the mechanism by which the stepping is coupled to ATP hydrolysis.In this work, we address the question: what is the stoichiometry of kinesin? In other words, how many steps does kinesin take for each ATP that it hydrolyzes? This question is important because it tests different models proposed to explain how motor proteins such as kinesin and myosin work. For example, one class of "thermal ratchet" models predicts that the stoichiometry is Յ0.5 steps/ATP (17, 18); these models postulate that ATP hydrolysis rectifies a diffusive motion in such a way that a step only occurs if the diffusion is toward the next binding site. Because there is an equal probability that the motor diffuses away from the next binding site (i.e. in the wrong direction), this model predicts that on average at least two molecules of ATP are hydrolyzed per forward step. A stoichiometry of less than one could also be due to "futile" hydrolysis cycles, those that fail to produce steps and lead to "slippage" between the mechanical and chemical cycles. Other models postulate a stoichiometry greater than 1 and have been...

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.

Summary Alignment of chromosomes at the metaphase plate is a signature of cell division in metazoan cells, yet the mechanisms controlling this process remain ambiguous. Here we use a combination of quantitative live cell imaging and reconstituted dynamic microtubule assays to investigate the molecular control of mitotic centromere movements. We establish that Kif18A (kinesin-8) attenuates centromere movement by directly promoting microtubule pausing in a concentration-dependent manner. This activity provides the dominant mechanism for restricting centromere movement to the spindle midzone. Furthermore, polar ejection forces spatially confine chromosomes via position-dependent regulation of kinetochore tension and centromere switch rates. We demonstrate that polar ejection forces are antagonistically modulated by chromokinesins. These pushing forces depend on Kid (kinesin-10) activity and are antagonized by Kif4A (kinesin-4), which functions to directly suppress microtubule growth. These data support a model in which Kif18A and polar ejection forces synergistically promote centromere alignment via spatial control of kinetochore-microtubule dynamics.

In cells, stable microtubules are covalently modified by a carboxy-peptidase which removes the C-terminal tyrosine residue of α-tubulin. The significance of this selective detyrosination of microtubules is not understood. Here, we report that tubulin detyrosination in fibroblasts inhibits microtubule disassembly. This inhibition is relieved by overexpression of the depolymerizing motor MCAK. Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated microtubules in fibroblasts. Detyrosination of microtubules suppresses the activity of MCAK in vitro, apparently due to a decreased affinity of the ADP-Pi and ADP bound forms of MCAK for the microtubule lattice. Detyrosination also impairs microtubule disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that microtubule depolymerizing motors are directly inhibited by detyrosination of tubulin, resulting in stabilization of cellular microtubules. Detyrosination of transiently stabilized microtubules may give rise to persistent subpopulations of disassembly-resistant polymers to sustain sub-cellular cytoskeletal differentiation.

The human genome has three unique genes coding for kinesin-13 proteins called Kif2a, Kif2b, and MCAK (Kif2c). Kif2a and MCAK have documented roles in mitosis, but the function of Kif2b has not been defined. Here, we show that Kif2b is expressed at very low levels in cultured cells and that GFP-Kif2b localizes predominately to centrosomes and midbodies, but also to spindle microtubules and transiently to kinetochores. Kif2b-deficient cells assemble monopolar or disorganized spindles. Chromosomes in Kif2b-deficient cells show typical kinetochore-microtubule attachments, but the velocity of movement is reduced approximately 80% compared with control cells. Some Kif2b-deficient cells attempt anaphase, but the cleavage furrow regresses and cytokinesis fails. Like Kif2a-deficient cells, bipolar spindle assembly can be restored to Kif2b-deficient cells by simultaneous deficiency of MCAK or Nuf2 or treatment with low doses of nocodazole. However, Kif2b-deficient cells are unique in that they assemble bipolar spindles when the pole focusing activities of NuMA and HSET are perturbed. These data demonstrate that Kif2b function is required for spindle assembly and chromosome movement and that the microtubule depolymerase activities of Kif2a, Kif2b, and MCAK fulfill distinct functions during mitosis in human cells.