Abstract, We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesininduced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP-sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility.V ARIOUS forms of intracellular transport are thought to depend upon microtubule-associated mechanochemical ATPases (Vale, 1987). One such enzyme, kinesin (Vale et al., 1985a), binds microtubules in a nucleotide sensitive fashion, and displays a microtubule-activated ATPase activity that drives movement of kinesin-coated objects towards the plus end of microtubules (MTs) ~ in vitro (Vale et al., 1985a, b;Scholey et al., 1985;Brady, 1985; Kuznetsoy and Gelfand, 1986;Porter et al., 1987;Cohn et al., 1987;Saxton et al., 1988;Gelles et al., 1988). Kinesin from a variety of animals contains a major polypeptide with an apparent molecular mass of 110-140 kD, encoded by a single gene in Drosophila (Yang et al., 1988), plus copurifying light chains of Mr = 40-80 kD (Vale et al., 1985a,b;Amos, 1987;Kuznetsov and Gelfand, 1986;Saxton et al., 1988). Bovine brain kinesin is an tt2132 heterotetramer consisting of two 120-kD heavy chains plus two 60-kD light chains, assembled into an elongated molecule (Amos, 1987;Kuznetsov et al., 1988;Bloom et al., 1988). Kinesin is proposed to participate in organelle/vesicle transport, in organizing the endomembrane system, and in mitosis (Vale et al., 1986;Vale, 1987;Leslie et al., 1987;Schroer and Sheetz, 1988), but direct evidence concerning the biological functions of kinesin is currently lacking.Antibodies that inhibit mechanochemical activity have been extremely useful in probing the functions of myosin and 1. Abbreviations used in this paper: MT(s), microtubule(s); PMEG, 0.9 M glycerol, 0.1 M Pipes, pH 6.9, 5 mM EGTA, 2.5 mM MgSO4, 0.5 mM EDTA, 1 mM DTT, 100 Ixg/ml soybean trypsin inhibitor, 1 mg/mlp-tosyl-Larginine methyl ester hydrochloride, 10 Ixg/ml leupeptin, pepstatin, and aprotinin; RT, room temperature. dynein. For example, an antiserum that inhibits the ATPase activity of sea urchin sperm dynein caused a corresponding decrease in the beat frequency of reactivated sea urchin sperm, supporting the hypothesis that the dynein ATPase drives axonemal motility (Ogawa and Mohri, 1975;O...

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.

Preparations of kinesin, a microtubule-based force-producing protein, have been isolated from Drosophila melanogaster embryos by incubation of microtubules with a nonhydrolyzable ATP analogue and gel filtration of proteins released from the microtubules by ATP. These preparations induced MgATP-dependent microtubule gliding in vitro with a Km for MgATP of 44 ,LM and a Vmax for gliding of 0.9 ,um/sec. Samples of Drosophila proteins that were active in motility assays possessed an average ATPase activity in solution of 17 nmol/min per mg that increased to an average of 106 nmol/min per mg in the presence of microtubules. The major polypeptides that copurified with these activities showed relative molecular masses of 115 kDa and 58 kDa. An antiserum raised against the 115-kDa polypeptide also recognized the 110-kDa component of squid kinesin preparations and the 130-kDa component of sea urchin kinesin preparations.

To better determine the ecological role of motility in pennate diatoms, we quantitatively characterized several motility and adhesion properties of four species of motile pennate diatoms (Craticula sp., Pinnularia sp., Nitzschia sp., and Stauroneis sp.) isolated from the same freshwater pond. Using computer‐assisted video microscopy, we measured speed, size/shape, functional adhesion, path curvature, and light sensitivity for these species, each of which shows a distinctive set of motile behaviors. The average speeds of Stauroneis, Pinnularia, Nitzschia, and Craticula cells are 4.6, 5.3, 10.4, and 10.0 μm · s−1, respectively. Craticula and Nitzschia cells move in a relatively straight path (<4 degrees rotation per 100 μm movement), Stauroneis exhibits minor rotation (about 7 degrees per 100 μm movement), and Pinnularia rotates considerably during movement (about 22 degrees per 100 μm moved). Functional adhesion (as measured by the release rate of attached cells from the underside of an inverted coverslip) shows a half time for cell release of approximately 50 min for Craticula, 192 min for Pinnularia, and >1 day for Nitzschia and Stauroneis. Direction reversal at light/dark boundaries, which appears to be the main contributor to diatom Phototaxis, is most responsive for Craticula, Pinnularia, and Nitzschia at wavelengths around 500 nm. Craticula and Nitzschia cells are the most sensitive in the photophobic response, with over 60% of these cells responding to a 30‐1x light/dark boundary at 500 nm, whereas Pinnularia cells are only moderately responsive at this irradiance, showing a maximal response of approximately 30% of cells at 450 nm. Stauroneis cells, in contrast, had a maximal photosensitive response at 700 nm, suggesting that this cell type may use a different response mechanism than the other three cell types. In addition, Craticula and Pinnularia show a net movement out of the light spot when illuminated at 650 nm, whereas Stauroneis shows a net movement out of the light spot when illuminated at 450 nm. Such quantitative characterizations of species‐specific responses to environmental stimuli should give us a firm foundation for future studies analyzing the behavior of interspecies diatom competition for limited light or nutrient resources.