The development of an enzyme-linked immunosorbent assay (ELISA) for the herbicide molinate (Ordram, S-ethyl hexahydroazepine-l-carbothioate) is described. By a thiol replacement reaction of thiocarbamate sulfones, several haptens were synthesized. These haptens were coupled to carrier proteins via mixed-anhydride and diazotization procedures. Antibodies raised against these antigens were screened for sensitivity and specificity for molinate. An assay is described with a limit of detectability of 3 ng/mL that is specific for molinate with some cross-reactivity (15%) to molinate sulfone. Other thiocarbamate pesticides had cross-reactivities of <1.4%. Propylene glycol, as a carrier solvent, interfered in the ELISA at concentrations of 12.5% and greater, whereas acetonitrile had no effect at 12.5% but had increased and then decreased absorbances relative to control at 5% and 25%, respectively. The half-lives of molinate in a laboratory study as measured by liquid scintillation counting and ELISA were 2.6 and 2.2 days, respectively. Paired t statistics on this data gave a significant correlation with 0.05 < p < 0.02. Techniques allowing current sample preparation methods to be easily adapted for use with ELISA analysis are also described.

Microtubules are essential for a wide range of cellular processes that vary between cell types. Katanin is a microtubule-severing protein that carries out an essential role in meiotic spindles in Caenorhabditis elegans and a non-essential role in mitotic spindles of vertebrates. In contrast to these M-phase associated roles, katanin is also essential for post-mitotic differentiation events in vertebrate neurons and in Arabidopsis. This diversity of function suggests that katanin's activity might be regulated by multiple mechanisms. Because katanin is active in M-phase Xenopus extracts but not in interphase extracts, we assayed for regulators of katanin's activity in these extracts. The microtubule-severing activity of purified katanin was inhibited by interphase Xenopus extracts. Fractionation revealed that this inhibition was due to at least 4 separable components, one of which contains the MAP4 homolog, XMAP230. Inhibition of katanin-mediated microtubule-disassembly activity by the XMAP230-containing fraction was reversible by cyclinB/cdk1, suggesting one possible mechanism for the increased severing activity observed in M-phase Xenopus extracts. In a previous study, spindle pole association by katanin was essential for its activity during mitosis suggesting that katanin's activity might also be regulated by co-localization with an activator. The polo-like kinase, Plx1, co-localized with katanin at spindle poles in vivo and purified Plx1 increased the microtubule-severing activity of katanin in vitro. These in vitro experiments illustrate the potential complexity of the regulation of katanin's activity in vivo and may explain how katanin can carry out widely different functions in different cell types.

Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartite tail [Scholey et al., 1989]. In the present, complementary study, we have used the monoclonal antikinesin, SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cytosol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitometry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equimolar quantities of heavy and light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.

The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm ∼20 minutes apart. Themei-1 and mei-2 genes of C. elegans are required for the establishment of the oocyte meiotic spindle but are not required for mitotic spindle function. mei-1 encodes an AAA ATPase family member with similarity to the p60 catalytic subunit of the heterodimeric sea urchin microtubule-severing protein, katanin. We report that mei-2 encodes a 280-amino acid protein containing a region similar to the p80-targeting subunit of katanin. MEI-1 and MEI-2 antibodies decorate the polar ends of meiotic spindle microtubules and meiotic chromatin. We find that the subcellular location of MEI-2 depends on wild-type mei-1 activity and vice versa. These experiments, combined with MEI-1 and MEI-2's similarity to p60 and p80 katanin, suggest that the C. elegans proteins function as a complex. In support of this idea, MEI-1 and MEI-2 physically associate in HeLa cells. Furthermore, co-expression of MEI-1 and MEI-2 in HeLa cells results in the disassembly of microtubules. These data lead us to conclude that MEI-1/MEI-2 microtubule-severing activity is required for meiotic spindle organization in C. elegans.

Katanin is a microtubule-severing protein that is concentrated at mitotic spindle poles but katanin's function in the mitotic spindle has not been previously reported. Inhibition of katanin with either of two dominant-negative proteins or a subunit-specific antibody prevented the redistribution of γ-tubulin from the centrosome to the spindle in prometaphase CV-1 cells as assayed by immunofluorescence microscopy. Becauseγ-tubulin complexes can bind to pre-existing microtubule minus ends,these results could be explained by a model in which the broad distribution ofγ-tubulin in the mitotic spindle is in part due to cytosolicγ-tubulin ring complexes binding to microtubule minus ends generated by katanin-mediated microtubule severing. Because microtubules depolymerize at their ends, we hypothesized that a greater number of microtubule ends generated by severing in the spindle would result in an increased rate of spindle disassembly when polymerization is blocked with nocodazole. Indeed,katanin inhibition slowed the rate of spindle microtubule disassembly in the presence of nocodazole. However, katanin inhibition did not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching. These results support a model in which katanin activity regulates the number of microtubule ends in the spindle.