Cytoskeleton: CLASPing the end to the edge.

Microtubules play a critical role in establishing asymmetry in many cell types. Recent work suggests how conserved protein complexes that bind specifically to the growing ends of microtubules may establish polarized links to special regions of the cell cortex.

MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis.

The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm approximately 20 minutes apart. The mei-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.

Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin.

The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle-pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin's activity in vivo.

Microtubule dynamics: Controlling split ends.

The rapid switching between growth and shrinkage at microtubule ends is important for many cellular processes. Recent studies on the structure of the microtubule and on the mechanism of action of the microtubule regulators XKCM1 and OP18 have revealed how these switching events are regulated.

An essential role for katanin in severing microtubules in the neuron.

Several lines of evidence suggest that microtubules are nucleated at the neuronal centrosome, and then released for transport into axons and dendrites. Here we sought to determine whether the microtubule-severing protein known as katanin mediates microtubule release from the neuronal centrosome. Immunomicroscopic analyses on cultured sympathetic neurons show that katanin is present at the centrosome, but is also widely distributed throughout the neuron. Microinjection of an antibody that inactivates katanin results in a dramatic accumulation of microtubules at the centrosome, indicating that katanin is indeed required for microtubule release from the centrosome. However, the antibody also causes an inhibition of axon outgrowth that is more immediate than expected on this basis alone. It may be that katanin severs microtubules throughout the cell body to keep them sufficiently short to be efficiently transported into developing processes. Consistent with this idea, there were significantly fewer free ends of microtubules in the cell bodies of neurons that had been injected with the katanin antibody compared with controls. These results indicate that microtubule-severing by katanin is essential for releasing microtubules from the neuronal centrosome, and also for regulating the length of the microtubules after their release.

Katanin is responsible for the M-phase microtubule-severing activity in Xenopus eggs.

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1alpha, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.

A role for katanin-mediated axonemal severing during Chlamydomonas deflagellation.

Deflagellation of Chlamydomonas reinhardtii, and other flagellated and ciliated cells, is a highly specific process that involves signal-induced severing of the outer doublet microtubules at a precise site in the transition region between the axoneme and basal body. Although the machinery of deflagellation is activated by Ca2+, the mechanism of microtubule severing is unknown. Severing of singlet microtubules has been observed in vitro to be catalyzed by katanin, a heterodimeric adenosine triphosphatase that can remove tubulin subunits from the walls of stable microtubules. We found that purified katanin induced an ATP-dependent severing of the Chlamydomonas axoneme. Using Western blot analysis and indirect immunofluorescence, we demonstrate that Chlamydomonas expresses a protein that is recognized by an anti-human katanin antibody and that this protein is localized, at least in part, to the basal body complex. Using an in vitro severing assay, we show that the protein(s) responsible for Ca2+-activated outer doublet severing purify with the flagellar-basal body complex. Furthermore, deflagellation of purified flagellar-basal body complexes is significantly blocked by the anti-katanin antibody. Taken together, these data suggest that a katanin-like mechanism may mediate the severing of the outer doublet microtubules during Chlamydomonas deflagellation.

Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.

Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60 and 80 kDa subunits, was previously purified and shown to localize to centrosomes in vivo. Here we report the sequences and activities of the katanin subunits. p60 is a new member of the AAA family of ATPases, and we show that expressed p60 has microtubule-stimulated ATPase and microtubule-severing activities in the absence of p80. p80 is a novel protein containing WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. These results indicate katanin's activities are segregated into a subunit (p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome.

Katanin, the microtubule-severing ATPase, is concentrated at centrosomes.

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

Modulation of microtubule dynamics during the cell cycle.

Microtubule dynamics change dramatically during the cell cycle, but the mechanisms by which these changes occur are unknown. Recent progress has been made in four areas: firstly, in the determination of changes in microtubule turnover and net tubulin polymer levels in vivo; secondly, in the elucidation of mechanisms of regulation of microtubule dynamics by microtubule-associated protein 4; thirdly, in the determination of the mechanisms by which Xenopus microtubule-associated protein regulates microtubule dynamics; and fourthly, in the elucidation of the structural basis of microtubule nucleation by gamma tubulin.

Origin recognition complex (ORC) in transcriptional silencing and DNA replication in S. cerevisiae.

In Saccharomyces cerevisiae, the HMR-E silencer blocks site-specific interactions between proteins and their recognition sequences in the vicinity of the silencer. Silencer function is correlated with the firing of an origin of replication at HMR-E. An essential gene with a role in transcriptional silencing was identified by means of a screen for mutations affecting expression of HMR. This gene, known as ORC2, was shown to encode a component of the origin recognition complex that binds yeast origins of replication. A temperature-sensitive mutation in ORC2 disrupted silencing in cells grown at the permissive temperature. At the restrictive temperature, the orc2-1 mutation caused cell cycle arrest at a point in the cell cycle indicative of blocks in DNA replication. The orc2-1 mutation also resulted in the enhanced mitotic loss of a plasmid, suggestive of a defect in replication. These results provide strong evidence for an in vivo role of ORC in both chromosomal replication and silencing, and provide a link between the mechanism of silencing and DNA replication.

Identification of katanin, an ATPase that severs and disassembles stable microtubules.

Eukaryotic cells rapidly reorganize their microtubule cytoskeleton during the cell cycle, differentiation, and cell migration. In this study, we have purified a heterodimeric protein, katanin, that severs and disassembles microtubules to tubulin dimers. The disassembled tubulin can repolymerize, indicating that it is not irreversibly modified or denatured in the reaction. Katanin is a microtubule-stimulated ATPase and requires ATP hydrolysis to sever microtubules. Katanin represents a novel type of enzyme that utilizes energy from nucleotide hydrolysis to break tubulin-tubulin bonds within a microtubule polymer, a process that may aid in disassembling complex microtubule arrays within cells.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

Isolation of the dorsal locus of Drosophila.

The establishment of embryonic polarity is a crucial step in pattern formation and morphogenesis. In the fruitfly Drosophila melanogaster, embryonic polarity depends primarily on genes expressed in the female during oogenesis. Mutations in these 'maternal effect' genes can lead to major disruptions in normal pattern formation. Two classes of maternal genes essential for the establishment of polarity in the embryo have been identified. Lesions in one class, the 'bicaudal' genes, disrupt the anterior-posterior axis; lesions in the other class disrupt dorsal-ventral polarity, and in the most extreme cases embryos fail to form any ventral or lateral structures. Genetic studies suggest that the anterior-posterior and dorsal-ventral axes may be independent as the defects observed in mutants from each class seem to be restricted to one axis only. The dorsal (dl) locus is one of the maternal effect genes involved in the establishment of dorsal-ventral polarity. Homozygous dl females produce embryos exhibiting the mutant phenotype--complete lack of dorsal-ventral polarity in the strongest alleles--irrespective of the genotype of the father. Although dl is a maternal effect locus and must be expressed during oogenesis, the gene product, or a substance depending on the normal function of the dl gene, seems to be active early in embryogenesis, as the dl phenotype can be partially rescued by injection of cytoplasm from wild-type cleavage-stage embryos. Here we report the molecular cloning of the dorsal locus and a study of its expression.