Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability.

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting ß-tubulin, suggesting Mgr function is required for tubulin stability. Instability of ß-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic ß-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.

Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine-arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10-Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.

Identification of a MAP65 isoform involved in directional expansion of plant cells.

MAP65 comprises a multigene family specific to plants. To see which isoform is utilised for the unique mechanism of cell expansion, uncomplicated by division structures, carrot cells were deprived of auxin whereupon they stopped dividing and elongated instead. During elongation, a MAP65 protein triplet reduced to a single band. Mass spectrometric analysis demonstrated that this corresponded to a single carrot cDNA; it also corresponded to the major protein previously shown to form filamentous cross-bridges between microtubules in vitro. This MAP65 isoform is concluded to have a major role in establishing the parallel microtubule arrays characteristic of cells undergoing directional expansion.

Klp10A, a microtubule-depolymerizing kinesin-13, cooperates with CP110 to control Drosophila centriole length.

Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules. In interphase, it promotes microtubule catastrophe; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux. Here we show that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. We also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation, in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. We discuss how loss of centriole capping by CP110 might have different consequences for centriole length in mammalian and insect cells and also relate these findings to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation.

Identification by high-throughput screening of viridin analogs as biochemical and cell-based inhibitors of the cell cycle-regulated nek2 kinase.

Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery.

The role of MAP65-1 in microtubule bundling during Zinnia tracheary element formation.

The MAP65 family of microtubule-associated proteins performs various functions at different stages of the cell cycle and differentiation. In this study, we have investigated the synchronous transdifferentiation of Zinnia mesophyll cells into tracheary elements in vitro. This allowed us to examine the role of the microtubule-associated protein MAP65 during the characteristic bunching of cortical microtubules that underlie the developing ribs of secondarily thickened cell wall. Immunofluorescence confirmed the microtubule bundles to be decorated with anti-MAP65 antibodies. Three Zinnia MAP65 genes were examined; the expression of ZeMAP65-1 was found to match that of the differentiation marker TED2 and both were found to be upregulated upon addition of inductive hormones. We cloned the full-length sequence of ZeMAP65-1 and found it to be most similar to other MAP65 isoforms known to bundle microtubules in other plant species. However, not all MAP65 proteins crosslink cortical microtubules and so, to confirm its potential bundling capacity, ZeMAP65-1 was transiently overexpressed in Arabidopsis suspension cells. This resulted in the super-bundling of microtubules in patterns resembling those in differentiating xylem cells. These findings establish that the MAP65-1 group of proteins is responsible for the bundling of cortical microtubules during secondary cell wall formation of xylogenesis as well as during the expansion of primary cell walls.

Modulated targeting of GFP-AtMAP65-1 to central spindle microtubules during division.

AtMAP65-1 bundles cortical microtubules and we examined how this property is regulated during division in time-lapse studies of Arabidopsis suspension cells expressing GFP-AtMAP65-1. Spindle fluorescence is diffuse during metaphase, restored to the central spindle at anaphase and then compacted at the midline during late anaphase/early telophase. However, mutagenesis of the microtubule-associated protein (MAP) consensus Cdk site to a non-phosphorylatable form allows premature decoration of microtubules traversing the central region of the metaphase spindle without affecting the timing of the subsequent compaction. This suggests that mutagenesis does not affect compaction but does affect a phosphorylation/dephosphorylation switch that normally targets AtMAP65-1 to the central spindle at the metaphase/anaphase transition. GFP-AtMAP65-1 continues to label the midline of the early phragmoplast, suggesting a structural continuity with the central spindle - both structures being composed of anti-parallel microtubules. However, once the cytokinetic apparatus expands into a ring the MAP becomes depleted at the midline. Despite this, cytokinesis is not arrested and membrane and callose are deposited at the cell plate. It is concluded that AtMAP65-1 plays a role in the central spindle at anaphase to early cytokinesis but is not essential at the midline of the phragmoplast at later stages.