(1/1179) Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.  (+info)

(2/1179) Dynacortin contributes to cortical viscoelasticity and helps define the shape changes of cytokinesis.

During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis.  (+info)

(3/1179) Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast.

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.  (+info)

(4/1179) The flagella connector of Trypanosoma brucei: an unusual mobile transmembrane junction.

Throughout its elongation, the new flagellum of the procyclic form of the African trypanosome Trypanosoma brucei is tethered at its tip to the lateral aspect of the old flagellum. This phenomenon provides a cytotactic mechanism for influencing inheritance of cellular pattern. Here, we show that this tethering is produced via a discrete, mobile transmembrane junction - the flagella connector. Light and electron microscopy reveal that the flagella connector links the extending microtubules at the tip of the new flagellum to the lateral aspect of three of the doublet microtubules in the old flagellar axoneme. Two sets of filaments connect the microtubules to three plates on the inner faces of the old and new flagellar membranes. Three differentiated areas of old and new flagellar membranes are then juxtaposed and connected by a central interstitial core of electron-dense material. The flagella connector is formed early in flagellum extension and is removed at the end of cytokinesis, but the exact timing of the latter event is slightly variable. The flagella connector represents a novel form of cellular junction that is both dynamic and mobile.  (+info)

(5/1179) Role of the midbody matrix in cytokinesis: RNAi and genetic rescue analysis of the mammalian motor protein CHO1.

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.  (+info)

(6/1179) A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast.

The Schizosaccaromyces pombe protein Flp1p belongs to a conserved family of serine-threonine-phosphatases. The founding member of this family, Saccharomyces cerevisiae Cdc14p, is required for inactivation of mitotic CDKs and reversal of CDK mediated phosphorylation at the end of mitosis, thereby bringing about the M-G1 transition. Initial studies of Flp1p suggest that it may play a different role to Cdc14p. Here we show that Flp1p is required for rapid degradation of the mitotic inducer Cdc25p at the end of mitosis, and that Cdc25p is a substrate of Flp1p in vitro. Down-regulation of Cdc25p activity by Flp1p may ensure a prompt inactivation of mitotic CDK complexes to trigger cell division. Our results suggest a regulatory mechanism, and a universal role, for Cdc14p like proteins in coordination of cytokinesis with other cell cycle events.  (+info)

(7/1179) Functional characterization of Dma1 and Dma2, the budding yeast homologues of Schizosaccharomyces pombe Dma1 and human Chfr.

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Delta dma2Delta cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.  (+info)

(8/1179) The nucleolus is involved in mRNA export from the nucleus in fission yeast.

To elucidate the mechanism of mRNA export from the nucleus, we isolated five novel temperature-sensitive mutants (ptr7 to ptr11) that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr11 mutation was found in the top2(+) gene encoding DNA topoisomerase II. In addition to the nuclear accumulation of poly(A)(+) RNA, ptr11 exhibited the cut (cell untimely torn) phenotype at the nonpermissive temperature, like the previously isolated mutant, ptr4. In these two mutants, cytokinesis occurred without prior nuclear division, resulting in cleavage of the undivided nuclei by the septum. To investigate the relationship between mRNA export defects and the cut phenotype observed in ptr4 and ptr11, we analyzed 11 other mutants displaying the cut phenotype and found that all these tested mutants accumulate poly(A)(+) mRNA in the aberrantly cleaved nuclei. Interestingly, nuclear accumulation of poly(A)(+) mRNA was observed only in the anucleolate nuclei produced by aberrant cytokinesis. In addition, nuc1, the S. pombe mutant exhibiting a collapsed nucleolus, trapped poly(A)(+) mRNA in the nucleolar region at the nonpermissive temperature. In ptr11 and nuc1, mRNA transcribed from the intron-containing TBP gene showed nuclear accumulation, but not transcripts from the intron-less TBP cDNA, suggesting that the export pathway differs between the spliced and unspliced TBP mRNAs. These findings support the notion that a subset of mRNAs in yeast is exported from the nucleus through transient association with the nucleolus.  (+info)