Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton during Drosophila oogenesis

The kinesin-like protein encoded by pavarotti (Pav-KLP) is essential for cytokinesis and associates with the central part of the late mitotic spindle and interphase nuclei in somatic cells (Adams et al., 1988). Here we define regions of the molecule that regulate its subcellular localisation and study the consequences of overexpressing mutant forms of the protein during oogenesis in Drosophila. Pav-KLP normally associates with the oocyte nucleus, but when over-expressed at moderate levels, its GFP tagged form also accumulates in nurse cell nuclei. At high expression levels this leads to loss of the microfilaments that tether these nuclei, so that they block the ring canals and prevent the 'dumping' of nurse cell cytoplasm into the oocyte, which results in sterility. Localisation to these nuclei is prevented by mutations in either the conserved ATP-binding site of the motor domain or the nuclear localisation sequences in the C-terminal domain. Both such mutations lead to the formation of stable arrays of cytoplasmic microtubules and the progressive disruption of the actin cytoskeleton. The latter is evident by a breakdown of the cortical actin causing disruption of cell membranes; this breakdown ultimately results in the accumulation of cytoplasmic aggregates containing tubulin, actin and at least some of their binding proteins. Pav-KLP is also found associated with the ring canals, actin-rich structures built from remnants of the cytokinesis ring. The stalk domain alone is sufficient for the exclusive association of Pav-KLP to these structures, and this has no consequences for fertility. We discuss whether disruption of actin structures by full-length cytoplasmic forms of Pav-KLP is a consequence of the resulting stabilised cytoplasmic microtubules per se or accumulation of the motor protein at ectopic cortical sites to sequester molecules that regulate actin behaviour

First operation and perspectives of the superconducting linac LISA

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Drosophila Polo Kinase Is Required for Cytokinesis

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis

Nessun Dorma, a novel centralspindlin partner, is required for cytokinesis in Drosophila spermatocytes

Nessun Dorma is a component of the ring canal with a polysaccharide-binding domain, which is important for cytokinesis during male meiosis

Localization of Pavarotti-KLP in Living Drosophila Embryos Suggests Roles in Reorganizing the Cortical Cytoskeleton during the Mitotic Cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation