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.
Minestrini
Gianluca
G
Cancer Research Campaign Cell Cycle Genetics Group, University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, UK.
Máthé
Endre
E
Glover
David M
DM
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D004330:Drosophila / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D029721:Drosophila Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005260:Female; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000032:analysis; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D008870:Microtubules / Q000648:ultrastructure; D009154:Mutation; D009866:Oogenesis; D017434:Protein Structure, Tertiary; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D014404:Tubulin; C490516:pav protein, Drosophila; D049452:Green Fluorescent Proteins