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Pavarotti Encodes a Kinesin-Like Protein Required to Organize the Central Spindle and Contractile Ring for Cytokinesis

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Pavarotti Encodes a Kinesin-Like Protein Required to Organize the Central Spindle and Contractile Ring for Cytokinesis Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis Richard R. Adams, Alvaro A.M. Tavares, Adi Salzberg,1,2 Hugo J. Bellen,1 and David M. Glover3 Cancer Research Campaign (CRC) Laboratories, Cell Cycle Genetics Research Group, Department of Anatomy and Physiology, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, UK; 1Howard Hughes Medical Institute, Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, Texas 77030 USA Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. We show that the septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV–KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. We show that Polo kinase associates with PAV–KLP with which it shows an overlapping pattern of subcellular localization during the mitotic cycle and this distribution is disrupted in pav mutants. We suggest that PAV–KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins. [Key Words: Cytokinesis; kinesin-like protein; Polo kinase; Drosophila] Received November 21, 1997; revised version accepted March 11, 1998. Cytokinesis is the ultimate act of mitosis, whereby seg- first model, the poles signal directly to the equatorial regated daughter nuclei are partitioned into two separate cortex of the cell, perhaps by the action of astral micro- cells. In eukaryotes, this is accomplished by an actin– tubules (Devore et al. 1989). Alternatively, the astral re- myosin contractile ring that forms around the cell equa- laxation model (White and Borisy 1983) proposes that tor during mitosis and constricts inwards at telophase spindle poles induce a relaxation of the cell cortex near- (Schroeder 1972). In higher eukaryotes the correct posi- est to the poles, leading to a tension differential between tioning and assembly of the contractile ring requires the the poles and the equator that results in equatorial con- mitotic spindle, although the mechanism by which this traction. However, this model has been discredited re- occurs is a matter of controversy (Swann and Mitchison cently by force measurements showing that tension in- 1956; Zhang and Nicklas 1996). The classical experi- creases at the equator without a concomitant decrease at ments of Rappaport (1961) suggest that the spindle poles the cell poles (Burton and Taylor 1997). are sufficient to stimulate cytokinesis. By generating On the other hand, there is growing evidence that the horseshoe-shaped single cell echinoderm embryos con- central spindle plays an essential role in the positioning taining two mitotic spindles, not only were cleavage fur- and assembly of the contractile ring. The central spindle rows formed in the central region of the mitotic spindles, is composed of a dense network of overlapping antipar- but an extra furrow was formed equidistant between two allel microtubules that forms between the separating asters lacking any intervening chromosomes or spindle daughter nuclei during anaphase (Mastronarde et al. microtubules. Similar phenomena have been observed in 1993). In cultured cells induced to develop multipolar cultured mammalian cells, and although this does occur spindles as result of treatment with low concentrations infrequently it suggests that this mechanism may act of the microtubule-destabilizing drug colcemid, the for- universally (Eckley et al. 1997; Rieder et al. 1997). Two mation of a cleavage furrow absolutely depends on the mechanisms have been put forward to demonstrate how presence of central spindle microtubules that are re- the spindle poles might induce cleavage furrows. In the quired throughout cytokinesis (Wheatley and Wang 1996). Creation of a barrier between the central spindle and the cell cortex prevents cleavage, indicating that a 2Present address: Department of Genetics, Bruce Rappaport Faculty of signal for cytokinesis may emanate from the central Medicine, Technion–Israel Institute of Technology, Haifa 31096, Israel. 3Corresponding author. spindle (Cao and Wang 1996). Manipulation of grasshop- E-MAIL [email protected]; FAX 44(0) 1382 344213. per neuroblast spindles results in the formation of a GENES & DEVELOPMENT 12:1483–1494 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org 1483 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Adams et al. cleavage furrow when the spindle is moved close to the Results cell cortex (Kawamura 1977). pav embryos show defects at cytokinesis In addition to the contribution made by the spindle, several proteins, known as the chromosomal passenger Preliminary characterization of the phenotype of pav proteins, dissociate from chromosomes at the metapha- mutants by staining embryos with the neuronal marker se–anaphase transition to be deposited at the cell equa- mAb 22C10 indicated the formation of fewer and larger tor. The inner centromere proteins (INCENPs), for ex- neurons than usual in the embryonic development of the ample, transfer to the central spindle and the cell cortex PNS, and an absence of support cells (Salzberg et al. and are necessary for completion of cytokinesis (Eckley 1994). To investigate whether the large cells seen in pav et al. 1997; Earnshaw and Cook 1991), whereas TD-60 mutant embryos could arise from cell division defects, forms a disc structure in the position of the metaphase we looked for mitotic defects in pav mutants from cycle plate anticipating the cleavage plane (Andreassen et al. 14 (the first cycle under the control of the zygotic ge- 1991). This disc is proposed to link the central spindle to nome) onward through cycles 15 and 16 (Materials and the cell cortex (Martineau et al. 1995). Methods). We could detect no differences between wild- In Drosophila, mutational analysis has identified sev- type and pav embryos in cycle 14 or 15, but in cycle 16 eral genes encoding structural elements of the contrac- we observed that although mitosis appeared to be normal tile ring essential for cytokinesis, such as myosin light within the ventral epidermis, interphase nuclei were fre- chain, cofilin, profilin, and Peanut, a homolog of the quently closely associated in pairs in both P-element and yeast septins (Karess et al. 1991; Neufeld and Rubin EMS-induced alleles of pav (Fig. 1B). The boundaries of 1994; Gunsalus et al. 1995; Giansanti et al. 1996). Re- the cells of the embryo shown in this field are defined cently, cloning of the gene defective in KLP3A mutants using an antibody against a-spectrin, a membrane-bound identified a kinesin-like protein (KLP) as having a role in structural protein. It can be seen that the cell membrane cytokinesis in male meiosis (Williams et al. 1995). KLPs does not invaginate during telophase in pav embryos as are microtubule motor proteins responsible for many of it does in wild type (Fig. 1A). Despite this failure of cy- the dynamic aspects of mitosis such as centrosome sepa- tokinesis, nuclei appear to enter interphase 17 success- ration, spindle assembly, and anaphase movement of fully, as nuclear laminae reform around both nuclei in chromosomes (Moore and Endow 1996). binucleate cells (Fig. 1D). Such binucleate cells are never In this paper we describe the phenotype of lethal mu- seen in wild type (Fig. 1C). tations in the essential gene pavarotti (pav),which en- In addition to the cytokinesis defects that can be ob- codes a fly homolog of the mammalian mitotic kinesin- served in essentially all cells at cycle 16, we also ob- like protein-1 (MKLP-1). MKLP-1 has been suggested to served other mitotic defects at a very low frequency in play a role in anaphase B spindle pole elongation based pavB200 and pavM187 embryos but not in the P-element on in vitro motility assays (Nislow et al. 1992). However, alleles. The affected cells show multipolar spindles and we show that in pav mutants anaphase B occurs nor- multiple centrosomes that are always associated with mally but cytokinesis is defective, due to a disruption of what appear to be tetraploid masses of chromatin (data the central spindle structure and subsequent failure to not shown). This would suggest that in the pavM187 and assemble a contractile ring. In mitosis, the KLP encoded pavB2004 alleles, binucleate cells are formed that can un- by pav (PAV–KLP) first associates with the centrosomes dergo another round of mitosis with double the usual and then becomes localized at the spindle equator at late number of centrosomes. This could occur if the maternal anaphase. We also show that PAV–KLP associates with supply of pav gene product were to become exhausted in Polo kinase and is required for its localization to the some cells during cycle 15. Such cells would fail to un- centrosome and central spindle. The Polo-like kinases dertake cytokinesis but then proceed to attempt a six- (plks) are known to be required for centrosome function: teenth division, the last cell cycle for the majority of Mutations in the Drosophila gene polo and its fission epidermal cells. yeast homolog plo1 lead to spindle defects including the formation of monopolar structures (Sunkel and Glover pav cells show defects in the central spindle 1988; Lamazares et al. 1991; Ohkura et al. 1995). Injec- at telophase, and septin and actin rings fail to form tion of antibodies against human plk also result in the formation of monopolar structures in HeLa cells (Lane To address how pav mutations might affect cytokinesis, and Nigg 1996).
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