Polo Kinase and Progression Through M Phase in Drosophila: a Perspective from the Spindle Poles

Polo kinase and progression through M phase in Drosophila: a perspective from the spindle poles

David M Glover*,1

1Cancer Research UK Cell Cycle Genetics Research Group, Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

Genes for the mitotic kinases Polo and Aurora A were ing to no more than 20 min to allow the onset of zygotic first identified in Drosophila through screens of maternal transcription from cycle 10 onwards. Individual somatic effect lethal mutations for defects in spindle pole cells are not formed until the 14th cycle at which point a behaviour. These enzymes have been shown to be highly G2 phase is introduced, the duration of which is conserved and required for multiple functions in mitosis. regulated by the accumulation and activation of String, Polo is stabilized at the centrosome by association with the fly cdc25 tyrosine phosphatase that activates cyclin- Hsp90. It is required for centrosome maturation on M- dependent kinase 1 (Cdk1). In order to achieve the phase entry in order to recruit the c-tubulin ring complex prodigious increase in nuclei from the single zygotic and activate the abnormal spindle protein, Asp. These complement to roughly 10 000 nuclei during syncytial events facilitate the nucleation of minus ends of micro- development, the egg is loaded with a dowry of tubules at the centrosome. The localization of Polo at the maternally provided cell cycle regulatory molecules. kinetochore and the mid-zone of the central spindle Such proteins are synthesized in the nurse cells of the together with the phenotypes of polo mutants point to female germ line during oogenesis. Thus, a class of functions at the metaphase to anaphase transition and in mutations was expected that when homozygous in the cytokinesis. The latter are mediated, at least in part, mother would cause her to produce eggs deficient in through the Pavarotti kinesin-like motor protein and its essential cell cycle regulatory proteins. In addition to conserved counterparts in other metazoans. such maternal effect lethal mutations, another class of Oncogene (2005) 24, 230–237. doi:10.1038/sj.onc.1208279 mutation was also known to result in lethality when homozygous in the zygote. In such cases, recessive lethal Keywords: Polo kinase; centrosome; spindle; microtu- mutations are carried out in each heterozygous parent. bule-organizing centre The heterozygous mother is able to produce a dowry for all her offspring, 25% of which will be homozygous for the recessive mutation. Such homozygous mutants can develop until a stage at which the essential maternally supplied cell cycle protein becomes limiting. This is Isolating cell cycle mutants in Drosophila usually either in the early cell cycles that follow cellularization of the embryo or later in larval develop- Genetic studies of mitotic progression in Drosophila ment when a burst of cell proliferation is required to have given valuable insight into the regulation of this produce the adult tissues during metamorphosis. string process in metazoan cells. In the 1980s, my own mutants, for example, arrest development during the G2 laboratory adopted two strategies to identify mitotic phase of cycle 14 (Edgar and O’Farrell, 1990). Mutants mutants: a search through existing collections of of dd4, a gene now known to encode a component of the maternal effect lethal mutants that had been generated g-tubulin ring complex (g-TuRC), show late larval with the primary objective of studying early embryonic lethality (Gatti and Baker, 1989; Barbosa et al., 2000). development, and the use of P-element transposon- The lethal phase is in part a reflection of the degree to mediated mutagenesis to isolate mutants that showed which the protein is turned over during the mitotic zygotic lethality. The rationale for such screens lay in cycles. the biology of Drosophila development and in particular the times during development at which the protein machinery required for cell division might become Attracted to the poles limiting. For its first two hours of development, the embryo is a syncytium within which nuclei undertake One of the first collections of maternal effect mutants highly simplified cell cycles of alternating S- and M- screened by my laboratory was kindly provided to us by phases at roughly 10-min intervals gradually lengthen- Christiani Nu¨ sslein-Volhard. This proved to be a treasure trove of cell cycle regulatory mutants. Some *Correspondence: DM Glover; E-mail: [email protected] of these identified genes encoded proteins strictly needed Roles of Polo kinase in Drosophila DM Glover 231 for the embryonic division cycles, such as gnu, a gene we by the tandem spindles of the second meiotic division found to be required to turn off DNA replication upon (Riparbelli et al., 2000). Moreover, the polar body nuclei the completion of female meiosis (Freeman et al., 1986; resulting from the meiotic divisions failed to arrest their Freeman and Glover, 1987), and that is now known to development and went on to undertake some mitotic encode a component of a complex of molecules required divisions. This was associated with a failure in the to regulate cyclin B levels (Lee et al., 2003; Renault et al., development of the sperm aster, a dramatic array of 2003). Others were weak hypomorphic alleles of genes microtubules (MTs) that normally facilitates migration whose function is required both in the syncytial of the female pronucleus to its male counterpart. In the embryonic divisions and for cell division at later absence of the sperm aster, the two pronuclei remained developmental stages. This category included the apart and both underwent a limited number of original alleles of both polo (Sunkel and Glover, 1988) independent cycles of haploid mitoses. Thus, a series and aurora (Glover et al., 1989; Leibowitz, 1990). In of defects, most of which are related to an inability of examining the defective syncytial embryos produced by MT-organizing centres to function correctly, led to a homozygous mutant mothers, we noticed several mu- variety of developmental problems. In addition to the tants that had defects at their spindle poles. This led us above functions of polo in the female germ line, the gene to adopt a series of names for the corresponding genes was also shown to function in the male germ line, where after the magnetic poles of the Earth or geo-magnetic it was required for correct chromosome segregation in phenomena associated with them. Whereas polo and meiosis (Sunkel and Glover, 1988). This first study and aurora subsequently proved to be highly appropriate the subsequent work of Llamazares et al. (1991) showed names, in other cases our nomenclature was no so that defects in the spindle poles were a characteristic but predictive of function; lodestar, for example (Girdham not exclusive aspect of the mutant phenotype. and Glover, 1991), encodes a protein actually not directly related to spindle pole function but required for chromatin structure and to release transcripts by Mitotic functions of Drosophila Polo and its conserved RNA polymerase II in an ATP-dependent manner! We counterparts were later able to isolate stronger hypomorphic alleles for both polo and aurora that showed zygotic lethality, The first realization that Polo kinase was highly suggesting the requirement for the two gene products in conserved came when Clay et al. (1993) serendipitously cell division throughout development (Glover et al., found a mouse homologue and when the budding yeast 1989, 1995; Llamazares et al., 1991). Indeed, the second CDC5 gene was also identified as a homologous protein allele of polo that we found originated in a collection of kinase (Kitada et al., 1993). The cdc5 mutant had been P-element mutants established by Roger Karess. This identified in a part of Hartwell’s seminal work to collection was also to liberate rough deal (Karess and identify cell division cycle mutants that defined all of the Glover, 1989) that we now know to encode a component central concepts of cell cycle control. cdc5 fell into a of the zest-white 10 checkpoint complex and abnormal group of mutants that showed a late mitotic phenotype. anaphase resolved (aar) that encodes a regulatory In such arrested cells, the bud did not separate from the subunit of protein phosphatase 1A, and that also mother cell and grew so as to form a ‘dumb-bell’-shaped participates in regulating the metaphase–anaphase pair of connected cells in which the spindle appeared to transition (Mayer-Jaekel et al., 1993). The P-element persist. This group of genes is now known as the mitotic tag allowed us to identify the polo gene and show for the exit network (MEN), a conserved cassette of genes that first time that it encoded a putative protein kinase function in late mitosis. Thus, there appeared to be a (Llamazares et al., 1991). Once the gene was cloned, paradox: on the one hand, the phenotype of polo in antibodies that were able to immunoprecipitate active Drosophila melanogaster seemed to suggest a function kinase were raised against it. Studies of carefully staged early in the mitotic cycle in spindle formation, whereas and individually selected syncytial embryos indicated that of cdc5 in Saccharomyces cerevisiae, a late anaphase that the enzyme was activated in mitosis, its activity function. This led us to ask what the phenotypes of a peaking slightly later than Cdk1 (Fenton and Glover, Polo-like kinase might be in another genetically 1993). tractable organism. This was the fission yeast Schizzo- The phenotype of the original polo1 allele gave a saccharomyces pombe that had been so valuable in number of clues to the function of the gene product. characterizing the mitotic roles of the p34cdc2 kinase that Multiple mitotic defects were evident in mutant we now know as Cdk1. Disruption of the fission yeast embryos, but characteristically the centrosomal antigen, Plk gene, plo1, led to two distinct phenotypes: the CP190 (at that time termed the Bx63 antigen), was not formation of monopolar spindles and a failure of recruited to discrete centrosomal bodies (Sunkel and septation (Ohkura et al., 1995). The former phenotype Glover, 1988; see also below). The study also showed was not dissimilar to aspects of the polo phenotype in that, although homozygous mutants of polo1 could flies and the latter was already known from other fission develop to adulthood, they still showed mitotic defects yeast mutants that define the septum-inducing network during the larval and pupal stages of development. Over (SIN). It had already become apparent at that time that a decade later, a detailed cytological examination of the the budding yeast MEN and fission yeast SIN were phenotype of polo1 in the female germ line revealed analogous regulatory cassettes. Thus, the fission yeast defects in the organization of the common pole shared study first clearly showed that a Polo-like kinase could

Oncogene Roles of Polo kinase in Drosophila DM Glover 232 function at two different mitotic stages and thereby by Polo is that they become recognizable by the pointed to the possibility of reconciling the apparently monoclonal antibody MPM2. This antibody raised disparate observations from Drosophila and budding against a mitotic phospho-epitope by Potu Rao’s lab yeast. Whether the MEN and SIN networks are truly in the early 1980s (Davis et al., 1983) recognizes scores replicated in metazoan cells is still by no means certain. of proteins in Western blots of mitotic but not However, as I will discuss below, a role for Polo in the interphase cells. It was shown by Kumagai and Dunphy late stages of mitosis was subsequently demonstrated. (1996) that the Xenopus plk1 could phosphorylate and participate in activating cdc25 and in so doing generated an MPM2 epitope. The Plks are not the only protein In the right place at the right time kinases able to generate such epitopes, but in Drosophila the level of cellular MPM2 immunoreactivity correlates The polo-like kinases have been shown to follow similar directly with the severity of polo mutant alleles, dynamic patterns of localization during progression suggesting that Polo kinase generates such epitopes on through mitosis essentially in all metazoans examined. a range of substrates (Logarinho and Sunkel, 1998). Perhaps the most dramatic illustration of the dynamics Moreover, the sites of immunostaining of mitotic cells of the protein kinase has come from studies by Claudio by MPM2 broadly correspond to the known localization Sunkel’s laboratory (Moutinho-Santos et al., 1999). patterns of plks. Thus, this antibody is likely to find These workers expressed a GFP-tagged form of Polo continued application in studying the cell biology of from its own promoter in a transgenic fly line. The Polo-mediated phosphorylation. tagged enzyme localized to centrosomes at mitotic entry and then became associated with the nuclear membrane until its breakdown. During prometaphase, Polo kinase Centrosome maturation associated with kinetochores and prior to cytokinesis with the mid-part of the central spindle, a characteristic Although much remains to be discovered, we now have structure that forms in late anaphase and that is a skeletal outline of the role of Polo kinase in essential for cytokinesis. centrosome maturation in Drosophila. The inability to This dynamic pattern of localization led to an recruit the centrosomal antigen, now known as CP190, expectation that there would be multiple substrates for seen in polo1-derived embryos appears to be a secondary the kinase at its multiple cellular sites. This prediction is consequence of a failure to recruit the g-TuRC, a being borne out, although to date still relatively few complex known to cap the minus ends of MTs on substrates have been determined. A characteristic centrosomes and that is required for their nucleation. feature of the polo-like kinases is the conserved Polo- CP190 has been shown to interact with the g-TuRC and boxes in the C-terminal domain of the protein. Studies indeed, when this complex is disrupted in mutants of from a variety of organisms have indicated that the Polo dd4, a gene encoding a 91 kDa component of the boxes are required to target the protein to specific complex, CP190 then becomes dispersed around the cell cellular sites (for example, Lee et al., 1998; Reynolds and (Barbosa et al., 2000). Strikingly, neither g-tubulin nor Ohkura, 2003). Recently, it has been shown that the CP190 are recruited to the mitotic centrosome either in Polo boxes bind to a phosphopeptide motif (Elia et al., strong hypomorphic polo mutants (Donaldson et al., 2003a, b; Barr et al., 2004). It seems likely that this 2001; Figure 1) or following depletion of the protein docking site is phosphorylated by either cdk1, MAPK or kinase by RNAi in cultured cells (Bettencourt-Dias et al., even plk itself to facilitate binding and perhaps 2004). A failure of centrosomes to recruit g-tubulin has explaining some evidence for co-operativity of Polo also been observed in HeLa cells following microinjec- with other mitotic kinases. tion of antibodies against Polo-like kinase (Lane and The search for bona fide substrates with the Droso- Nigg, 1996). phila Polo was initiated by Alvaro Tavares, who Some understanding of centrosome maturation in compared the proteins phosphorylated by exogenous, Drosophila has been gained through the use of an in vitro bacculovirus-expressed Polo kinase added to extracts of system developed to study MT nucleation (Figure 2a). cytoplasm from polo1 mutant or wild-type embryos Partially purified centrosomes prepared from extracts of (Tavares et al., 1996). Endogenous Polo phosphorylated Drosophila embryos are capable of nucleating asters of a similar series of proteins. These included b-tubulin and MTs on a microscope slide. Such preparations lose their two proteins of 85 kDa and approximately 200 kDa. nucleating ability if stripped with potassium iodide, but Subsequently, it appears that these last two proteins this can be restored upon ‘add-back’ of a high-speed are likely to be an 85 kDa MT-associated protein supernatant of cytoplasmic extract. If the cytoplasmic released from MTs following phosphorylation by Polo extract is depleted of the g-TuRC, then the cytoplasmic (Cambiazo et al., 2000) and Asp, the product of the extract is no longer able to restore MT-nucleating abnormal spindle gene (Saunders et al., 1997; Avides and activity (Moritz et al., 1998). A second MT-associated Glover, 1999; Avides et al., 2001; see below). protein (MAP) Asp, product of the abnormal spindle As I will detail below, some other Polo substrates gene, is also essential for the cytoplasmic extract to be have been identified in Drosophila. However, without able to restore MT-nucleating activity (Avides and doubt, there remain many more to be determined. One Glover, 1999). A deficiency in the ability to restore characteristic of proteins that have been phosphorylated MT nucleation is thus seen with cytoplasmic extracts

Oncogene Roles of Polo kinase in Drosophila DM Glover 233 Wild-type polo mutant

Centrosomin

γ-tubulin Figure 1 Polo kinase is required for the recruitment of g-tubulin to the centrosome. Mitotic ﬁgures from wild-type and polo9 mutant cells in which MTs are stained green and DNA, red. The upper panels are stained to reveal the core centrosomal protein, centrosomin (blue). Note that this core antigen is present at the centrosomes of both wild-type and polo mutant cells. The lower panels are stained to reveal g-tubulin (blue). Note that this antigen is present at the poles of the wild-type spindle but is not detectable at the centrosomes of the polo mutant. Immunostaining was carried out by Mary Donaldson

depleted of Asp protein either through the experimental density that is seen at the centrosome upon the entry use of antibodies or as a result of asp mutations. This into mitosis (Figure 2b). The finding in other organisms functional deficiency can be rescued by adding a purified of MT-associated protein substrates of the plks suggests preparation of Asp protein to the mutant cytoplasm. that these enzymes may have further roles in regulating Typically, asp mutants have mitotic spindles with MT behaviour during mitosis (Budde et al., 2001; Yarm, highly unfocused poles and likely show a high mitotic 2002). index. The finding that polo1 and aspE3 double mutants Many questions remain unanswered. What is the Polo showed a further synergistic increase in mitotic index substrate whose phosphorylation facilitates recruitment could be interpreted if Asp were a substrate of the Polo of the g-TuRC? What is the centrosomal docking site for kinase (Gonzalez et al., 1998). This was later shown Polo kinase itself? Is there a specific docking molecule or directly by Avides et al. (2001), who found that are its centrosomal substrates sufficient? How is Polo phosphorylation of Asp by Polo in vitro generated a kinase activated at the centrosome? Is there a functional mitotic phospho-epitope recognized by the monoclonal equivalent of the spindle pole body component Cut12 of antibody MPM2. Although Asp still localizes to the fission yeast that binds Plo1 and promotes its activity, minus ends of MTs in polo mutants, it appears that the and are kinases of the NIMA family required for this Asp protein has to be phosphorylated by Polo for it to process (Grallert and Hagan, 2002; MacIver et al., be active. Cytoplasmic extracts from polo mutants 2003)? The ability to apply a concerted biochemical and would not rescue the nucleating activity of salt-stripped genetic approach in Drosophila may well enable some of centrosome preparations, but this could be restored by these questions to be approached. We are beginning to adding back phosphorylated Asp protein, and further glimpse some aspects of the regulation of Polo kinase enhanced by active Polo kinase (Avides et al., 2001). activity at the centrosome. It appears in both Drosophila Therefore, it seems that, in Drosophila, Polo kinase plays and human cells that the Plks require the chaperone at least two roles in the mitotic maturation of protein Hsp90 to maintain their stability (de Carcer centrosomes: a role both in recruiting the g-TuRCs et al., 2001). In Drosophila, this interaction is par- and in activating Asp protein that is recruited indepen- ticularly important at the centrosome, where the two dently of Polo activity. Together, these two minus end proteins co-localize. In either Hsp90 mutants or in MT-associated proteins then enable the increase in MT cells treated with the Hsp90 inhibitor, geldanamycin,

Oncogene Roles of Polo kinase in Drosophila DM Glover 234 a geldanamycin geldanamycin treated + control treated Polo kinase

hsp90 Polo

hsp90 Polo P P hsp90 Polo

Figure 2 Studies of MT nucleation by Drosophila centrosomes in vitro have led to a model for Polo function in the mitotic maturation of the centrosome. (a) Puriﬁed preparations of centrosomes lose their ability to nucleate asters of MTs following treatment with KI. This can be restored by incubation in extracts of untreated cytoplasm (control), whereupon centrosomes (green–yellow in overlap) then nucleate rhodamine-labelled tubulin (red). Treatment with geldanamycin destroys this ability of cytoplasmic extracts to rescue MT nucleation (centre panel). Addition of exogenous active Polo kinase overcomes the effect of geldanaycin (right-hand panel). Similar experimentation has shown that the abnormal spindle protein is required and that to be active is must be phosphorylated by Polo (see text for detail). These are taken from experiments of Carmo Avides. (b) A model for the mitotic maturation of the Drosophila centrosome. The dark grey cylinders represent the centrioles and the large light grey sphere, the pericentriolar material. Polo and associated hsp90 are represented in green, Asp in red and the g-tubulin ring complex in yellow. Active Polo kinase is required to recruit the g-tubulin ring complex and to activate Asp. These molecules then co-operate to nucleate MTs (red)

centrosome segregation and maturation are affected (de (Descombes and Nigg, 1998), budding yeast (Charles Carcer et al., 2001). The ability of the cytoplasmic et al., 1998; Shirayama et al., 1998) and mammalian cells extracts to restore the nucleating ability of salt-stripped (Kotani et al., 1998, 1999). The phosphorylation centrosomes is also lost following geldanamycin treat- patterns of APC/C components have been carefully ment, and this can be restored by addition of active Polo studied by Golan et al. (2002), who have shown that kinase. This indicates the potential importance of such Plk1 and cdk1-cyclin B have additive effects in chaperone proteins for processes in which complex phosphorylating and activating the APC/C, the former molecular structures need to be rapidly assembled and preferentially phosphorylating Cdc16 and Cdc23 and disassembled during the mitotic cycle. Thus, it seems the latter, Cdc27. It was also recently shown that the likely that other chaperone protein complexes will also ﬁssion yeast Polo-like kinase, Plo1, physically interacts be required at the centrosome. with the Cut23 (Apc8) component of the APC/C (May et al., 2002). These observations have prompted a study of genetic interactions between alleles of polo and Polo and the metaphase–anaphase transition makos, a gene encoding the Drosophila Cdc27 (Deak et al., 2003). However, this did not cast any light on the A number of indirect observations have implicated the potential function of Polo in activating APC/C, but Plks as having a role in activating the anaphase- instead led to the unexpected ﬁnding that the weakly promoting complex or cyclosome (APC/C) in Xenopus hypomorphic phenotype of polo1 in larval neuroblasts

Oncogene Roles of Polo kinase in Drosophila DM Glover 235 was enhanced by mks with respect to its effect upon phenotype, to have a role in organizing the central centrosome structure and function. The polo1 mutant spindle late in mitosis in order for the correct execution shows some limited recruitment of the g-TuRC to the of cytokinesis. The failure of cytokinesis in embryos centrosome in mitosis. In an mks background, however, results in the formation of large cells, hence the name the centrosomal phenotype of polo1 resembles the assigned to the locus, pavarotti. We now know that this stronger polo hypomorphs with no detectable g-TuRC, family of motor proteins plays a conserved role in suggesting that the APC/C is required either directly or organizing the central spindle in anticipation of cytokin- indirectly to fully activate Polo kinase function. More- esis and that one of the functions of the motor is to over, the mks1 polo1 double mutants also showed absence deliver an essential activating protein for a small of MPM2 reactivity at the centrosome consistent with GTPase (MgcRacGAP in mammalian cells; Rac- reduced Polo kinase activity. This has led to the GAP50C in Drosophila) to the equator of the cell. Just speculation that an early mitotic function of the APC/ as in mammalian cells, Polo kinase and Pav-KLP co- C may be required for the full activation of Polo kinase immunoprecipitate, and furthermore they co-localize in and the recruitment of the g-TuRC to the centrosome. the central part of the spindle. One possibility is that the APC/C is needed to degrade A functional role for Polo in cytokinesis in metazoan cyclin A or some other critical regulatory molecule that cells was again first revealed through the phenotype remains present in mks1polo1 cells. of polo1 mutants in spermatogenesis (Carmena et al., An indication that Polo may facilitate aspects of 1998; Herrmann et al., 1998). This cell type has proved sister chromatid separation at the metaphase–anaphase to be extremely valuable in assessing whether proteins transition comes from a report that downregulation of that function early in mitosis or meiosis have later Polo kinase function will enhance the effects of roles in cytokinesis. This is because primary spermato- stabilized Securin. It is generally thought that Securin cytes of Drosophila are unusual in having a very weak is destroyed by the APC/C to release separase and spindle integrity checkpoint. Thus, mitotic defects that promote sister separation. In Drosophila, however, a would cause a significant metaphase arrest in somatic Securin stabilized by a D-box mutation alone is cells only delay the progress of male meiosis by a matter insufficient to prevent separase activity, but the effects of some minutes. Consequently, polo mutant spermato- of this mutation can be enhanced either by reduced Polo cytes are able to undertake the metaphase–anaphase kinase or by stabilized cyclin A (Leismann and Lehner, transition and then begin to prepare for cytokinesis. 2003). The mechanisms of this interaction are presently As polo1 is a weak hypomorph, some cells within the a mystery. It seems likely to involve some interplay cysts of primary spermatocytes are able to undertake between Polo kinase and the Cyclin A : cdk1, as this cytokinesis, but in many cells the central spindle cyclin has only been found in complexes with cdk1 in fails to form (Figure 3) and Pav-KLP is not recruited Drosophila. to a ring-like structure at the equator as would Although the above genetic interactions give some normally occur. tantalizing glimpses of Polo’s participation in regulatory Together, these pointers suggested a role for the Plks networks, they do not yet permit any mechanistic in directing the function of this Pavarotti-KLP family of insight. Thus, much remains to be done to clarify the motor proteins. The molecular interactions between roles of Polo kinase at the metaphase–anaphase transi- Plk1 and MKLP1 have now been characterized in some tion both in Drosophila and other organisms. detail by Liu et al. (2004). However, the exact function of the Plks in regulating the Pav-KLP class of motors is still uncertain and in mammalian cells is further Roles for Polo in cytokinesis complicated by the finding of another related motor protein, MKLP2, also phosphorylated by Plk1 (Neef As I have discussed above, studies in fission yeast et al., 2003). pointed to the major role played by Plo1 kinase in The precise roles for Polo in the emerging network directing septum formation. Indeed, when overex- that regulates cytokinesis are not yet clear. It is likely pressed, it could drive cells into the septation pathway that numerous substrates have to be phosphorylated by irrespective of whether cells were arrested in G1, G2 or Polo to ensure the correct execution of this process. undergoing repeated rounds of S phase (Ohkura et al., Moreover, it appears important to inactivate Plk1 in a 1995). Indeed, it has been suggested that Plo1 acts timely manner to complete cytokinesis correctly (Lindon upstream of the SIN to induce septation (Tanaka et al., and Pines, 2004). It is curious as to how many proteins 2001). This raised the question of whether Plks could play roles in organizing the centrosome and the early have similar functions late in mitosis in metazoan cells? mitotic spindle have roles in cytokinesis. Studies in A hint of this possibility first came from an observation Drosophila have revealed that not only Polo but also of Lee et al. (1995) that the kinesin-like motor protein Asp (Wakefield et al., 2001; Riparbelli et al., 2002) and MKLP1 was associated with immunoprecipitates of the g-TuRC (Sampaio et al., 2001; Barbosa et al., 2003) murine Plk1. At that time, it was believed that the main are required for the correct organization of the central function of this motor was in anaphase B spindle spindle and thus cytokinesis. Perhaps Polo kinase elongation (Nislow et al., 1992). However, when the ensures the phosphorylation of a set of familiar counterpart of MKLP1 was identified in Drosophila substrates both at the onset and at the end of the (Adams et al., 1998), it was shown, through its mutant mitotic process.

Oncogene Roles of Polo kinase in Drosophila DM Glover 236 microtubules DNA

metaphase

anaphase

late anaphase - telophase

Figure 3 Polo is required to form the central spindle for cytokinesis. The main panels show meiotic ﬁgures from cysts of spermatocytes from polo1 males. The insets represent single meiotic ﬁgures from equivalent wild-type cysts. The left-hand column shows staining of MTs and the right-hand column, DNA. The spindle MTs of wild-type and polo mutant cells are virtually indistinguishable at metaphase (upper panels). At anaphase the distinctive central spindle structure forms in anticipation of cytokinesis in wild-type cells (arrowhead, left-hand central inset). This structure is lacking in the polo mutant cells at this stage (arrow, main left- hand central panel). At late anaphase/telophase, the central spindle has begun to condense in the early stages of mid-body formation in the wild-type cell (arrowhead, left-hand lower inset). No such structure is present in the polo mutant cells at a similar stage (arrow, main left-hand lower panel). Immunostaining carried out by Maria Giovanna Riparbelli and Giuliano Callaini

Concluding remarks to identify genes that participate in regulatory networks. Knowledge gained in this way should be readily Further understanding of the regulation, both in applicable to the study of the polo-like kinases in time and space, of the interplay between the plks human cells. This is of particular interest in the and their docking proteins and/or substrates will long term as these enzymes, their regulators and be important to fully appreciate the intricacies of substrates are attractive drug targets in the treatment mitotic control. It seems likely that Drosophila will of human cancer. continue to play a role in unravelling these networks. It is an ideal organism in which to apply proteomics to study the interactions of Polo with other proteins. Acknowledgements I would like to thank Cancer Research UK, MRC, BBSRC Its fully sequenced genome and the ease of applying and the EU for research funding, and all past and present RNAi in cultured cells and in the whole organism members of my research group and collaborators for their provides the opportunity to test the functions of contributions to this work. I am particularly grateful to proteins identiﬁed in this way. Moreover, it still offers Monica Bettencourt-Dias for her comments on the manu- the opportunity of applying classical genetic approaches script.

Oncogene Roles of Polo kinase in Drosophila DM Glover 237 References

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