Fluorescence Microscopy

Immunostaining of testes from pharate adults or young (0–1-d-old) flies was performed either by the methanol/acetone fixation method as described by Glover and Gonzalez (1993) or by the ethanol/formaldehyde fixation method as described by Hime et al. (1996). After counterstaining of DNA with either Hoechst 33258, propidium iodide (1 mg/ml) or TOTO-3 iodide (1:200), the samples were rinsed in PBS and mounted in either 90% glycerol containing 2.5% N-propyl gallate (Giloh and Sedat, 1982) or in Vectashield mounting medium H-1000. Fluorescence observations were made on a Leitz Aristoplan microscope equipped with FITC, TRITC, and UV filters, or on a MRC 1024 Confocal Imaging Head (Bio-Rad Laboratories, Hercules, CA) on a Nikon Optiphot microscope. Pho-tomicrographs were taken with Kodak Tri-X 400 Pro and developed in Kodak HC110 developer for 7 min at 20°C. Confocal images were processed using Photoshop (Adobe Systems, Mountain View, CA) and printed using an Epson Stylus Photo color printer.

Ring Canal Formation Is Defective in Polo¹ Spermatocytes

In both male and female gametogenesis in Drosophila a founder cell, produced by the asymmetric division of a germ line stem cell, first undertakes four rounds of mitosis to produce a cell of either 16 spermatocytes or 15 nurse cells and a single oocyte (for review see Fuller, 1993; Spradling, 1993). Cytokinesis is incomplete in these divisions and the cells remain connected by ring canals. Additional ring canals are formed in the male at each meiotic division such that the resulting cyst contains 64 spermatids connected by 63 cytoplasmic bridges. A phospho-tyrosine epitope has been described to accumulate early in ring canal formation in both males and females (Robinson et al., 1994; Hime et al., 1996). The ring canals in the male have been shown to contain three septins, polypeptides first identified as being required to form filaments in the Saccharomyces cerevisiae bud neck, one of which is encoded by the gene peanut, a gene essential for cytokinesis (Neufeld and Rubin, 1994). These proteins are indeed also present during cytokinesis in the contractile ring, from which the ring canals are derived (Hime et al., 1996). The normal developmental progression of spermatogenesis is diagrammed in Fig. ​Fig.9.9.

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Figure 9

Transmission electron micrographs of polo¹ (A, C, E) and wild-type (B, D, F) cysts of spermatids at sequential stages of elongation. (A) Cross section of an early elongating polo¹ spermatid showing three axonemes (large arrowheads) associated with only one mitochondrial derivative (small arrowhead). The structure of one axoneme appears abnormal (large arrowhead). A fourth axoneme is outside the frame of the picture. (B) In contrast, in wild-type early elongating spermatids one axoneme (large arrowhead) is normally associated with a pair of mitochondrial derivatives (small arrowheads). (C) Cross section of elongating cysts at a later stage shows that spermatids in polo¹ males may contain four axonemes (cell indicated by arrowhead). (D) Wild-type spermatids at a similar stage contain single axonemes. (E) Section of mature sperm from polo¹ males showing irregular numbers of axonemes (two in the cell indicated by the large arrowhead, three in the cell indicated by the small arrowhead). (F) Mature wild-type sperm contain single axonemes. Although the occasional abnormal axoneme is seen in the polo¹ mutant (A), the majority of axonemes from polo¹ and wild-type males have an identical structure throughout the sequential stages of elongation: they comprise one pair of central tubules surrounded by nine doublets, and nine accessory tubules in later stages.

The appearance of the cell displayed in Fig. ​Fig.11 would suggest that it has undergone the pre-meiotic cell cycles in which the γ-tubulin–containing MTOCs have increased geometrically in number in the absence of cytokinesis. It has the characteristics of a multinuclear cell that has entered meiosis. Examination of cysts of primary spermatocytes revealed them to vary in appearance from those containing single enlarged cells to others of wild-type appearance. Many cysts were intermediate between these extremes and contained both normal and polyploid cells. One such cyst is shown in comparison with a wild-type cyst in Fig. ​Fig.2.2. Although the gonial mitoses are difficult to study because these cells are very small, the process of cytokinesis leaves its “footprint” on these cells in the form of ring canals. Cytokinesis is normally incomplete in the gonial cysts and the 16 discrete primary spermatocytes are connected by 15 cytoplasmic bridges. The ring canals are structures formed by stabilization of contractile rings following cytokinesis through which these cytoplasmic bridges pass. The kinesin-like protein encoded by pavarotti (Pav-KLP) is essential for cytokinesis, and associates with the central region of the spindle at telophase. In the embryonic mitoses it is discarded from the cell as part of the mid-body upon the completion of cell division (Adams et al., 1998). In male meiosis it persists to become incorporated into the 15 ring canals of the primary spermatocyte cyst (Fig. ​(Fig.22 A, red stain). The polo¹ mutant cyst (Fig. ​(Fig.22 B) has just 12 ring canals connecting 13 cells, one of which (arrow) is considerably larger than the others. The reduction in the total number of ring canals in this particular cyst indicates that the large cell has failed to undertake the previous two rounds of cytokinesis.

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Figure 2

Cytokinesis defects in the pre-meiotic divisions are indicated by reduced numbers of ring canals, and enlarged cells in cysts of primary spermatocytes. Optical sections of a cyst of (A) wild-type and (B) polo¹ mutant primary spermatocytes. DNA is stained blue, microtubules in green and Pav-KLP in red. The wild-type cyst contains 16 cells inter-connected through 15 ring canals that contain Pav-KLP. The polo¹ mutant cyst contains 13 cells connected by 12 ring canals. One cell (arrow) is considerably larger than its neighbors. Bars, 20 μm.

The polo¹ Mutation Prevents Formation of the Spindle Mid-zone at Late Anaphase

To look directly for defects in centrosome behavior or cytokinesis during cell division, we studied the intracellular distribution of both γ- and β-tubulins. During the transition into the first meiotic division, γ-tubulin becomes organized as a compact body at the centrioles where it remains until completion of meiosis II (Wilson et al., 1997). The majority of polo¹ spermatocytes showed a pattern of γ-tubulin localization at the spindle poles indistinguishable from wild-type during meiosis I (Fig. ​(Fig.3).3). As previously described, the γ-tubulin condenses into orthogonal rod-like structures during prophase I (Fig. ​(Fig.33 A), which transforms into more diffuse bodies during metaphase and anaphase (Fig. ​(Fig.3,3, B and C). At metaphase, γ-tubulin also becomes localized perpendicular to the axis of the meiotic spindle, delimiting the central spindle region. In late anaphase– telophase, however, many polo¹ mutant cells show defects in the appearance of the central region of the spindle (Fig. ​(Fig.3,3, C and D). Whereas in wild-type, a distinct mid-zone structure develops in late anaphase that shows pronounced staining for β-tubulin and moderate staining for γ-tubulin (Fig. ​(Fig.33 C, inset, arrowheads), no such γ-tubulin structure is seen at this stage in bipolar spindles in the polo¹ mutant. However, γ-tubulin still localizes to the midzone in the absence of this central spindle structure (Fig. ​(Fig.33 C, arrows). Many bipolar spindles fail to constrict at telophase in polo¹ mutants (Fig. ​(Fig.33 D, arrows) to form the characteristic mid-bodies seen in wild-type meiocytes (Fig. ​(Fig.33 D, insets, arrows). The localization of γ-tubulin must be dependent upon correct mid-body formation at this stage since we observe it to have an abnormal distribution, remaining in the middle region instead of “migrating” polewards to the dividing nuclei as in the wild type. Mutations in genes encoding two kinesin-like motor proteins, Klp3A and pavarotti, show similar defects in the structure of the mid-zone region of the spindle in late anaphase–telophase and also ultimately result in a failure of cytokinesis (Williams et al., 1995; Adams et al., 1998).

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Figure 3

polo¹ spermatocytes show spindle mid-zone defects at late anaphase. Progression through the first meiotic division in polo¹ is shown in the main panels, and in wild-type in the insets. Testes preparations have been stained with antibodies against β-tubulin (left-hand panels), γ-tubulin (middle panels), and Hoechst to reveal DNA (right-hand panels) (see Materials and Methods). (A) Mature spermatocytes. Note the orthogonal rod-like γ-tubulin structures at the focus of the cytoplasmic microtubules. (B) Metaphase spermatocytes. γ-Tubulin is transformed into more diffuse bodies at the poles of the spindles. Bivalents congregate in single masses. (C) Late anaphase spermatocytes. A prominent mid-zone structure strongly stained for β-tubulin (arrowhead in insets) is present in wild-type anaphase spindles, whereas no such structure is found in polo¹ spindles (arrows). γ-Tubulin is present in this region in both mutant and wild-type. (D) Telophase spermatocytes. Wild-type spermatocytes show characteristic mid-bodies with a distinct gap in β-tubulin staining between the two halves of the spindles (arrowheads in insets); in contrast the spindle microtubules of polo¹ mutant spermatocytes fail to organize similar structures during telophase (arrows). Whereas in the wild-type, the γ-tubulin in the central region of the spindle begins to redistribute towards the poles, in the mutant cell it remains in the central region.

Secondary spermatocytes that have failed to undergo cytokinesis in the first division enter the second meiotic cycle and undergo centrosome separation. Such cells typically contain four MTOCs (Fig. ​(Fig.4,4, arrows). Chromosomes frequently appear to undergo non-disjunction on such tetra-polar structures (Fig. ​(Fig.4,4, arrowheads). This is consistent with the diverse sizes of spermatid nuclei (see also Table ​TableI),I), and the non-disjunction seen by following the segregation of marked chromosomes (Sunkel and Glover, 1988).

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Figure 4

Tetrapolar cells in which cytokinesis has failed in both meiotic divisions in the polo¹ mutant. Testes have been stained to reveal microtubules (left), γ-tubulin (middle), and chromatin (right). Each cell contains four astral microtubules and four foci of γ-tubulin (arrows). Chromosomes frequently appear to undergo non-disjunction (arrowheads).

In testes from polo¹ mutants we typically found 3–4 cysts of cells undergoing meiosis, in comparison with wild-type testes where there are 5–6 meiotic cysts. We scored 1,965 meiotic figures from 20 homozygous polo¹ mutant testes, and found 69% to show normal meioses. Meiotic abnormalities were very rare at metaphase of the first division, but defects of the types illustrated in Figs. ​Figs.44–6 were found in a total of 31% of cells from anaphase I onwards.

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Figure 6

Abnormal actin ring formation during cytokinesis in polo¹ mutants. DNA is stained blue; microtubules are stained green; and actin is stained red. Actin rings at equivalent stages can be seen in the telophase figures from the wild-type cyst. In the polo¹ mutant cyst, actin forms a ring in late telophase cells with a normal spindle mid-body (small arrow), but not in cells in which the mid-body has not formed (large arrow). Ring-like structures of actin may (small arrowhead) or may not (large arrowhead) form in cells with tripolar spindles. Bars, 10 μm.

The polo¹ Mutation Affects the Relocalization of the Pav-KLP to the Mid-zone of the Spindle and Prevents Contractile Ring Formation

To correlate the spindle defects described above with abnormal cytokinesis, we studied the distribution of a number of proteins that associate with the contractile furrow. We have previously shown that the kinesin-like protein encoded by pavarotti can associate with Polo kinase. In mitotically dividing cells, both proteins can be found at spindle poles until anaphase, but become associated with the most central part of the spindle in the region of the cleavage furrow at telophase. This pattern of subcellular localization of both proteins is disrupted in pavarotti mutants (Adams et al., 1998). We therefore decided to examine the distribution of Pav-KLP in meiosis in both wild-type and polo¹ mutant males. We found it difficult to visualize Pav-KLP at the spindle poles in wild-type meiosis, although we could readily observe the protein in the cleavage furrows at the telophase of both meiotic divisions (Fig. ​(Fig.55 A, large arrow), and to the ring canals (Fig. ​(Fig.55 A, arrowhead). polo¹ mutant cysts contain ~30% of cells of mutant phenotype alongside cells of wild-type appearance. In such a mutant cyst displayed in Fig. ​Fig.55 B, Pav-KLP can be seen associated with the cleavage furrow of the wild-type appearing cell indicated by the arrow. In contrast, the protein is not found between the telophase nuclei in the cell showing mutant phenotype indicated by the large arrowhead, but instead appears to accumulate in the region of the poles (Fig. ​(Fig.5,5, small arrowheads).

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Figure 5

Mislocalization of Pav-KLP and Peanut at telophase in polo¹ mutants. In all of these panels, DNA is stained blue, and Pav-KLP red. The third immunostain (green) in C and D reveals microtubules, and in E and F, the septin Peanut. (A) A wild-type cyst at late anaphase/early telophase I in which Pav-KLP can be seen to be present in the ring canals (arrowhead) and the contractile rings (arrow). (B) In an equivalent polo¹ mutant cyst, Pav-KLP (red) is localized in the contractile ring (arrow) and remains in the ring canal in normal cells. In spermatocytes showing the mutant phenotype, Pav-KLP accumulates in the polar regions (small arrowhead) and does not redistribute to the midbody. (C) A telophase I cyst showing the distribution of Pav-KLP (red) relative to microtubules (green). Large arrow, a cleavage ring; and small arrowhead, a ring canal. In addition, Pav-KLP is associated with some (putatively immature) axonemal microtubules (small arrow), but not with others (large arrowhead). (D) Telophase figures in polo¹ in which Pav-KLP is associated with early (arrow) and late (small arrowhead) cleavage rings in cells undergoing apparently normal late meiotic events. Cleavage rings fail to form correctly in the multipolar figure (large arrow). (E) Immunolocalization of Pav-KLP (red) and Peanut (green) in a wild-type late anaphase/early telophase I cyst. The large arrow indicates an early cleavage ring in which the staining for Peanut is more pronounced than for Pav-KLP. The two proteins colocalize in the later cleavage ring (small arrow) with Pav-KLP being more concentrated towards the inner side. Ring canals appear to contain Pav-KLP and not Peanut (small arrow). (F) polo¹ mutant cyst with Pav-KLP and Peanut colocalizing in both early (large arrow) and late cleavage rings. Punctate staining of Pav-KLP is also seen in the regions of the spindle poles (small arrows).

The other striking feature of cysts from polo¹ mutant testes is the loss of synchrony of the meiotic divisions in comparison with wild-type meiosis. All of the cells in the wild-type cyst in Fig. ​Fig.55 C, for example, have progressed to approximately the same stage of telophase. Pav-KLP is present in the rings of the cleavage furrows (Fig. ​(Fig.55 C, large arrow) and in the ring canals (Fig. ​(Fig.55 C, small arrowhead). In addition, Pav-KLP is found associated with some (putative immature) axonemes (Fig. ​(Fig.55 C, small arrow), but not with others (Fig. ​(Fig.55 C, large arrowhead). We speculate that this points to another function of this motor protein in transporting flagellar components to its growing distal tip, or to facilitate the elongation of organelles such as mitochondria in the sperm tail. On the other hand, the polo¹ mutant cyst (Fig. ​(Fig.55 D) contains cells at a variety of late meiotic stages. Many cells have a wild-type appearance in which case Pav-KLP may be seen in large cleavage rings at late anaphase-early telophase (Fig. ​(Fig.55 D, arrow), or more compact rings at late telophase (Fig. ​(Fig.55 D, arrowhead). In other cells such rings of Pav-KLP never seem to form even though the nuclei have moved to the poles of the spindle. This is frequently the case with multipolar cells (Fig. ​(Fig.55 D, large arrowhead). The polo¹ mutation does not affect the association of Pav-KLP with the flagellar axonemes (not shown).

As septins are known to become incorporated into the contractile ring, we attempted to localize the Drosophila septin Peanut in telophase cells from polo¹ mutant cysts during meiosis (Fig. ​(Fig.55 F). In wild-type cysts, Peanut appears in the cleavage furrow rings at anaphase (Fig. ​(Fig.55 E, green stain, large arrow), slightly ahead of Pav-KLP (red stain). The two proteins colocalize in the furrow at telophase (Fig. ​(Fig.55 E, overlap shown by yellow staining, small arrow), with Pav-KLP appearing more concentrated on the inner side of the ring. The ring canals appear to contain Pav-KLP but Peanut is less easily detected (Fig. ​(Fig.55 E, small arrowhead). It is our impression that in mutant cysts, there is a tendency for both proteins to accumulate in the larger cleavage furrow rings (Fig. ​(Fig.55 F, large arrow). This particular mutant cyst, which has a reduced number of nuclei, has paired dots of Pav-KLP staining that has accumulated at the spindle pole regions (Fig. ​(Fig.55 F, small arrows) even though the cleavage furrows are forming.

The loss of synchrony of the meiotic divisions within a cyst is also apparent in the late meiotic cyst shown in Fig. ​Fig.6,6, that illustrates the formation of actin rings at the cleavage furrow. In the wild-type cyst shown in this Fig. ​Fig.6,6, cells are uniformly at telophase, and show actin rings of comparable sizes, in contrast to the cells of the mutant cyst that are at a variety of meiotic stages. Late telophase cells can be seen in which the spindle mid-body is well formed and is associated with a compact actin ring (Fig. ​(Fig.6,6, small arrow). Other bipolar cells in which the chromatin has migrated fully to the poles have no mid-zone structure to the spindle microtubules, and are lacking any actin ring (Fig. ​(Fig.6,6, large arrow). This directly links the defect in the central spindle to a failure to establish a contractile ring. A number of cells with tripolar spindles are also seen in this particular cyst. In some of these cells there is no indication of any actin ring formation (Fig. ​(Fig.6,6, large arrowhead), whereas in others a large somewhat misshapen actin ring has formed (small arrowhead). Such cells could arise as a result of cytokinesis failure in the first meiotic division, and failure of one of the centrosomes to separate in the second division.

Cyclin B Is Degraded in polo¹ Mutant Meioses

A number of recent papers have reported that polo-like kinases are required for mitotic exit and cyclin destruction, functions that are mediated through activation of the anaphase-promoting complex (APC) (Charles et al., 1998; Descombes and Nigg, 1998; Kotani et al., 1998; Shirayama et al., 1998). Chromatid separation at anaphase is also thought to be under control of the APC. Thus our observations that anaphase could occur in polo¹ mutant cells did not seem to accord with these results. We therefore examined whether cyclin B degradation could take place in polo¹ mutant testes. In wild-type meiotic cysts, a gradient of cyclin B degradation can be seen as cells progress through the late stages of meiotic division (Fig. ​(Fig.77 B). Cyclin B degradation is also seen late in the meiotic cycle in polo¹ cells that display either multipolar spindles (Fig. ​(Fig.77 A, tetrapolar spindle indicated by the large arrow), or bipolar spindles in which the central spindle is abnormal (Fig. ​(Fig.77 A, small arrow). We therefore conclude either that polo kinase is not required to activate the APC to mediate cyclin B degradation in these cells even though cytokinesis has been affected, or that these processes (together with chromosome disjunction) are differentially sensitive to the polo¹ mutation.

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Figure 7

Cyclin B is degraded upon meiotic exit in polo¹ mutants. Metaphase–telophase mutant figures in the polo¹ mutant. This cyst has several multipolar cells at stages both before and after cyclin B degradation. The large and small arrows indicate cells with a tetrapolar and abnormal bipolar spindles respectively in which cyclin B has undergone degradation. Wild-type cyst with cells showing progressive cyclin B degradation at various stages of the metaphase-anaphase transition.

The Earliest Defects Before Cytokinesis Are in the Structure of the Central Spindle

It seems likely that defects in the organization of the central region of the spindle at anaphase anticipate the failure of the contractile actin ring to form and the failure of cytokinesis in polo¹ meiosis. The central spindle shows an abnormal distribution of mid-zone microtubules from late anaphase–telophase. This region normally shows some staining of γ-tubulin that recedes polewards as the mid-body structure matures. In the polo¹ mutant a mid-body does not form and some γ-tubulin remains in the mid-zone region. γ-Tubulin at the central region of the spindle has previously been suggested to be required to form the mid-body, and for the process of cytokinesis (Julian et al., 1993; Shu et al., 1995). Thus a common function of γ-tubulin in both the centrosome and spindle mid-zone might be to act as a microtubule organizing center, a property that might require to be activated by the Polo-like kinases.

The central spindle defects are accompanied by a failure to correctly localize the kinesin-like protein encoded by pavarotti, a gene shown to be required for cytokinesis in developing embryos (Adams et al., 1998). Moreover, rings of the septin Peanut and of actin fail to form in the mid-region of the cell. These defects are similar to those seen in other cytokinesis mutants. The formation of a defective spindle mid-zone at late anaphase and failure to form a mid-body is seen with mutations in Klp3A and pavarotti (Williams et al., 1995; Adams et al., 1998). These genes each encode kinesin-like proteins, KLP3A and Pav-KLP, respectively, that have been postulated to have a direct role in organizing the central spindle in preparation for cytokinesis.

An additional role has been suggested for Pav-KLP by its association with Polo kinase, namely that it might also serve as a means of localizing this enzyme first to the centrosomes, and subsequently at the central spindle to choreograph formation of the bipolar spindle and then cytokinesis (Adams et al., 1998). The two proteins also appear to be mutually dependent for their correct localization. Polo kinase fails to localize to spindle poles or the spindle mid-zone in pavarotti mutants (Adams et al., 1998) and we now show that Pav-KLP accumulates at the spindle poles in meiosis in polo¹ males and often fails to become associated with the spindle mid-zone. This suggests a model whereby the motor properties of Pav-KLP might be changed as a consequence of phosphorylation by Polo kinase, a hypothesis that is currently under investigation.

Polo kinase could also phosphorylate other cytoskeletal components, such as septins or actin-associated proteins to directly modify the structure of the contractile apparatus. Such modifications may in turn lead to changes in the structure of the central spindle. It appears that cooperative interactions occur between the central spindle and the acto–myosin ring to effect cytokinesis. The acto–myosin contractile ring fails to form following mutation of the chickadee gene, which encodes the actin binding protein profilin (Giansanti et al., 1998), and consequently the morphology of the central spindle is affected during late meiosis. Thus it would seen that there is a mutual dependence for the correct formation of the cleavage ring and the central spindle region.

Why Should It Be Easier to Observe Cytokinesis Defects in polo Mutants during Spermatogenesis?

One possible answer to this question could be that there are different demands upon polo function in mitosis and meiosis. In such a case one might imagine that Polo kinase is less important for correct centrosome behavior in the meiotic cycle, so emphasizing the requirement for cytokinesis. This may be accentuated by the hypomorphic nature of the polo¹ mutation, the residual function being sufficient to form a functional meiotic spindle, but insufficient for its subsequent role. Examination of spermatogenesis in an allelic series of polo mutants (White-Cooper et al., 1996) reveals a range of meiotic defects consistent with the strength of the mutant allele. The large primary spermatocytes we previously reported in the stronger alleles (polo⁴, polo⁶, polo⁷) are very similar to those we now describe in detail for polo¹, but occur at a higher frequency. However, the asynchrony in the passage through spermatogenesis within a cyst is much more pronounced in these strong alleles. Few cells develop to spermatids suggesting a block to pre-meiotic or meiotic divisions. The spermatids that are formed show multiple nuclei and enlarged Nebenkern indicative of cytokinesis defects.

It is also possible that the cytokinesis defects seen in male meiosis in polo¹ could reflect the absence of the checkpoint controls that monitor the structure of the spindle during the male meiotic divisions. The continuation of the meiotic cycle in the absence of a functional spindle is reported for the β2tⁿ mutant that lacks a functional male specific tubulin isoform. The gene for this isoform is expressed at high levels in the growing stage of primary spermatocytes, and the protein is the major γ-tubulin present in the microtubules of the meiotic spindle, and subsequently in the flagellar axoneme. Although these mutants lack a meiotic spindle, their chromosomes are described to undergo normal meiotic condensation–decondensation cycles, and although homologues separate, they fail to segregate resulting ultimately in the formation of tetraploid spermatids that begin to differentiate (Fuller, 1993). Thus in the absence of a spindle integrity checkpoint in male meiosis, if meiotic spindle defects develop in polo mutants, chromosome segregation may be able to continue. However, meiosis may fail subsequently at cytokinesis. In contrast, if spindle defects occur in the mitotic divisions of larval neuroblasts, then the cycle is arrested at metaphase by checkpoint controls and so cytokinesis is never attempted.

A Unifying Role for polo-like Kinases in the Early Events of Cytokinesis

The central importance of our present findings is that they clearly establish a role for Polo kinase in the early events of cytokinesis in animal cells, as has previously been established for its fission yeast counterpart plo1 (Ohkura et al., 1995). Thus there appears to be a unifying role for the Polo-like kinases in both establishing the bipolar spindle and in regulating late mitotic events from the yeasts to the metazoans. It is of interest that the Polo-like kinases of fission yeast and animal cells show some common aspects of localization. In flies and mammals, Polo-like kinases are found associated with centrosomes and centromeres early in mitosis and then subsequently at the spindle mid-zone and cleavage furrow before cytokinesis (Goldsteyn, 1995; Adams et al., 1998). The fission yeast Plo1p like its animal cell counterparts localizes to the spindle poles at the onset of mitosis, but structures analogous to the central spindle and cleavage furrow are not found in fission yeast cytokinesis (Mulvihill, D., H. Ohkura, I. Hagan, and D.M. Glover, manuscript in preparation). Indeed, other proteins required for septation such as the protein kinase encoded by cdc7, that lies at the head of the hierarchy regulating septum formation, have also been found to accumulate at the spindle poles (Sohrmann et al., 1998). This establishes the spindle pole bodies as a potential source for signaling molecules that regulate the onset of cytokinesis in the fission yeast. Animal cells may show a variation in this theme by which the Polo-like kinases become redistributed from the spindle poles and centromeres to the central region of the spindle. This could be an evolutionary adaptation that has paralleled the increase in size and complexity of the metazoan mitotic spindle in comparison with its yeast counterpart. Thus the association of Polo kinase with a kinesin-like protein, recently shown to be essential for cytokinesis (Lee et al., 1995; Adams et al., 1998), may be required both to regulate the change in structure of the central spindle immediately before cytokinesis and to correctly localize Polo kinase to its site of action late in cell division. Significantly mutations in Polo and in the associated Pav-KLP both lead to defects in the structure of the central spindle in late anaphase and telophase, and the two proteins appear to be mutually dependent upon each other for their correct subcellular localization.

Our findings have implications towards the existing models for the origins of the signals for cytokinesis in animal cells. Rappaport (1961) has provided compelling evidence that in Echinoderm embryos asters can dictate the position of the cleavage furrow. On the other hand, Wheatley and Wang (1996) found that when tripolar cells were induced to undergo mitosis, the position of cytokinesis was determined by the position of mid-zone microtubules. Moreover, Cao and Wang (1996) found that cytokinesis could be blocked by placing a barrier between the central spindle and the cell cortex suggesting a signal for cytokinesis emanates from the central spindle. These two general hypotheses could be reconciled if the signaling molecule(s), for which Polo-like kinase could be one prime candidate, were initially localized at the poles, and subsequently at the central spindle anticipating the position of the cleavage furrow, and if the extent of this relocalization were to vary between different cell types.

We thank two unknown referees for their helpful criticism of the paper.