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A role for a novel centrosome cycle in asymmetric cell division; Asymmetric localization of Polo kinase

Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. It has been shown that their mitotic spindle orientation is established before spindle formation. This study investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a 'dominant centrosome.' This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. A two-step mechanism is proposed to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment (Rusan, 2007).

By prophase, NBs contain two MTOCs that are almost fully separated and aligned along the NB/GMC axis, but analysis of fixed NBs revealed a single MTOC positioned opposite the GMCs before mitotic entry. Thus MTOC behavior was examined throughout the cell cycle as an initial approach to test the hypothesis that fixing the position of one MTOC through successive divisions helps ensure persistent spindle orientation. Live NBs expressing GFP-G147, an MT-associated protein were examined, revealing a striking temporal difference in MTOC activity. During interphase, a single detectable MTOC persists opposite the previous division site; this is referred to as the dominant MTOC. As NBs approach mitosis, this MTOC increases activity (matures; empirically judged by size and MT fluorescence intensity), forming an MT basket around the nucleus. This is referred to as preprophase; this stage is also seen in fixed samples stained for tubulin. Soon after, sometime before the dominant MTOC fully matures, something striking happens: a second MTOC appears distant from the first. This is the second MTOC and this stage is referred to as prophase onset. The second MTOC increases activity, maturing ~10 min before nuclear envelope breakdown (NEB). Using 4D imaging, the possibility was excluded that the second MTOC was present earlier in another focal plane. To further assess this, forming spindles were imaged end on. It is clear that the second MTOC did not emerge from the dominant MTOC or travel around the nucleus. Instead, the second MTOC appeared roughly opposite the dominant MTOC. MTOC separation began immediately, and by NEB, they were 146 ± 20° apart. Thus, NBs form two distinct MTOCs: an MTOC persisting from the previous division and another only activated at mitotic entry (Rusan, 2007).

This distant activation of the second MTOC raised questions about the centrosome cycle. One possibility is that NBs have two MT nucleating centrosomes, but only one can retain MTs and act as an MTOC during interphase, whereas the second acquires MT retention ability during mitotic entry, explaining the second MTOC's sudden appearance. There is precedent for this: mouse L929 cells have two γtub-bearing centrosomes that can nucleate MTs, but only one contains Ninein and can retain MTs to form an MTOC (Rusan, 2007).

To test this hypothesis in NBs, EB1-GFP was used. This binds growing MT plus ends and reliably identifies MT nucleation sites. Only one nucleation site was present in interphase and preprophase, and a new nucleation site appeared distant from the first, consistent with spatially and temporally distinct second MTOC activation. Thus, NBs regulate MT nucleation and not just MT retention (Rusan, 2007).

To examine how the new nucleation center forms, centrosomes were imaged using a PCM protein, GFP-Cnn. NBs contain a single detectable centrosome during interphase. When NBs reenter mitosis, a second Cnn-positive centrosome appears distant from the first, mimicking activation of the second MTOC. To verify that these occur simultaneously, NBs expressing mCherry-Tubulin (chTub) and GFP-Cnn were imaged. This revealed perfect temporal and spatial correlation between the appearance of the second centrosome and activation of the second MTOC. Physical separation of two centrosomes/MTOCs was never seen. This is the first example of asynchronous and physically distant centrosome maturation, suggesting that NBs use a novel centrosome cycle (Rusan, 2007).

Higher temporal/spatial resolution imaging revealed that two GFP-Cnn spots separate during mitotic exit. One GFP-Cnn spot persists as the NB interphase centrosome, forming the dominant MTOC, whereas the other spot disappears. The persistent Cnn spot (centrosome) remains relatively stationary in interphase, consistent with the hypothesis that coarse spindle alignment begins in interphase by anchoring the dominant centrosome (Rusan, 2007).

Centrosome fate was examined in the two daughters (new NB and GMC). They differ dramatically in PCM retention, in contrast to mammalian cells, where both daughters' centrosomes retain PCM. The GMC centrosome sheds all PCM. The new NB centrosome (that becomes the dominant MTOC) retains PCM throughout interphase and further accumulates PCM during the next mitosis. The complete shedding of PCM in GMCs appears to be the normal behavior of interphase centrosomes in most fly cells, whereas in syncytial early embryos, both daughters retain PCM foci through the cell cycle. In contrast to both cell types, the NB daughters exhibit differential PCM retention (Rusan, 2007).

These data suggest that NBs have a novel centrosome cycle in which the second centrosome matures distant from the dominant centrosome/MTOC. One hypothesis to explain this would be the distal positioning of a differentially regulated centriole that is blocked from recruiting PCM in interphase and thus cannot form an MTOC until 'activated' during mitosis. If this centriole is always inherited by the GMC, it might also explain complete PCM loss as GMCs exit mitosis (Rusan, 2007).

Centrioles were examined live to test this hypothesis, using the centriole marker GFP-PACT and Histone-GFP Mother/daughter centrioles disengaged in late telophase, as in mammalian cells and fly embryos. Thus, two NB centrioles are present throughout interphase despite the presence of only one MTOC (Rusan, 2007).

The two centrioles then exhibit different behaviors. One remains fairly stationary, whereas the second moves to roughly the other side of the nucleus. Disengagement perfectly correlates with separation of Cnn spots, suggesting that the stationary centriole retains PCM to form the dominant MTOC and the mobile centriole completely sheds PCM. To test this, NBs were imaged expressing chTub and GFP-PACT. The stationary centriole retained MTs, whereas the mobile centriole did not. Upon reentering mitosis, the mobile centriole regained nucleation activity, forming the second MTOC (Rusan, 2007).

This suggests that full separation of the MTOCs that organize the spindle is biphasic. It begins in interphase, when one centriole retains PCM, remains stationary, and forms the dominant MTOC, whereas the second centriole sheds PCM and becomes mobile. Movement of the second centriole away from the dominant MTOC in interphase accounts for ~70% of the separation needed to form a spindle. Mechanisms of transporting the mobile centriole remain to be identified, but it is nonrandom, as in 26/30 NBs, the second MTOC emerged more than 90° from the dominant MTOC. After the second MTOC is activated, the two separate the last 30%, most likely via MT sliding forces. This might explain defects in lissencephaly1 mutants, where MTOCs are only separated by 124° at NEB. Perhaps interphase centriole movement is normal, but MT-based MTOC separation is defective (Rusan, 2007).

These data suggest that NBs differentially regulate the activity of their two centrioles within the same cytoplasm. Interestingly, a similar observation was made in clam eggs, which have three centrosomes just after fertilization. The sperm centrosome is functionally inactivated, whereas female centrosomes organize the meiotic spindle (Rusan, 2007).

Next NB centrosome regulation was examined. In preprophase, one centriole (marked by anti-DPLP) formed the dominant MTOC, whereas the second centriole had no associated MTs and was randomly positioned, confirming the live-cell data. Thus whether γtub is recruited asymmetrically was examined. Fixed preprophase NBs had two centrioles; only that opposite the GMCs accumulated γtub. Further, both γtub and Cnn are absent from the NB centriole nearest the GMCs in interphase/preprophase (Rusan, 2007).

Polo kinase promotes centrosome maturation by promoting γtub recruitment during mitotic entry. Differences in Polo localization/activity might underlie differences in timing of NB centrosome maturation. NBs expressing Polo-GFP and the centriole marker mCherry-DSAS-6 were examined. Only the centriole pair that forms the dominant centrosome was Polo-GFP positive during preprophase. Polo-GFP accumulated on the mobile centriole pair as the NB entered mitosis, increased on both centriole pairs through prophase, and moved on to kinetochores. When Polo-GFP was examined in cells exiting mitosis, it was retained at low levels on the dominant centrosome. In the future, it will be interesting to examine the localization of Aurora A, another centrosome regulator (Rusan, 2007).

Unlike the distal appendages of mammalian mother centrioles, fly mother and daughter centrioles have been thought to have no known ultrastructural or molecular differences. These data suggest that differences exist. It is unlikely that this differential regulation is a result of location, as both centrioles are initially adjacent after disengagement. The differences may be due to centriole age or procentriole maturation state (Rusan, 2007).

The NB spindle is largely aligned by NEB. Based on the current data, the hypothesis was tested that the dominant centrosome helps define one spindle pole before prophase. The angle between the dominant centrosome/MTOC axis and the anaphase axis was calculated, using the nuclear centroid as a fixed reference. This revealed two phases in defining the future spindle axis. Through prophase onset, the dominant centrosome remains fairly stationary roughly opposite the GMCs, agreeing with fixed images, whereas the second centriole moves to a distal position (to within 46 ± 33° of the anaphase axis). This is consistent with the hypothesis. The dominant centrosome may be immobilized by aster–cortex interactions or by absence of an active displacement mechanism. In the second phase, alignment is refined in prophase and prometaphase (the angle between the NB centrosome and anaphase axes decreases from 31 ± 29° to 15 ± 12° (Rusan, 2007).

To further test whether anchoring the dominant centrosome helps roughly align the spindle, asl mutant NBs were imaged live. They lack functional centrosomes and astral MTs. Mutant NBs lack a dominant interphase centrosome, allowing assessment of its role in spindle orientation and asymmetric cell division. Live imaging revealed robust chromatin-mediated MT nucleation and spindle assembly producing fairly normal spindles. Spindle poles emerge from a disorganized MT array near the chromosomes that focuses as the spindle lengthened. Spindles do not rotate during formation, always forming along the initial pole separation axis, but do rotate during metaphase, suggesting that rotation can occur without astral MTs or that asl mutants have a reduced astral array sufficient for rotation. Surprisingly, consecutive divisions in asl mutants usually produce adjacent or near-adjacent daughters, as in wild type. In a few cases, however, spindles form parallel to the GMC cap and, presumably, the polarity axis (2/13; ~15%); these NBs divide symmetrically. This suggests that the second phase of spindle alignment can occur without a dominant centrosome and can rescue misalignment, as long as it is not too extreme, but occasional atypical symmetric divisions occur. This results in defective brain anatomy, with ectopic paired, smaller NBs, presumably progeny of symmetric divisions (Rusan, 2007).

These data reveal two new aspects of asymmetric division in this stem cell model. First, cells can adjust the canonical centrosome cycle to allow novel cell behaviors, as was observed during clam meiosis. Central brain NBs also alter this cycle: rather than both centrosomes maturing in synchrony and proximity, the two centriole pairs are differentially regulated, maturing asynchronously and distant from one another. One retains MT nucleating activity throughout the cell cycle, forming the dominant MTOC during interphase, whereas the second is initially inactive, only forming a functional centrosome and nucleating MTs at mitotic entry. One speculative possibility is that these are mother and daughter centrioles and that one is preferentially retained in the stem cell, a hypothesis that will now be tested. It is also of interest to ask whether other stem cells use this mechanism (Rusan, 2007).

Second, the data suggest that this novel centrosome cycle helps ensure high-fidelity spindle positioning and thus asymmetric division. A model is proposed in which NB mitotic spindles are aligned in two phases to ensure that GMC daughters are born next to the previous GMC. Rough alignment is achieved by confining the dominant MTOC to a relatively fixed position from the previous division and moving the second centriole to the other side of the cell. As spindles form, a second process refines this initial alignment. In asl mutants, without centrosomes, the first mechanism is inactive, but the second mechanism can align the spindle unless initial alignment is wildly off axis. In mud mutants, centriole separation must occur normally, as prophase MTOCs are nearly fully separated, but alignment of spindle poles to cortical polarity cues is defective. The normal two-step process is a robust mechanism ensuring successful asymmetric divisions and reproducible brain anatomy (Rusan, 2007).

RNAi screening for kinases and phosphatases identifies Polo as a FoxO regulator

FoxO transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. This study reports a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays several enzymes were identified that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of the approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3beta, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. These results contribute to a comprehensive understanding of FoxO regulatory processes (Mattila, 2008).

By using a combination of transcriptional reporter and localization assays, twenty one dFoxO regulators were discovered. Some positive hits from the screen had an effect in dFoxO activity, localization, and protein stability, whereas other hits affected only transcriptional activity, suggesting that more mechanisms beyond subcellular localization and degradation are used by cells to regulate dFoxO activity. In addition to the 18 proteins that affected dFoxO transcriptional activity, the screening produced three more hits. Two of them seem to affect only dFoxO localization (dgkd and ptp69d), and one, neurospecific receptor kinase (nrk), affected exclusively dFoxO protein stability. It is possible that these proteins regulate dFoxO transcription on specific promoters in conjunction with other activators and that such factors are missing in Drosophila S2 cells. This would explain their lack of effect on the dInR promoter. Alternatively, they could affect dFoxO stability resulting in a net effect of dFoxO protein accumulation in the nucleus (Mattila, 2008).

Initially, the screening strategy was designed to identify both positive and negative regulators of dFoxO activity; however, no dFoxO repressors were found. Putative dFoxO repressors were present in the primary hit list of 31 targets, but those were later excluded in the secondary screen. This surprising observation suggests that the screen may be biased against dFoxO repressors. dFoxO is a well known inhibitor of protein biosynthesis in vivo, so under conditions of increased dFoxO activity, a reduction of general translation is expected that could affect GFP and luciferase translation too. Therefore, it is hypothesized that in the case of enhanced dFoxO activity it is possible that the concomitant inhibition of protein biosynthesis overruled a slight increase in reporter accumulation. This would explain the lack of dFoxO repressors among the targets of the screen. Moreover, the design of the screening based on S2 cells excludes the identification of regulatory mechanisms specific for other cell types, and instances where dFoxO is acting as a cofactor thereby regulating transcription indirectly (Mattila, 2008).

The results demonstrate that Drosophila PKC53E isoform is a dFoxO activator. Similar results were obtained in mammalian cells pointing out that the interaction is conserved. PKC isoforms have very important roles in insulin signaling, and each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Importantly, PKC isoforms can both activate or inhibit insulin signaling: Atypical PKC isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. In contrast, certain conventional and novel PKC isoforms are known to antagonize insulin signaling in vertebrates. This interaction is implicated in the pathogenesis of free fatty acid mediated insulin resistance. Drosophila possesses six PKC isoforms whose role in this context has not yet been addressed. PKC53E homolog is closest to human conventional PKCα. Interestingly, it has been shown that PKCα inhibits insulin signaling through binding and phosphorylation of IRS1. Thus, PKCα would serve as a constitutively active inhibitory regulator of the insulin cascade through its association with IRS1. On stimulation with insulin, PKCα would dissociate from IRS1, thus releasing this protein from its down-regulated state. This would open the 'gate' for transmission of the insulin signal. It has been found that dFoxO/FOXO1 increases insulin sensitivity by up-regulating insulin receptor transcription. The observation that Drosophila PKCα activates dFoxO adds an additional twist in the complex regulatory network that dFoxO has on insulin signaling. Interestingly, in the experimental system used in this study AKT dependent dFoxO bandshift and AKT Ser-505 phosphorylation was not affected by PKC53E, indicating that PKC53E regulation of dFoxO is independent of AKT signaling (Mattila, 2008).

Another well known enzyme implicated in the control of metabolism identified as a regulator of dFoxO transcriptional activity is the Drosophila ortholog of Glycogen synthase kinase 3β (GSK-3β, Shaggy). GSK-3β is a regulator of glucose metabolism through the phosphorylation and inhibition of glycogen synthase, the rate limiting enzyme of glycogen deposition. GSK-3β is inhibited by AKT, so it was not surprising to see that GSK-3β activates dFoxO. GSK-3β protein level and activity is elevated in type II diabetic skeletal muscle cells reflecting the impairment of whole body glucose uptake characteristic to this disease. In addition, selective inhibition of GSK-3β by lithium chloride represses the expression of g6pase and pepck in rat hepatoma cells, both known targets of FoxO. Taken together, these observations suggest that some of the metabolic effects of GSK-3β are achieved by directly modulating dFoxO activity (Mattila, 2008).

An interesting dFoxO regulator is Polo-like kinase. Polo-like kinases (Plks) are known regulators of cell cycle progression. In addition, Plks have a role in the protection against cellular stress through the transcription factor HSF1. Recently it was proposed that an intricate tradeoff between lifespan and cancer results from opposing effects of enzymes regulating FoxO and p53 activity. Plks are known to inhibit p53 transcriptional activity, so the results raise the possibility that Plks mediate the common but opposing regulators of p53 and FoxO. Interestingly, FoxOs are necessary in the completion of the cell cycle, which is partly mediated by cell cycle dependent activation of Plk expression by FOXO3a. The results show that Drosophila dFoxO is regulated by Polo, suggesting the existence of a positive feedback mechanism that has a role in achieving periodic M-phase gene expression and proper cell cycle exit (Mattila, 2008).

dFoxO localization was affected by eight modulators; however, band shifts demonstrated that none of these proteins phosphorylated dFoxO in the three conserved Ser/Thr amino acids known to regulate nuclear/cytoplasmic status through AKT. This observation raises the possibility that some of the newly identified dFoxO regulators could affect dFoxO nuclear/cytoplasmic localization by phosphorylating dFoxO in additional residues that do not alter its electrophoretic mobility, or that dFoxO regulation by these proteins is indirect. Further studies will be needed to clarify this point (Mattila, 2008).

In summary, this study has identified 21 dFoxO modulators. The results underscore the complexity underlying dFoxO regulation and establish dFoxO as a transcription factor controlled exquisitely by an intricate network of kinases and phosphatases achieving a perfect balance of activity. This balance ensures the correct execution of key cellular processes in metabolism, response to stress, and life span (Mattila, 2008).

Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells

Drosophila larval brain neuroblasts divide asymmetrically to generate a self-renewing neuroblast and a ganglion mother cell (GMC) that divides terminally to produce two differentiated neurons or glia. Failure of asymmetric cell division can result in hyperproliferation of neuroblasts, a phenotype resembling brain tumors. This study has identified Drosophila Protein phosphatase 2A (PP2A) as a brain tumor-suppressor that can inhibit self-renewal of neuroblasts. Supernumerary larval brain neuroblasts are generated at the expense of differentiated neurons in PP2A mutants. Neuroblast overgrowth was observed in both dorsomedial (DM)/posterior Asense-negative (PAN) neuroblast lineages and non-DM neuroblast lineages. The PP2A heterotrimeric complex, composed of the catalytic subunit (Mts), scaffold subunit (PP2A-29B) and a B-regulatory subunit (Tws), is required for the asymmetric cell division of neuroblasts. The PP2A complex regulates asymmetric localization of Numb, Pon and Atypical protein kinase C, as well as proper mitotic spindle orientation. Interestingly, PP2A and Polo kinase enhance Numb and Pon phosphorylation. PP2A, like Polo, acts to prevent excess neuroblast self-renewal primarily by regulating asymmetric localization and activation of Numb. Reduction of PP2A function in larval brains or S2 cells causes a marked decrease in Polo transcript and protein abundance. Overexpression of Polo or Numb significantly suppresses neuroblast overgrowth in PP2A mutants, suggesting that PP2A inhibits excess neuroblast self-renewal in the Polo/Numb pathway (Wang, 2009).

Mammalian PP2A is a tumor suppressor that participates in malignant transformation by regulating multiple pathways. However, it is unknown whether PP2A controls neural stem cell self-renewal. These data explicitly show that the Drosophila PP2A trimeric complex confers brain tumor-suppressor activity and controls the balance of self-renewal and differentiation of neural stem cells. This study shows that PP2A mutation leads to neural stem cell overproliferation in Drosophila larval brains, which is associated with dramatically reduced neuronal differentiation. Cell cycle genes including CycE, and phospho-Histone H3 and growth factor Myc are upregulated in PP2A mutants, consistent with the neuroblast overgrowth phenotype. Neuroblasts overproliferate in PP2A mutant MARCM clones. When these mutant clones that were generated at larval stages are kept until adulthood, neural stem cells continue to proliferate in adult brains, which is never observed for wild-type clones. Therefore, PP2A can inhibit excess self-renewal and promote neuronal differentiation of neural stem cells (Wang, 2009).

This overgrowth of neural stem cells in PP2A mutants is a consequence of defects in the asymmetric division of neural stem cells. PP2A regulates asymmetric protein localization as well as mitotic spindle orientation. It has been shown that Polo kinase is a brain tumor-suppressor that regulates Numb/Pon and aPKC asymmetric localization, as well as mitotic spindle orientation. Although polo mutants displayed pleiotropic phenotypes during asymmetric divisions, Polo primarily regulates asymmetric division of neural stem cells by regulating Numb asymmetry. Polo directly phosphorylates Pon on Ser611, which leads to the asymmetric localization of Pon and subsequently Numb (Wang, 2007). Strikingly similar to Polo, PP2A also regulates the asymmetric localization of aPKC, Pon and Numb, and is required for Pon phosphorylation on Ser611. Interestingly, both PP2A and Polo are required for Numb phosphorylation, which may be important for Numb asymmetric localization or activity on the cortex. Thus, Numb is a major downstream factor for both PP2A and Polo in regulating neural stem cell self-renewal. Consistent with this, overexpression of Numb, but not PonS611D, a phospho-mimetic form of Pon, in polo mutants significantly rescues the neuroblast overgrowth phenotype (Wang, 2009).

It was further discovered that PP2A functions upstream of Polo/Numb in the same pathway to control self-renewal of neuroblasts. Polo transcript and protein abundance is dependent on PP2A function. The expression of several other genes, including numb, baz and lgl, are not affected by PP2A knockdown, suggesting that the downregulation of polo in PP2A mutants appears to be specific. Moreover, overexpression of GFP-Polo or Numb can largely suppress neuroblast overgrowth in PP2A mutants, suggesting that PP2A primarily acts in the Polo/Numb pathway to inhibit neuroblast overgrowth. This discovery suggests that PP2A and Polo, both of which are crucial brain tumor-suppressors and cell cycle regulators, can function in the same pathway to regulate stem cell self-renewal and tumorigenesis. Currently, it is not clear how PP2A, which is a protein phosphatase, promotes polo expression. It is conceivable that PP2A dephosphorylates a transcription factor and consequently activates it to allow polo transcription. Alternatively, PP2A may dephosphorylate a protein that is required for polo mRNA stabilization (Wang, 2009).

PP2A is involved in a broad range of cellular processes including signal transduction, transcriptional regulation and cell cycle control. PP2A regulates the Wnt/Wingless signaling pathway and affects the degradation of β-catenin, a transcription factor and the central molecule of this pathway. Two of the components of Wnt/Wingless signaling pathway, Adenomatous polyposis coli (APC) and Shaggy (also known as GSK3), do not regulate neuroblast polarity. So it remains to be determined whether Wnt/Wingless signaling plays a role in neuroblast polarity. Mammalian PP2A directly dephosphorylates oncogene cMyc and tumor suppressor p53, both of which are transcription factors. Future studies should identify potential substrate(s) of PP2A that can promote polo expression and control neural stem cell self-renewal (Wang, 2009).

Interestingly, it was also observed that cMyc protein levels are increased in PP2A mutants, suggesting that PP2A may have a conserved role in modulating cMyc protein and suppressing its function. However, ectopic expression of cMyc alone does not induce brain tumor formation in Drosophila, suggesting that PP2A can regulate multiple pathways to affect neural stem cell self-renewal. However, the PP2A/Numb pathway appears to be one of the major pathways by which PP2A controls the balance of self-renewal and differentiation in Drosophila, as overexpression of Polo or Numb can largely suppress neural stem cell overgrowth in PP2A mutants. Furthermore, PP2A may regulate Numb function and activity by both promoting polo expression and antagonizing aPKC phosphorylation of Numb. Whether mammalian PP2A also regulates stem cell polarity will be of great interest for future study (Wang, 2009).

RNA polymerase II kinetics in polo polyadenylation signal selection

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, it was shown that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. These results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation (Pinto, 2011).

Since many genes in flies and other eukaryotes possess alternative pA sites in terminal exons, the possibility that general transcription kinetics might also have a role in alternative polyadenylation was investigated. To do this, the well-characterized C4 fly mutant, which has a 50% decrease in Pol II elongation rate, was used. Interestingly, it was shown that this results in increased utilization of the polo pA1 site. In particular, in C4 flies, polo pA1 is used 3.5-fold more efficiently than in the wild type. It was further demonstrated that the effect of Pol II kinetics in alternative pA site selection is likely to be general as analysis of five additional Drosophila genes possessing alternative terminal exon pA sites showed a similar switch to upstream pA site usage in the C4 mutant. This clearly indicates that Pol II kinetics has an important role in alternative polyadenylation site selection. Significantly, human Pol II carrying the equivalent mutation to the Drosophila C4, when transfected into human cells, affects alternative splicing of a FN minigene. Inclusion of the alternative EDI exon in this system increases ~4-fold, a similar level to that obtained for alternative polo pA signal selection. C4 flies also show a difference in Pol II occupancy across the polo gene. Thus, in wild-type flies Pol II levels are reduced downstream of pA1, while in C4 flies this pattern is disrupted. Presumably, the slow Pol II enables the pA1 signal on the nascent transcript to be exposed to the polyadenylation machinery for a longer time before Pol II transcribes pA2. Therefore, pA1 will be processed before pA2 is transcribed pointing to a mechanism that relies on the rule of 'first come, first served'. When the Pol II elongation rate is higher, as in wild-type flies, it transcribes through pA1 and pA2 more efficiently, so that the polyadenylation machinery processes both pA signals on the nascent transcript. This resembles the mechanism described above for EDI alternative splicing where slow Pol II preferentially includes the alternative EDI exon which is normally excluded, because it allows the machinery time to assemble on the spliceosome. The results now indicate that both alternative polyadenylation and alternative splicing depend on Pol II kinetics. In view of recent findings that highlight the importance of alternative pA signal selection, the results now suggest a general molecular mechanism for this process (Pinto, 2011).

The physiological consequences of correct polo pA signal choice in vivo as shown in this study are profound: pA2 is essential for abdominal histoblast proliferation, development of the adult epidermis and viability of the transgenic flies. The lethality and strong abdominal phenotype observed in gfp-poloΔpA2;polo9 flies are due to the fact that these flies lack polo pA2 mRNA and consequently cannot express sufficient levels of Polo protein (from polo pA1 mRNA) for flies to survive the pupa stage. This phenotype is in agreement with earlier studies showing that polo1/polo2 individuals express low levels of Polo protein and present an abnormal development of the abdomen. It is anticipated that the C4 mutant would display a similar phenotype to gfp-polo ΔpA2 flies since it downregulates pA2 usage. However, a developmental defect was observed similar to the so-called 'Ubx effect'. It is noted that slow Pol II elongation will impact on many genes so that any phenotype observed is likely to derive from complex genetic effects. It is also noted that polo pA2 mRNA is still produced at significant levels in the C4 mutant, which may produce sufficient protein for development of the abdomen, as opposed to the ΔpA2 transgenic flies that lack polo pA2 mRNA and show a decrease in Polo protein production in the histoblasts (Pinto, 2011).

Two genes in humans (hap and Bzw1) where alternative distal pA site usage results in increased levels of protein production have been previously described. In T cells and cancer cell lines, proximal pA site selection in the 3′UTR results in a relief from microRNA repression with the same final effect of an increase in protein production. However, a considerable proportion of genes do not follow this pattern, pointing to the existence of other regulatory elements in the different 3′UTRs. Presumably, polo is one such gene since it is shown that its expression is not regulated by dme-mir-8 and dme-mir-1016 overexpression. Instead regulated polo expression relies on the fact that the choice of pA1 by the transcriptional/processing machinery leads to a decrease in the translation of Polo. This indicates that Polo protein expression is modulated by pA signal selection and by translational control through the 3′UTR, suggesting the presence of regulatory elements in the different 3′UTRs. As Polo is a master regulator of the cell cycle, it is predicted that the consequences of this type of control will be critical to the cell (Pinto, 2011).

Histoblasts have a very high proliferation rate and go through very rapid cell cycles at the onset of metamorphosis, therefore would be predicted to be especially sensitive to Polo levels. The lack of polo pA2 mRNA in ΔpA2 flies leads to a reduction in Polo protein levels with the consequent block of histoblast proliferation. This will in turn result in a subsequent failure to correctly develop the adult epidermis. Consistent with these studies, loss of Polo has been shown to cause G2 arrest (Pinto, 2011).

Interestingly, it was also shown that, in flies where Polo is overexpressed, the shorter mRNA produced by pA1 usage is more abundant than the longer mRNA produced by pA2 selection. This suggests that Polo controls its own expression levels by an auto-regulatory loop, where higher levels of Polo lead to preferential recognition of pA1. As this mRNA is not efficiently translated, this will lead to a decrease in the protein levels produced. It has been shown that upregulation of su(f) leads to an auto-regulatory feedback loop through the recognition of a weak intronic pA site. This results in the production of a truncated polypeptide leading to a shut down in gene expression. In the case of polo, a more subtle process is evident, as pA1 selection generates a functional transcript, even though it is not efficiently translated into protein (Pinto, 2011).

Taken together, these results suggest that Pol II kinetics have an important role in pA site selection. Moreover, the results reinforce the view that tight regulation at the level of pA signal selection is necessary for the cell. The importance of precisely choosing the correct pA signal to control cell viability during development is underlined by these studies (Pinto, 2011).

Regulation of centromere localization of the Drosophila Shugoshin MEI-S332 and sister-chromatid cohesion in meiosis

The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. This study investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported in this study by localization and function studies in meiosis. Phospho-resistant and phospho-mimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phospho-mimetic mutation in the AuroraB phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly (Nogueira, 2014).

Protein Interactions

The polo gene product immunoprecipitated from extracts of single Drosophila embryos can phosphorylate casein in vitro. The kinase activity peaks cyclically at late anaphase/telophase. This contrasts with the cyclical activity of cyclin B-associated p34cdc2 kinase, which is maximal upon entry into mitosis during the rapid cycles of mitosis in the syncytium (Fenton, 1993).

Polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts. Wild type Polo protein migrates as a tight doublet of 67 kDA. By phosphatase treatment, which also inactivates the kinase, this is converted to a single band. Putative Polo substrates include proteins of sizes 220 kDa, 85 kDa and 54 KDa. Monoclonal antibody to ß-tubulin precipitates the phosphorylated 54 kDa protein, together with an associated 85 kDa protein also phosphorylated by Polo. Moreover, Polo binds to an 85 kDa protein that is enriched in microtubule preparations (Tavares, 1996).

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. 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. Polo kinase associates with PAV-KLP, with which it shows an overlapping pattern of subcellular localization during the mitotic cycle. This distribution is disrupted in pav mutants. It is suggested 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. PAV-KLP may be responsible for transporting Polo kinase from one set of centrosome-associated substrates to a second set of substrates in the midzone of the spindle as mitosis progresses (Adams, 1998).

Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants abnormal spindle (asp) and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which gamma-tubulin has an abnormal distribution. Moreover, the synergism of polo1;aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with gamma-tubulin to organize microtubule asters. Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25-38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase (do Carmo Avides, 2001).

These findings offer a route to understanding the role of phosphorylation in regulating the behaviour of the centrosomal microtubule-organizing centre. Thus, of the three microtubule-associated Polo substrates in Drosophila embryos, one was identified as beta-tubulin and another is now shown to be Asp. Asp becomes an MPM2 epitope when phosphorylated by Polo in vitro, suggesting that its MPM2 reactivity in wild-type but not asp mutant embryos is likely to be a direct consequence of a Polo-mediated phosphorylation event. The phosphorylation of Asp by Cdk1 and MAP kinases might also have physiological significance and, in fact, a genetic interaction has been reported between asp and rolled, a MAP kinase mutant, in Drosophila. Asp and Polo immunoprecipitate together and seem to be components of a complex that sediments at 25-35 S and has an estimated Mr of 3,000K by gel filtration. The size of the complex suggests that it either contains multimers of Asp and/or Polo or has additional components. It is not know where in the cell this complex might be located but it is known that both Asp and Polo localize to the centrosomes. Polo is likely to be associated with many proteins as it is dispersed both on sucrose gradients and following gel filtration. Polo-like kinases can associate with Cdc25 and the MKLP1/PAV-KLP motor protein. The position at which Cdc25 fractionates by these methods has not been checked, although it is known that PAV-KLP sediments at a position distinct from Asp, suggesting that it is part of a different complex (do Carmo Avides, 2001).

Although Polo, Cdk1 and MAP kinases all phosphorylate Asp in its amino-terminal region, this phosphorylation does not seem to be required for the binding of this segment of Asp to microtubules. Moreover, since Asp is found at spindle poles in polo mutants, it would seem not to require phosphorylation, at least by Polo kinase, for this aspect of its localization. Nevertheless, phosphorylated Asp protein is required for cytoplasmic extracts from polo1-derived embryos to be able to restore microtubule-nucleating activity to preparations of salt-stripped centrosomes. This provides an explanation of why mutations in both polo and asp result in broad spindle poles at which the gamma-tubulin ring complex is not focused, and why polo:asp double mutants have a synergistic interaction. It is proposed that, upon association of Asp and Polo with the centrosome at the onset of prophase, Polo phosphorylates Asp and thereby stimulates its activity to organize microtubules into asters that will later form the spindle poles (do Carmo Avides, 2001).

Polo kinase regulates the Drosophila centromere cohesion protein MEI-S332

Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein Mei-s332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. Mei-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. A mechanism has been elucidated for controlling release of Mei-S332 from centromeres via phosphorylation by POLO kinase. Polo antagonizes Mei-S332 cohesive function and full Polo activity is needed to remove Mei-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. Polo phosphorylates Mei-S332 in vitro; Polo and Mei-S332 bind each other, and mutation of Polo binding sites prevents Mei-S332 dissociation from centromeres (Clarke, 2005).

The results demonstrate that Polo regulates Mei-S332 localization and aspects of its function. polo mutants dominantly suppress the mei-S3328 nondisjunction phenotype and wild-type Mei-S332 is retained at centromeres past the metaphase II/anaphase II transition in these polo mutants. Mei-S332 appears to be phosphorylated in mitosis at the metaphase/anaphase transition, and in polo mutants, Mei-S332 persists on centromeres into interphase, consistent with phosphorylation being a signal for Mei-S332 to delocalize. Polo kinase binds to Mei-S332, and this is partly dependent on two PBD binding site motifs. Furthermore, in vitro phosphorylation of Mei-S332 is dependent on at least one motif and dependent on Polo. These two PBD binding site motifs in vivo are likely required for chromosomal dissociation of Mei-S332, similar to the effects observed in polo mutants. Together, these data point to Polo as a key regulator of Mei-S332 centromere localization in both mitosis and meiosis (Clarke, 2005).

Polo function is required for Mei-S332 delocalization from centromeres in mitosis and meiosis, but the ability of polo mutants to dominantly suppress mei-S332 mutants additionally shows that Polo antagonizes Mei-S332 function. This is important because the results indicate that cohesion can be released even if Mei-S332 remains localized in polo mutants. Mei-S332 remains on centromeres after the metaphase II/anaphase II transition in polo/+ mutants, yet reasonably normal disjunction of chromosomes occurs during meiosis II in these heterozygotes. In mitosis, Mei-S332 can remain on the centromeres of the fourth chromosomes in polo mutants even when sister chromatid cohesion is released and they segregate to the poles. Thus, Polo phosphorylation is necessary for delocalization of Mei-S332, but there must exist a mechanism to inactivate Mei-S332 to release cohesion that is independent of Mei-S332 dissociation. This pathway is not entirely dependent on Polo, although Polo may contribute (Clarke, 2005).

The idea that Mei-S332 can remain localized to centromeres without cohesion between sister chromatids is supported by several examples. In double parked mutants, Mei-S332 localizes to unreplicated, single chromatids on which cohesion has never been established (Lee, 2004). This shows that the presence of sister chromatid cohesion is not a prerequisite for Mei-S332 localization to centromeres. Similarly, Mei-S332 localizes to single sister chromatids in ord mutants, in which sister chromatids separate prematurely early in meiosis I. Finally, Sgo1, a yeast homolog of Mei-S332, can localize to centromeres in early anaphase II when the 3' UTR of Sgo1 is disrupted, yet no interference of the release of sister chromatid cohesion is observed (Rabitsch, 2004). This result suggests that Sgo1 can promote sister chromatid cohesion in meiosis and can subsequently be inactivated yet remain at centromeres (Clarke, 2005).

It is proposed that Mei-S332 centromere localization is regulated by phosphorylation. In this model, the assembly of Mei-S332 onto centromeres in prometaphase I is controlled by the action of an unknown phosphatase. Mei-S332 remains localized to centromeres until the metaphase II/anaphase II transition when Polo kinase binds to Mei-S332, via the phosphorylated T331 PBD binding site, and phosphorylates Mei-S332 elsewhere, initiating Mei-S332 dissociation from centromeres. The data suggest that both S234 and T331 contribute to Polo binding and to Mei-S332 centromere dissociation, but in vitro T331 plays the predominant role in Plx1-dependent phosphorylation. Further, it is suggested that Polo functions to antagonize Mei-S332 activity in meiosis, thereby affecting the release of sister chromatid cohesion. This may be either through phosphorylation of Mei-S332 or by affecting another component of sister chromatid segregation. Based on these results, it cannot be distinguished whether phosphorylation of Mei-S332 by Polo antagonizes Mei-S332 activity directly (Clarke, 2005).

It is proposed that Polo directly phosphorylates Mei-S332 because the proteins can bind each other. Importantly, this binding is reduced by disruption of the PBD binding site motifs, and these mutations abolish Plx1-dependent phosphorylation of Mei-S332 and prevent Mei-S332 from dissociating from centromeres in S2 cells. PBD binding sites are required to be phosphorylated in order for Polo to bind to its substrates. Given that Plx1-dependent phosphorylation of Mei-S332 was disrupted by mutating this site, it would seem that T331 and S234 need to be phosphorylated prior to Polo binding to Mei-S332, and then subsequent unknown sites on Mei-S332 can be phosphorylated by Polo kinase, thereby dissociating Mei-S332 from centromeres. In support of this idea, it has recently been proposed that once Polo binds to a substrate via an interaction with a PBD binding site, its kinase activity toward that substrate is stimulated (Clarke, 2005).

Which kinase is responsible for phosphorylating S234/T331 initially? Candidate kinases are a cyclin-dependent kinase, with specificity for sites with a proline in the +1 position as at the T331 and S234 sites, or a kinase such as Aurora B, which is localized to centromeres at the metaphase/anaphase transition in mitosis. The control of this precise phosphorylation event would effectively prevent Polo from phosphorylating Mei-S332 and releasing it from centromeres until the appropriate time. Correlating with this possibility, during both mitosis and meiosis, Polo is poised at centromeres yet does not act to remove Mei-S332 until the proper time. In mitosis, Mei-S332 becomes localized to centromeres in prometaphase, and Polo kinase is also localized to centromeres at this time. In meiosis, from metaphase I to metaphase II Polo localizes to centrosomes and centromeres, yet Mei-S332 is not released until metaphase II/anaphase II (Clarke, 2005).

Mei-S332 is the founding member of a family of proteins required for maintaining centromere cohesion between sister chromatids. This study has define Polo kinase as crucial for delocalization of Mei-S332. It was also shown that Polo antagonizes Mei-S332 activity. These results strongly indicate that Polo directly phosphorylates Mei-S332 and this leads to delocalization. It will be interesting to identify the anchor for Mei-S332 centromere binding and to decipher how phosphorylation of Mei-S332 affects this interaction. The results additionally uncover a mechanism distinct from delocalization to inactivate Mei-S332 (Clarke, 2005).

Interaction between Polo and BicD proteins links oocyte determination and meiosis control in Drosophila

Meiosis is a specialized cell cycle limited to the gametes in Metazoa. In Drosophila, oocyte determination and meiosis control are interdependent processes, and BicD appears to play a key role in both. However, the exact mechanism of how BicD-dependent polarized transport could influence meiosis and vice versa remains an open question. This article reports that the cell cycle regulatory kinase Polo binds to BicD protein during oogenesis. Polo is expressed in all cells during cyst formation before specifically localizing to the oocyte. This is the earliest known example of asymmetric localization of a cell-cycle regulator in this process. This localization is dependent on BicD and the Dynein complex. Loss- and gain-of-function experiments showed that Polo has two independent functions. On the one hand, it acts as a trigger for meiosis. On the other, it is independently required, in a cell-autonomous manner, for the activation of BicD-dependent transport. Moreover, Polo overexpression can rescue a hypomorphic mutation of BicD by restoring its localization and its function, suggesting that the requirement for Polo in polarized transport acts through regulation of BicD. Taken together, these data indicate the existence of a positive feedback loop between BicD and Polo, and it is proposed that this loop represents a functional link between oocyte specification and the control of meiosis (Mirouse, 2006).

This paper describes the localization of the Polo protein and its genetic control in the Drosophila germline during early oogenesis. Polo has a peculiar subcellular localization in cytoplasmic dots that do not correspond to any well-known structures of germline cysts or to microtubule minus-ends where BicD accumulates. Polo has previously been described as colocalizing with several subcellular structures depending on cell cycle phase, but none of these corresponds to the localization observed in this study. Similar cytoplasmic dots were observed in the primordial germline cells of the Drosophila embryo as soon as they were formed, suggesting that this unusual localization could be a specific feature of the germline (Mirouse, 2006).

From region 2a onward, Polo dots are present mostly in the cells containing SCs. This is the first report of a cell-cycle regulator whose localization is spatially and temporally correlated with meiotic progression during early oogenesis. Moreover this correlation is still conserved in mutants that affect polarized transport and the restriction and maintenance of meiosis. This indicates that Polo localization is dependent on polarized transport. One possibility is that Polo itself is directly transported to the oocyte. This hypothesis is reinforced by the physical interaction between BicD and Polo proteins, according to the proposed function of BicD as adapter for Dynein cargos. However, the BicD-dependent localization of Polo is not sufficient to explain its expression profile. Polo is strongly expressed in region 1 of the germarium, and the overall amount of the protein in the cyst progressively decreases, becoming undetectable after stage 2. This degradation seems to be compensated in meiotic cells and then in the oocyte by the polarized transport. The progressive degradation of Polo is also observed in egl and BicD null mutants. Degradation in association with a complete absence of Polo transport may explain why all the cells of a cyst enter into meiosis in these mutants (all the cells contain the same amount of Polo), and then exit meiosis simultaneously (none of the cells preferentially accumulates enough Polo). Alternatively, rather than by direct transport of Polo to the oocyte, its asymmetric distribution in the cyst could be due to a differential control of its stability between nurse cells and oocyte under the control of the BicD-dependent polarized transport (Mirouse, 2006).

BicD and egl null mutants showed a very similar phenotype, in which all 16 cells of a cyst first enter into meiosis but subsequently lose the synaptonemal complexes (SCs). This phenotype cannot be compared with null mutants of the dhc, since Dynein is required at earlier steps of cyst formation. The human homolog of BicD interacts directly with Dynamitin, and this interaction is thought to mediate the interaction of BicD with the Dynein complex. In contrast to BicD, Dynamitin is not involved in the initial restriction of meiosis, showing that the interaction of BicD with Dynamitin, and thus probably Dynein, is not required for the initial restriction of meiosis. In a similar way, LC8 (cut up) null mutants or egl mutants that specifically block the interaction between Egl and LC8 do not interfere with the initiation of meiosis in only four cells. Transport of the BicD protein between the cyst cells is apparently not required for this first step, sinc the BicDPA66 allele or drug-induced microtubule depolymerization does not affect this initial restriction, although BicD is diffuse throughout the entire cyst. Finally, a null mutant for the plakin shot, which has been proposed to be an essential upstream component of the Dynein function in centrosome migration, exhibits variable meiotic phenotypes but allows a normal initial restriction of meiosis to four cells. These data are consistent with a function of BicD and Egl independent of Dynein in the initial restriction of meiosis (Mirouse, 2006).

Polo is involved in many crucial steps of the cell cycle, including the G2/M transition of mitosis and meiosis processes. This study shows that hypomorphic polo alleles lead to a delay in meiotic entry and that Polo overexpression can trigger meiosis in more than four cells per cyst in region 2a. These phenotypes could be related to the function of Polo in the G2/M transition. In vertebrates, Polo is an activator of the String/CDC25 phosphatase, and it has also been proposed that Polo can repress the kinases Myt1 and Wee1. String is the main activator of the cyclinB/CDC2 complex, the activity of which triggers the G2/M transition, whereas Myt1 and Wee are repressors of this complex. However, the role of the cyclin B and CDC25 in meiosis in Drosophila oogenesis is not yet well understood because, for example, CDC25 seems to act as a negative regulator of meiotic oocyte cell fate. Further investigations will be needed to determine how Polo triggers meiotic entry during early oogenesis (Mirouse, 2006).

This study has shown that in mutants with partial loss of polo function, SCs start to disassemble in region 3 but are well formed again in stage 2/3 before disappearing in the following stages. One possible hypothesis to explain how meiosis is finally properly maintained in polo hypomorphic mutants is that the repression of cyclin E by Dacapo during stage 2/3 represses endoreplication, and thus allows meiotic progression. This is consistent with the finding that the specific localization of Dacapo to the oocyte and its requirement for meiosis maintenance begins only in region 3. Moreover, null mutations of dacapo do not lead to a fully penetrant 16-nurse-cell phenotype, confirming the existence of a partially redundant control. Therefore, it is proposed that the balance in favor of meiosis is initially due to the localized activation of meiosis by Polo, and later to the localized inhibition of endoreplication by Dacapo, and that both mechanisms partially overlap (Mirouse, 2006).

It was also observed that Polo is required for the normal restriction of meiosis. Moreover, the defects in the restriction of meiosis caused by both loss and gain of polo function are correlated with defects in oocyte determination. Meiosis restriction and oocyte specification both depend on the Dynein complex and the BicD polarized transport system. Thus, it is assumed that these Polo phenotypes indicate that Polo is involved in polarized transport. This role may be indirect and thus reveals the influence of meiosis and cell-cycle control on oocyte differentiation. Such influence has been observed in situations where there is activation of the meiotic checkpoint due to a failure in DNA double-stand break repair. However, at least two results argue for a direct role of Polo in polarized transport, independently of its meiotic function. First, in mosaic germline cysts, nonmeiotic cells mutant for polo retain BicD protein. Thus, this phenotype cannot be due to the activation of the meiotic checkpoint. This strongly suggests that Polo is required in each cell of the cyst to initiate BicD-dependent transport to the presumptive oocyte. Second, the overexpression of Polo is able to restore the localization and therefore the function of BicDPA66 protein. Interestingly, this mutant allele is due to a single amino acid substitution (A40V) that leads to a hypophosphorylation of BicD, and genetic evidence indicates that this phosphorylation is crucial for BicD function. Polo overexpression might restore a functional level of BicDPA66 phosphorylation. Therefore, even if no significant changes were observed in the gel mobility of BicD in polo hypomorph mutants, it is tempting to propose that the function of Polo in the polarized transport could be to activate, directly or indirectly, BicD by phosphorylation (Mirouse, 2006).

Taken together, these results lead to a model that can explain a reciprocal requirement between the control of meiosis and oocyte specification. This model is based on four major points: (1) BicD is required for the Dynein-dependent polarized transport of oocyte determinants; (2) BicD is also required for the progressive localization of Polo to the oocyte; (3) Polo appears to trigger meiosis in the germarium; (4) Polo is required to activate the BicD and Dynein-dependent polarized transport. These findings suggest the existence of a positive feedback loop between Polo and BicD proteins, and therefore between oocyte specification and meiosis (Mirouse, 2006).

Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon

Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced. A failure of this balance could lead to diseases such as cancer. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila, where it inhibits self-renewal. The localization and function of Numb is cell-cycle-dependent. This study shows that Polo acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast overproliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. These results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb (Wang, 2007).

Asymmetric localization of Numb depends on its adaptor protein Pon. The Pon localization domain (Pon-LD) is located at the carboxy terminus of the protein. The Ser 611 (S611) residue in this domain matches the consensus phosphorylation site for Polo. Because the localization of Pon is cell-cycle-dependent, tests were perfomred to see whether Polo can directly phosphorylate Pon. Pon-LD, but not Pon(S611A)-LD, in which S611 was mutated to Ala, was readily phosphorylated by mammalian Polo-like kinase 1 (Plk1) in vitro, demonstrating that Pon S611 is a Polo phosphorylation site (Wang, 2007).

To test whether Pon S611 is normally phosphorylated in vivo, an antibody was generated against S611-phosphorylated (p-S611) Pon. The specificity of this antibody was shown by its ability to recognize a glutathione S-transferase-Pon-LD fusion protein (GST-Pon-LD) only after the fusion protein was pre-phosphorylated by Plk1. It did not recognize GST-Pon(S611A)-LD in the same assay. Next, larval brain extracts prepared from wild type as well as heterozygotes [polo9(+/-) and polo10(+/-)], and homozygotes [polo9(-/-) and polo10(-/-)] of two different polo loss-of-function alleles were analysed by western blotting using this p-S611-specific antibody. p-S611-positive Pon was clearly detected in wild-type animals and in heterozygotes, but was barely detectable in homozygous mutant animals, demonstrating that Pon is phosphorylated at S611 in vivo in a Polo-dependent fashion (Wang, 2007).

Immunohistochemistry was used to verify S611 phosphorylation in vivo and to visualize phospho-Pon localization. p-S611-positive endogenous Pon was detected in wild-type larval brains as a crescent in metaphase neuroblasts, and was segregated to the ganglion mother cell (GMC, the daughter committed to the differentiation pathway) after division. In the polo9 mutant, however, p-S611-positive Pon was undetectable. The p-S611 antibody also reacted with Pon-LD, but not with Pon(S611A)-LD, in transgenic larval brain (Wang, 2007).

To test for a functional role of S611 phosphorylation, S611 was mutated to a non-phosphorylatable Ala residue (S611A) or to a phospho-mimetic Asp residue (S611D). Wild-type and phospho-mutant Pon-LD were fused to green fluorescent protein (GFP) and expressed in embryonic neuroblasts. Both GFP-Pon-LD and GFP-Pon(S611D)-LD showed the expected basal localization. In contrast, the localization of GFP-Pon(S611A)-LD was defective. At prometaphase and metaphase, it showed either uniformly cortical (80%) or basally enriched but apically detectable cortical (20%) distribution. At anaphase and telophase, however, it formed basal crescents in most neuroblasts. This 'telophase rescue' seemed to be partially mediated by endogenous Pon, because less rescue was observed in pon mutant neuroblasts, with 58 neuroblasts mis-segregating GFP-Pon(S611A)-LD at late anaphase/telophase. It is unlikely that the S611A mutation affects Pon localization by altering the charge or global conformation and folding of the protein, because mutation of an adjacent Ser residue (S616) or triple mutations at three potential atypical protein kinase C (aPKC) phosphorylation sites (S644A/S648A/S652A) had no effect on Pon-LD localization, suggesting that S611 represents a unique regulatory point in Pon localization (Wang, 2007).

To assess whether Polo has a role in neuroblast self-renewal and/or asymmetric division, central brain neuroblast numbers were quantified in two strong hypomorphic alleles, polo9 and polo10, using Deadpan (Dpn) and Miranda (Mira) as neuroblast markers. Wild-type larval central brains averaged 37 neuroblasts 24 h after larval hatching (ALH) and 10 neuroblasts 96 h ALH. polo9 larval central brains averaged 36 neuroblasts 24 h ALH. However, the number increased dramatically to 254 96 h ALH. Consistent with this increase in neuroblast number, the numbers of BrdU-labelled, CycE-positive or phospho-histone-H3-positive proliferating cells were also increased in polo9 mutant brains compared to wild type. Concomitantly, a dramatic reduction of differentiated cells expressing neuronal markers, Embryonic Lethal Abnormal Vision (Elav) or nuclear Prospero (Pros), was observed in polo9 mutant brains. A similar neuroblast overproliferation phenotype was observed in polo10 and in trans-heterozygotes between polo9 and a deficiency that deletes polo. A Polo-GFP genomic construct fully rescued this polo mutant phenotype, verifying that these defects are caused by polo loss-of-function. Excess self-renewal and proliferation at the expense of neuronal differentiation was also observed in MARCM (mosaic analysis with a repressible cell marker) clones derived from single polo9 mutant neuroblasts. These results indicate that Polo behaves like a tumour suppressor to inhibit neuroblast self-renewal and to promote differentiation. polo mutant GMCs may revert to neuroblast-like cells, as has been shown for brat (brain tumor) mutants (Wang, 2007).

The physiological role of Polo in regulating Pon localization and function was analyzed. Most larval neuroblasts were found at metaphase in polo9 mutant brains, and both Pon and Numb were uniformly distributed. In late anaphase/telophase neuroblasts, Pon and Numb were mis-segregated to both daughter cells. Defective Pon and Numb localization in the polo mutant is unlikely to be a secondary consequence of cell cycle arrest, because arrest of wild-type neuroblasts at metaphase with microtubule-depolymerizing drugs actually increased the number of cells possessing a Numb crescent (Wang, 2007).

To test whether Polo is specifically required for Pon/Numb localization, other apical and basal proteins were analyzed. In polo9 mutant neuroblasts, the basal localization of Brat and Pros was relatively normal. Moreover, double-labelling of the same mutant neuroblast showed that the localization of Mira, the adaptor protein for Pros and Brat, was normal, whereas Pon localization was abnormal. Introduction of a Polo-GFP transgene into polo9 effectively rescued the Pon localization and cell-cycle defects. Apical proteins such as Insc, Baz, Pins and Discs Large 1 (Dlg1) were localized normally in polo9 mutant neuroblasts. The only apical protein showing abnormal localization was aPKC, which became distributed uniformly on the cortex and showed cytoplasmic localization. During telophase, aPKC could be mis-segregated into both daughter cells (Wang, 2007).

Polo is localized to centrosomes and is required for centrosome organization and separation. Whether Polo has a role in orienting neuroblast mitotic spindles was tested. The tight coupling of spindle orientation with crescent formation seen in wild-type neuroblasts was disrupted in polo9 metaphase neuroblasts with two centrosomes. Therefore, Polo kinase is also required for coupling mitotic spindle orientation with cortical protein asymmetry. This spindle phenotype was fully rescued by the Polo-GFP transgene (Wang, 2007).

Next the role of Pon phosphorylation in mediating Numb localization was probed. Full-length Pon containing the S611A or S611D mutation was used to assess the effects of S611 phosphorylation. In pon mutant neuroblasts, Numb localization was defective. Introducing wild-type Pon restored Numb asymmetric localization at metaphase and later stages. Most pon mutant neuroblasts expressing Pon(S611D) also showed rescue. In contrast, pon mutant neuroblasts expressing Pon(S611A) showed largely abnormal Numb localization. Polo-mediated phosphorylation is therefore important for Pon to localize Numb. The function of Pon in neuroblast self-renewal was tested by generating pon MARCM clones. Interestingly, compared to wild-type clones, ectopic neuroblast self-renewal was observed in pon mutant clones (Wang, 2007).

This study has shown that polo mutations affect Numb and aPKC localization as well as spindle orientation -- processes known to affect neuroblast self-renewal to various degrees. Strikingly, overexpression of either Numb-GFP or Numb effectively suppressed the ectopic neuroblast self-renewal phenotype seen in the polo9 mutant. This effect was not caused by increased neuroblast apoptosis, and overexpression of Numb-GFP or Numb in a wild-type background did not affect the neuroblast number. These results indicate that Numb is a principal player downstream of Polo in regulating neuroblast self-renewal. Numb overexpression did not rescue the aPKC mislocalization and spindle misorientation phenotypes of polo mutants. These defects could also contribute to the neuroblast overgrowth phenotype of polo mutants, but their effects might have been masked by Numb overexpression. Consistent with this, introduction of Pon(S611D) into a polo mutant neuroblast did not significantly rescue the neuroblast overgrowth phenotype, despite the partial restoration of Numb localization. Because aPKC localization and spindle orientation defects were not rescued by Pon(S611D), these defects may account for the inability of Pon(S611D) to rescue the overgrowth phenotype of polo. aPKC has been shown to phosphorylate Numb. Delocalized aPKC at the basal side may be sufficient to inactivate endogenous Numb, but not overexpressed Numb, owing to titration by the overexpressed protein (Wang, 2007).

Numb was previously shown to inhibit neuroblast self-renewal by antagonizing Notch signalling. Reducing Notch significantly suppressed the neuroblast overgrowth phenotype of the polo9 mutant. Reducing Notch in a wild-type background also led to a partial loss of neuroblasts, consistent with Notch being required for progenitor self-renewal. It is envisioned that in polo or pon mutants, owing to the symmetric distribution of Numb, the GMCs receive insufficient Numb to inhibit Notch, thereby causing them to adopt a neuroblast-like fate. To test further the importance of Numb asymmetric localization in neuroblast self-renewal versus differentiation, the numbS52F mutation, which apparently affects Numb asymmetric localization but not its stability or activity, was tested. In numbS52F neuroblast clones, ectopic neuroblast self-renewal similar to that seen in polo or pon clones was observed. Thus, loss of Numb asymmetric localization is sufficient to cause neuroblast overgrowth (Wang, 2007).

These results indicate that Polo controls the self-renewal versus differentiation decision of neural progenitors by regulating the localization and activity of Numb and the orientation of mitotic spindles. Polo regulates the localization of Numb by means of Pon. Although immunofluorescence shows that Polo is primarily localized to the centrosomes, a cytosolic pool of Polo probably exists because Polo exhibits dynamic in vivo association with the mitotic apparatus and many non-centrosomal Polo substrates have been identified. Cytosolic localization of the centrosomal kinase Aurora-A has been demonstrated. How Polo regulates the localization of aPKC, the activity of Numb and the orientation of mitotic spindles awaits further investigation. In addition to the Numb/Notch pathway, other factors such as Pros and Brat are necessary for preventing GMCs from undergoing self-renewing divisions. Because these factors are segregated normally in polo neuroblasts, it seems that they are not sufficient to prevent progenitor self-renewal or that activation of Notch is able to override their effects. Intriguingly, some Plks behave as tumour suppressors in mammals, and loss of Numb has also been implicated in the hyperactivation of Notch signalling in breast cancer cells. These results and future studies in models like Drosophila will provide mechanistic insights into these observed tumour-suppressor roles of Polo and Numb (Wang, 2007).

The inhibition of polo kinase by matrimony maintains G2 arrest in the meiotic cell cycle

Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. These data suggest a model in which the eventual activation of Cdc25 (Drosophila twine) by an excess of Polo at stage 13 triggers NEB and entry into prometaphase (Xiang, 2007).

The mechanism of the lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB) (which is a characterization of many female meiotic systems) has remained a mystery. One can imagine that both the maintenance and the termination of this arrest might involve either or both of two mechanisms: the transcriptional or translational repression of a protein that induces NEB, and thus meiotic entry, or the presence of an inhibitory protein that precludes entry into the first meiotic division. Because Drosophila females exhibit a prolonged G2 arrest and are amenable to both genetic and cytological analyses, they provide an ideal system in which to study this problem (Xiang, 2007).

The ovaries of Drosophila females are composed of a bundle of ovarioles, each of which contains a number of oocytes arranged in order of their developmental stages. For the purposes of this study, the process of oogenesis may be said to consist of three separate sets of divisions: the initial stem cell divisions, which create primary cystoblasts; four incomplete cystoblast divisions, which create a 16-cell cyst that contains the oocyte; and the two meiotic divisions. Although a great deal is known regarding the mechanisms that control cystoblast divisions and oocyte differentiation, relatively little is known about the mechanisms by which the progression of meiosis is controlled (Xiang, 2007).

As is the case in many meiotic systems, female meiosis in Drosophila involves preprogrammed developmental pauses. The two most prominent pauses during Drosophila meiosis are an arrest that separates the end of pachytene at stages 5-6 from NEB at stage 13, and a second pause that begins with metaphase I arrest at stage 14 and continues until the egg passes through the oviduct. It is the release of this second preprogrammed arrest event that initiates anaphase I and allows the completion of meiosis I followed by meiosis II. The end of meiotic prophase by dissolution of the synaptonemal complex (SC) at stages 5-6 is separated from the beginning of the meiotic divisions, which is defined by NEB at stage 13, by approximately 40 h to allow for oocyte growth (Xiang, 2007).

Elucidating the mechanisms that arrest meiotic progression at the end of prophase, but then allow onset of NEB and the initiation of meiotic spindle formation some 40 h later, was of great interest. One intriguing possibility is that during this period of meiotic arrest, the oocyte actively blocks the function of cell cycle regulatory proteins such as cyclin dependent kinase 1 (Cdk1), the phosphatase Cdc25, and Polo kinase (Polo), all of which promote meiotic progression just as they do during mitotic growth. Recently, Polo was shown to be expressed in the germarium and required for the proper entry of Drosophila oocytes into meiotic prophase, as defined by the assembly of the SC. Decreased levels of Polo resulted in delayed entry into meiotic prophase, whereas overexpression of Polo caused a dramatic increase in the number of cystocyte cells entering meiotic prophase, indicating that Polo is involved both in the initiation of SC formation and in the restriction of meiosis to the oocyte. How then is Polo, which is known to play multiple roles in promoting meiotic and mitotic progression, prevented from compelling the differentiated oocyte to proceed further into meiosis? One component of this regulation may well lie in the fact that Polo is not expressed during much of oogenesis. Polo is clearly visible in the germarium but is then absent until stage 11, when it begins to accumulate to high levels in the oocyte. A second component of Polo regulation is mediated by binding to the protein product of the matrimony (mtrm) gene, which occurs from stage 11 until the onset of NEB at stage 13. This binding serves to inhibit Polo in the early stages of its expression, and thus prevents precocious nuclear envelope breakdown (Xiang, 2007).

The mtrm gene was first identified in a deficiency screen for loci that were required in two doses for faithful meiotic chromosome segregation (Harris, 2003). mtrm/+ heterozygotes display a significant defect in achiasmate segregation (the meiotic process that ensures the segregation of those homologs that, for various reasons, fail to undergo crossingover). As a result of this defect, mtrm/+ heterozygotes exhibit high levels of achiasmate nondisjunction. As homozygotes, mtrm mutants are fully viable but exhibit complete female sterility. This study shows that the Mtrm protein prevents precocious NEB. Indeed, the effects of reducing the dose of mtrm on meiotic progression and on chromosome segregation are easily explained as the consequence of precocious NEB at stages 11 or 12, and can be suppressed by simultaneously reducing the copy number of polo+. In addition, the effects of heterozygosity for loss-of-function alleles of mtrm can be phenocopied by increasing the copy number of polo+. These genetic interactions suggest that Mtrm negatively regulates Polo in vivo (Xiang, 2007).

Interestingly, Mtrm was shown to interact physically with Polo by a global yeast two-hybrid study. This yeast two-hybrid finding reflects a true physical interaction in vivo by both coimmunoprecipitation studies and by multidimensional protein identification technology (MudPIT) mass spectrometry experiments, which indicate that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Moreover, ablating one of the two putative Polo binding sites on Mtrm by mutation prevents the physical interaction between Polo and Mtrm and renders the mutated Mtrm protein functionless. This experiment, along with genetic interaction studies, provides compelling evidence that the function of the binding of Mtrm to Polo is to inhibit Polo, and not vice versa (Xiang, 2007).

The analysis of mtrm mutants allows examination of the effects of premature Polo function during oogenesis. The evidence shows that in the absence of Mtrm, newly synthesized Polo is capable of inducing NEB from stage 11 onward. As a result of this precocious NEB, chromosomes are not properly compacted into a mature karyosome and they are released prematurely onto the meiotic spindle. In many cases, the centromeres of achiasmate bivalents subsequently fail to co-orient (Xiang, 2007).

The mtrm gene was first identified as a dosage-sensitive meiotic locus; heterozygosity for a loss-of-function allele of mtrm specifically induced the failed segregation of achiasmate homologs (Harris, 2003). The mtrm gene encodes a 217-amino acid protein with two Polo-Box Domain (PBD) binding sites (STP and SSP) and a C-terminal SAM/Pointed domain. The studies reported in this paper rely primarily on a null allele of mtrm (mtrm126), which removes 80 bp of upstream sequence and the sequences encoding the first 41 amino acids of the Mtrm protein (Xiang, 2007).

Western blot analysis using an antibody to Mtrm reveals that Mtrm can be detected only in ovaries. This is consistent with a previous report by Arbeitman (2002), which showed that the expression profile of the mtrm gene product was strictly maternal and that its expression was reduced greater than 10-fold over 0-6.5 h of embryonic development. The specificity of this antibody is demonstrated by the fact that no signal was detected by either Western blotting or by immunofluorescence of ovarioles homozygous for the mtrm126 mutant. Immunofluorescence studies using the same antibody reveal that Mtrm is expressed as a diffuse nuclear protein in the oocytes and nurse cells beginning at stage 4-5. The Mtrm signal was not restricted to the karyosome itself; but rather Mtrm seems to fill the space in the entire nucleus. Although Mtrm is restricted to the nucleus until approximately stage 10, it localizes throughout the oocyte in later stages. Mtrm brightly stains both the oocyte nucleus and cytoplasm between stage 11 and stage 12, but staining is greatly reduced at stage 13, the stage at which NEB occurs (Xiang, 2007).

The data presented in this study that Mtrm serves to inactivate newly synthesized Polo during the period of meiotic progression that precedes NEB. An excess of functional (unbound) Polo, produced by reducing the amount of available Mtrm, causes the early onset of NEB. This early entry into prometaphase releases an immature karyosome into the cytoplasm, which then fails to properly align the centromeres of achiasmate chromosomes on the prometaphase spindle. These observations raise a number of questions ranging from the role of Polo in mediating the G2/M transition in oogenesis to the role of the karyosome structure in facilitating the proper segregation of achiasmate chromosomes (Xiang, 2007).

The trigger for the G2/M transition is activation of Cdk1 by Cdc25, and multiple lines of evidence suggest that Polo can activate Cdc25. First, in Caenorhabditis elegans, RNAi experiments demonstrate that ablation of Polo prevents NEB. Second, the Xenopus Polo homolog Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25. Additionally, microinjection of Plx1 accelerates the activation of both Cdc25 and cyclinB-Cdk1. Moreover, microinjection of either an antibody to Plx1 or kinase-dead mutant of Plx1 inhibits the activation of Cdc25 and its target cyclinB-Cdk1. Another demonstrated that injection of a constitutively active form of Plx1 accelerated Cdc25 activation. These studies support "the concept that Plx1 is the 'trigger' kinase for the activation of Cdc25 during the G2/M transition." Finally, a small molecule inhibitor of Polo kinase (BI 2536) also results in extension of prophase (Lenart, 2007). These data are consistent with the view that the presence of functional (unbound) Polo plays a critical role in ending the extended G2 that is characteristic of oogenesis in most animals (Xiang, 2007).

In light of these data, it is tempting to suggest that in wild-type Drosophila oocytes, the large quantity of Mtrm deposited into the oocyte from stage 10 onward inhibits the Polo that is either newly synthesized or transported into the oocyte during stages 11-12. However, at stage 13, an excess of functional Polo is created when the number of Polo proteins exceeds the available amount of inhibitory Mtrm proteins. This unencumbered and thus functional Polo then serves to activate Cdc25, initiating the chain of events that leads to NEB and the initiation of prometaphase. In the absence of a sufficient amount of Mtrm, an excess of Polo causes the precocious activation of Cdc25, and thus an early G2/M transition. A model describing this hypothesis is presented. Based on this model, one can visualize that decreasing the dose of Mtrm or increasing the dose of Polo will hasten NEB, whereas simultaneous reduction in the dose of both proteins should allow for proper timing of NEB (Xiang, 2007).

Mtrm's first PBD binding site (T40) is required for its interaction with Polo. Mtrm T40 has to be first phosphorylated by a priming kinase, such as one of the Cdks or MAPKs, and was indeed detected as phosphorylated in the mass spectrometry dataset. The NetPhosK algorithm predicts T40 to be a Cdk5 site, and the serines immediately distal to T40 (S48 and S52), which were also detected as phosphorylated are sites for proline-directed kinases such as Cdk or MAPK sites as well. The other prominent phosphorylation event occurs at S137, which could be a Polo phosphorylation site since it falls within a Polo consensus (D/E-X-S/T-Ø-X-D/E). Although the combined sequence coverage for Mtrm was 44%, indicating that some phosphorylated sites might have been missed, Mtrm S137 is a suitable binding site for activated Polo, in agreement with the processive phosphorylation model. At this point of these studies, Mtrm T40 priming kinase or the kinase responsible for Polo activating phosphorylation on T182 has not been identified (Xiang, 2007).

The finding that Polo not only is able to bind to Mtrm in vivo in a 1:1 ratio, but also is fully phosphorylated on T182 in its activation loop suggests a method by which Mtrm serves to inhibit Polo. In general, enzymes are usually not recovered from affinity purifications at levels similar to their targets. They do not form stable complexes, but rather form transient interactions with their substrates, which is how efficient catalysis is achieved. In this instance, Mtrm is able to sequester activated Polo away in a stable binary complex over a long period of time. It is only when this equilibrium is disturbed at the onset of stage 13 by the production of an excess of Polo or by degradation of Mtrm that Polo can be released. The molecular determinants of the Mtrm::Polo sequestration event are not clear, but it would be interesting to test whether the serines found phosphorylated in the vicinity of Mtrm PBD binding sites play a role in locking the binary complex into place (Xiang, 2007).

The data demonstrate that a reduction in the levels of Mtrm results in the release of an incompletely compacted karyosome that rapidly dissolves into individual bivalents during the early stages of spindle formation. For chiasmate bivalents, this is apparently not a problem, because they still co-orient correctly. However, the nonexchange bivalents frequently fail to co-orient properly, such that both homologs are oriented toward the same pole (but often occupy two different arcs of the spindle). This initial failure of proper co-orientation leads to high frequencies of nondisjunction as demonstrated by the genetic studies and analysis of metaphase I images (Xiang, 2007).

Although achiasmate homologs are properly co-oriented in wild-type oocytes, it has been noted that such homologs can often vacillate between the poles such that two achiasmate homologs are often found on the same arc of the same half-spindle during mid to late prometaphase (Gilliland, 2007). These chromosomes are often observed to be physically associated. This situation is quite different from the defect observed in mtrm/+ heterozygotes, where the homologs are neither physically associated nor on the same arc of the spindle (Xiang, 2007).

It is tempting to suggest that the chromosome segregation defects observed in mtrm/+ heterozygotes are simply the result of precocious release of an incompletely re-compacted karyosome. According to this explanation, the defects observed in meiotic chromosome segregation are solely the consequence of premature NEB. (Implicit in this model is the assumption that it is the events that occur during karyosome re-compaction, at stages 11 and 12, that serve to initially bi-orient achiasmate chromosomes, and there is no direct evidence to support such a hypothesis) (Xiang, 2007).

Alternatively, Polo plays multiple roles in the meiotic process (Lee, 2003a; Lee, 2003b), and it is possible that the chromosome segregation defects seen represent effects of excess Polo that are un-related to the precocious breakdown of the nuclear envelope. Such a view is supported by two observations. First, the bivalent individualization observed after NEB in mtrm/+ oocytes does not disrupt FM7-X pairings. Second, although heterozygosity for twine in mtrm126/+ heterozygotes suppresses the frequency of precocious NEB from 42%, two alleles of twine tested (twe1 and twek08310) failed to suppress the levels of meiotic nondisjunction observed in FM7/X; mtrm126/+ heterozygotes. These data suggest that the effects of excess Polo on nondisjunction may not be regulated via Cdc25/Twine, but rather by the effects of excess Polo on some other as-yet-unidentified Polo target. This suggests that the effects of Mtrm on the level of Polo might affect multiple Polo-related processes (Xiang, 2007).

Support for such an idea that Mtrm can inhibit Polo-regulated proteins that are unrelated to NEB comes from the observation that the ectopic expression of Drosophila Mtrm in Schizosaccharomyces pombe blocks karyokinesis, producing long multi-septate cells with only one or two large nuclei. This phenotype is similar, if not identical to, that exhibited by mutants in the S. pombe Polo homolog plo1 (Plo1), which fail in later stages of mitosis due to the role of Plo1 in activating the septation initiation network to trigger cytokinesis and cell division. However, Plo1 also plays a role in bipolar spindle assembly that might also be inhibited in the Mtrm expressing cells, but this function of Plo1 is less well understood (Xiang, 2007).

Thus the possibility exists that the effect of mtrm mutants on meiotic chromosome segregation may well not be the direct consequence of early NEB, but rather may be due to the role of Polo in other meiotic activities, such as spindle formation or the combined effects of these defects with precocious NEB. Efforts to identify such processes and their components are underway (Xiang, 2007).

Finally, it should be noted that while Mtrm is the first known protein that is able to inactivate Polo by physical interaction with Polo itself; there is certainly additional mechanisms of Polo regulation. For example, mutants have been described in the gene that encodes Greatwall/Scant kinase, that have both late meiotic and mitotic defects. Although there is no evidence for a physical interaction between these two kinases, the authors speculate that the function of the Greatwall kinase serves to antagonize that of Polo. The Scant mutations create a hyperactive form of Greatwall, which might be expected to lower the dosage Polo, and thus perhaps partially suppress the defects observed in mtrm/+ heterozygotes. Indeed, exactly such a suppressive effect has been observed in Scant homozygotes (however, this suppression is much weaker than that obtained by heterozygosity for loss of function alleles of polo) (Xiang, 2007).

The data presented in this study demonstrate that Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest during meiosis. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. These data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase. Although the data do shed some light on the mechanism by which Mtrm inhibits Polo, it is not entirely clear whether Polo's ability to phosphorylate targets other than Cdc25 might be blocked by Mtrm::Polo binding. These issues will need to be addressed in the future studies. Finally, it is noted that although small molecule inhibitors of Polo have been identified, Mtrm represents the first case of a protein inhibitor of Polo. It would be most exciting to identify functional orthologs of Mtrm outside of the genus Drosophila. Perhaps that might best be accomplished through a screen for oocyte-specific Polo-interacting proteins (Xiang, 2007).

Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis

The conserved Polo kinase controls multiple events in mitosis and cytokinesis. Although Polo-like kinases are regulated by phosphorylation and proteolysis, control of subcellular localization plays a major role in coordinating their mitotic functions. This is achieved largely by the Polo-Box Domain, which binds prephosphorylated targets. However, it remains unclear whether and how Polo might interact with partner proteins when priming mitotic kinases are inactive. This study shows that Polo associates with microtubules in interphase and cytokinesis, through a strong interaction with the microtubule-associated protein Map205. Surprisingly, this interaction does not require priming phosphorylation of Map205, and the Polo-Box Domain of Polo is required but not sufficient for this interaction. Moreover, phosphorylation of Map205 at a CDK site relieves this interaction. Map205 can stabilize Polo and inhibit its cellular activity in vivo. In syncytial embryos, the centrosome defects observed in polo hypomorphs are enhanced by overexpression of Map205 and suppressed by its deletion. It is proposed that Map205-dependent targeting of Polo to microtubules provides a stable reservoir of Polo that can be rapidly mobilized by the activity of Cdk1 at mitotic entry (Archambault, 2008).

How Plks are controlled in space and time during the cell cycle is not completely understood, despite three known levels of control. First, hPlk1 has been shown to be ubiquitinated and degraded by the proteasome in mammalian cells. However, the importance and extent of conservation of this mechanism are still to be determined. For example, although the destruction box identified in hPlk1 appears to be conserved in Drosophila Polo, Polo does not disappear (or even decrease significantly in abundance) between mitosis and interphase. Moreover, even in mammalian cells, a substantial fraction of the hPlk1 pool remains in G1. Second, human Plk1 and Xenopus Plx1 are activated in mitosis by phosphorylation at a threonine residue in the T-loop (T210 in hPlk1, T201 in Plx1). As this site is conserved, this likely acts as a universal activation mechanism. In human cells, Aurora A and its adaptor protein Bora have been shown to activate hPlk1 by phosphorylation at T210. A third control mechanism lies in docking of Plks to prephosphorylated targets using the PBD. This provides an efficient way to target Plks to their mitotic targets. It has also been shown that PBD-dependent sequestration of Polo to a strong binding partner, a female germline-specific protein named Matrimony (Mtrm), can keep this kinase inactive in meiotic prophase I (Xiang, 2007). Polo later overcomes this inhibition, and is thought to trigger germinal vesicle breakdown (Xiang, 2007). This interaction depends on phosphopriming of Mtrm. In contrast, this study describes a mechanism for regulating Polo through its cell cycle-regulated sequestration to MTs by strong binding to a MT-associated protein in a manner that is not primed by phosphorylation but nonetheless requires the PBD (Archambault, 2008).

The current results shed new light on how Drosophila Polo is regulated at mitotic entry and exit. Based on the results, a model is suggested whereby Polo is sequestered on MTs (and can be stabilized) in interphase by interacting with Map205, and where this interaction is negatively regulated by Cdk1 activity in early mitosis. Although Polo kinases have been reported to interact with tubulins in vitro, this does not appear to provide an efficient mechanism of Polo targeting to MTs, which is instead strongly dependent on Map205. The cellular concentration of Map205 is very similar or possibly higher than that of Polo, consistent with a role for Map205 in sequestering Polo. Such sequestration of Polo onto MTs could serve to store and stabilize the kinase in an innocuous location, until needed at mitotic entry (Archambault, 2008).

While phosphorylation of Map205 at S283 is sufficient to block its interaction with Polo and the localization of Polo to mitotic MTs, it is not known if this phosphorylation event is necessary for inhibiting the targeting of Polo to MTs. For example, phosphorylation of Map205 at other sites may also contribute to negatively regulate the interaction. This possibility has not yet been rigorously tested. However, the fact that overexpression of Map205-S283A in embryos enhances polo-dependent defects more strongly than overexpression of Map205-wt suggests that phosphorylation of Map205 at S283 is necessary for full inhibition of the Map205-Polo interaction in vivo (Archambault, 2008).

The single centrosome detachment from prophase nuclei observed in polo mutants is enhanced by overexpression of Map205 and suppressed by the loss of Map205 function. Centrosome detachment appeared more sensitive to a reduction in Polo activity in the preblastoderm than in the early blastoderm embryo. It is speculated that this may be due to a difference in the forces that the centrosome-nuclear envelope linkage must withstand at those different stages. The organismal and cellular phenotypes observed in polo mutants after Map205 overexpression were dependent on the ability of Map205 to interact with Polo and identical to those caused by an increase in the activity of Greatwall, a recently identified antagonist of Polo (Archambault, 2007). Importantly, the suppression of the polo-dependent centrosome-detachment phenotype by deletion of map205 is consistent with the notion that endogenous Map205 functions to regulate Polo in vivo. Sequestering of Polo by Map205 could prevent it from accessing its targets prematurely on centrosomes and kinetochores, whether or not Polo's kinase activity is inhibited at the structural level. Antibodies against Polo and Map205 did not allow visualization of the endogenous proteins in the embryo for technical reasons, but the genetic, functional, and biochemical results obtained in this system support the model presented here. Exploring what other tissues rely on this Polo-control pathway should be the subject of future studies (Archambault, 2008).

In addition, the interaction with Map205 appears to stabilize Polo. In this way, the Map205-Polo complex on MTs (or even if partly present as a cytosolic pool) could constitute a reservoir of Polo, making Polo available for rapid, Cdk1-triggered mobilization at mitotic entry, and resequestration at mitotic exit. The prevention of hPlk1 from binding PRC-1 by Cdk1-dependent phosphorylation was recently identified as a mechanism for timing hPlk1 localization to the midbody (Neef, 2007). Tbe current findings augment the general model whereby the choice of Polo kinase binding partners is controlled by Cdk1 activity during the cell cycle (Neef, 2007). The model does not exclude the possibility that Polo is targeted to MTs for another function. For example, Polo could regulate Map205. Indeed, Polo can phosphorylate Map205 in vitro, and the physiological relevance of this phosphorylation remains to be investigated. Additionally, targeting of Polo to MTs could facilitate its regulation of motor proteins or other MT-associated proteins with roles in interphase or cytokinesis (Archambault, 2008).

Orthologs of Map205 were identified in the 12 sequenced Drosophila genomes. An alignment between these protein sequences shows that the N-terminal Polo-binding region of Map205 (within amino acids 254-416 in Drosophila melanogaster) is very well conserved. Importantly, the Cdk1 phosphorylation site (S283) and the sequence immediately surrounding it (VAESPRK) are perfectly conserved between all 12 species. In fact, the Polo-binding region appears to be even better conserved than the C-terminal MT-binding region (amino acids 723-955 in D. melanogaster). This suggests that the Polo-binding function of Map205 may be at least as important as its MT-binding function, and that the molecular mechanisms reported in this study are likely to be conserved between species (Archambault, 2008).

RNAi depletion of Map205 in cultured Drosophila cells leads to perturbations in the cell cycle profile, including an increase in the percentage of cells in mitosis, and more specifically in prometaphase. This may be a consequence of the Polo destabilization, since partial loss of Polo function also results in an accumulation of cells in prometaphase. By replacing endogenous Map205 with a form of Map205 that cannot bind Polo (Map205-S283E), it was verified that the destabilization of Polo following Map205 depletion in D-Mel cells results from the loss of interaction between Polo and Map205 and not from other perturbations arising from the loss of Map205. In contrast, removal of Map205 from the early embryo did not result in a destabilization of Polo. Budding yeast Cdc5 and human Plk1, both orthologs of Polo, undergo cell cycle-regulated degradation that is thought to depend on Cdh1. If Drosophila Polo is degraded by the same pathway, the results can be explained by the absence of Fizzy related (Drosophila Cdh1) in the early embryo (Archambault, 2008).

The experiments reveal that Map205 can strongly alter the subcellular localization of Polo (by targeting Polo to MTs), stabilize Polo in D-Mel cells, and inhibit Polo's cellular activity in the embryo (probably by sequestering of the enzyme away from its substrates). Yet, despite the ability of Map205 depletion to delay progression through prometaphase in cultured cells, map205-null flies are viable and fertile, and no obvious mitotic defects are detected in their embryos or dividing neuroblasts. Because of the existence of at least three more, partially overlapping levels of control of Polo activity (proteolysis, activating phosphorylation, phosphopriming of Plk docking) it is difficult to assess the consequences of a total failure to down-regulate and stabilize Plks in interphase. Other mechanisms regulating Polo may compensate for the loss of the Map205-dependent pathway in most tissues. Indeed, one would need to combine disruptions of the multiple regulatory mechanisms for Polo or hPlk1 to determine the effects of a full failure to down-regulate or up-regulate these kinases in the cell cycle, and these experiments may become possible as the pathways controlling these kinases continue to be dissected. The existence of multiple, partially redundant control systems is seen for other cell cycle regulators with functions at key stages of cell cycle progression (for example, Cdk1, the APC, or the prereplicative complex). Such redundancy in regulatory mechanisms can provide robustness to the system that can become crucial in situations of stress (Archambault, 2008).

The Polo-Map205 interaction does not depend on priming phosphorylation. Interestingly, the PBD is essential for this interaction, but it is not sufficient, and therefore some elements of the kinase domain appear to be also needed. This is the first time that such requirements have been demonstrated for a Plk interaction in vivo. This phosphorylation-independent mode of interaction is well suited to control Polo in interphase, when cellular kinase activities are typically low. Since hPlk1, like Drosophila Polo, can also bind Map205, this mode of interaction is likely to be conserved for other Plks and could potentially involve other substrates. It was recently shown that the PBD of hPlk1 is capable of interacting with Cdc25C peptides with similar affinities for the phospho- and nonphospho forms in vitro (Garcia-Alvarez, 2007). The full biological significance of these modes of interaction and how they affect the activity of the kinase should become clearer as the molecular details of more Plk-target interactions are investigated (Archambault, 2008).

The strong interaction between hPlk1 and Map205 in vitro also raises the possibility that the type of regulatory mechanism identified in this study may be conserved beyond the Drosophila genus. There is relatively poor sequence conservation between Map205 and its human ortholog, Map4, but this is not unusual among MT-associated proteins. The relative importance of the different mechanisms for regulating the location and activity of Plks (activating phosphorylation, proteolysis, and protein binding) will almost certainly depend on the organism and may even vary between cell and tissue types. As different types of cancer cells may undergo deregulation of different hPlk1 control mechanisms, it will be of considerable interest to further explore the relative contributions of these alternative mechanisms for regulating hPlk1 in different disease situations (Archambault, 2008).

α-Endosulfine is a conserved protein required for oocyte meiotic maturation in Drosophila

Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. This study shows that Drosophila α-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. Twine and Polo kinase levels are reduced in endos mutants, and Early girl (Elgi), a predicted E3 ubiquitin ligase, was identified as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human α-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and α-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function (Von Stetina, 2008).

These studies demonstrate previously unknown roles for α-endosulfine in meiotic maturation. Endos is required to ensure normal Polo kinase levels and, perhaps indirectly, to stabilize Twine/Cdc25 phosphatase. A generalized effect of endos on protein translation or stability is unlikely, given that Cyclin B and actin protein levels are both unaffected by the loss of endos function. Owing to problems in maintaining high levels of Twine or Polo transgenes in endos mutants, however, it could not be demonstrated that the low levels of Twine and/or Polo do indeed cause the endos meiotic maturation defects. In addition, the data suggest that Endos has a separate role during meiotic maturation, through the negative regulation of Elgi. The function of α-endosulfine in meiotic maturation potentially may be conserved because ENSA, the human homolog, can efficiently rescue the endos mutant phenotype, and because α-endosulfine is expressed in mammalian oocytes. It would be interesting and informative to determine whether elimination of α-endosulfine function in the mouse germline results in similar meiotic maturation defects to those seen in Drosophila and in sterility (Von Stetina, 2008).

Levels of Cdc25 phosphatases are tightly regulated during the cell cycle by the balance of protein synthesis and degradation. Phosphorylation of Ser18 and Ser116 residues by Cyclin B/Cdk1 results in mouse Cdc25A stabilization, thereby creating a positive-feedback loop that allows Cdc25A to dephosphorylate and activate Cyclin B/Cdk1. Evidence from Xenopus studies indicates that phosphorylation and activation of Cdc25 by Polo-like kinase generate MPM2 epitopes, which reflect high Cyclin B/Cdk1 activity. Moreover, a recent study in C. elegans demonstrates a role for Polo-like kinase in meiotic maturation. It is therefore likely that the low Twine levels observed in endos mutants are an indirect consequence of reduced Polo levels, which may result in impaired Cdk1 activity. It remains a formal possibility, however, that endos regulates Twine and Polo levels independently of each other. In either case, there are clear differences between the endos and twine phenotypes: only endos mutant oocytes show a severe reduction in Polo and a slight reduction in Cdk1 levels; twine but not endos mutants show slightly elevated Cyclin B levels; the phosphorylation status of Cdk1 seems differently altered in endos (appears to be hypophosphorylated) and twine (appears to be hyperphosphorylated, as expected) mutants relative to in control oocytes; and in vitro Cdk1 activity is reduced in immunoprecipitates from twine but not endos oocytes (Von Stetina, 2008).

It is possible that the wild-type levels of in vitro phosphorylation of histone H1 of endos00003 immunoprecipitates accurately reflect Cdk1 kinase activity levels in living endos00003 oocytes, in which case it would be concluded that the reduction in Twine and Polo levels observed in endos00003 mutants is not sufficient to affect Cdk1 activity, and that the observed MPM2 epitope level reduction is simply due to low Polo levels. Another possibility is that in vitro phosphorylation of histone H1 is not reflective of the in vivo Cdk1 kinase activity levels in endos mutants. For example, Cdk1 substrate specificity may be altered in endos mutants such that endogenous substrates other than histone H1 are not properly phosphorylated, or Cdk1 kinase activity may be reduced in specific subcellular pools in these mutant oocytes, perhaps via local alterations in phosphorylation or Cyclin B levels. In fact, the spatial regulation of Cyclin B has been reported during meiosis and syncytial mitotic cycles in Drosophila (Von Stetina, 2008).

Although the Endos-Elgi interaction could not be confirmed in vivo, their strong interaction in vitro, combined with the premature meiotic maturation phenotype of elgi mutants, suggest that these genes function in the same pathway. The mammalian Elgi homolog Nrdp1 has been shown to act as an E3 ubiquitin ligase in vitro to promote degradation of the Erbb3 and Erbb4 receptor tyrosine kinases, and of the inhibitor-of-apoptosis protein BRUCE. It would be interesting to determine whether Elgi also has E3 ligase activity in flies and to identify its direct targets. Nrdp1 mRNA is expressed in multiple human tissues, including the ovary; however, a role for NRDP1 in meiotic maturation or the modulation of Cdk1 has not been examined. The strong degree of amino acid similarity between human NRDP1 and Elgi is suggestive of functional conservation (Von Stetina, 2008).

The premature entry into metaphase I observed in elgi null mutants in the absence of effects on Polo or Twine levels suggests that Endos uses a separate mechanism that involves Elgi function to control the timing of Cdk1 activation and, ultimately, that of meiotic maturation, without necessarily affecting the final levels of Cdk1 activation. The premature meiotic maturation phenotype of elgi mutants is reminiscent of the phenotype recently reported for matrimony heterozygous mutants. In these studies, Matrimony was reported to interact with Polo kinase in vivo and to function as a Polo inhibitor, with a suggested role in finely controlling the timing of meiotic maturation. One possible model to explain the premature meiotic maturation of elgi mutant oocytes is that Elgi positively regulates the interaction between Matrimony and Polo, and that Endos controls the precise timing of meiotic maturation by inhibiting this E3 ubiquitin ligase, in addition to having a key role in promoting high Polo (and Twine) protein levels. It will be very interesting to experimentally address this possibility in future studies (Von Stetina, 2008).

In addition to having key roles in meiosis, this study found that Drosophila α-endosulfine is required during early embryonic mitoses. These findings are consistent with recent studies showing, as part of a large-scale screen for genes required for mitotic spindle assembly in Drosophila S2 cells, that disruption of α-endosulfine expression by RNA interference produces defects such as chromosome misalignment and abnormal spindles. It is conceivable that α-endosulfine uses similar mechanisms in both meiosis and mitosis. Further characterization of the role of α-endosulfine in mitosis will help to address this question (Von Stetina, 2008).

Given the central role for Endos in meiotic maturation and the fact that Endos is expressed throughout oogenesis, it will next be essential to investigate how Endos activity is regulated as the oocyte develops and becomes competent to undergo meiotic maturation. Intriguingly, Endos contains a highly conserved protein kinase A (PKA) phosphorylation site. Indeed, mammalian homologs can be phosphorylated by PKA at this site, and, in vertebrate oocytes, high levels of cyclic adenosine monophosphate (cAMP) and PKA activity inhibit the resumption of meiosis by inhibiting Cyclin B/Cdk1 activity. Although the evidence suggests that PKA-dependent phosphorylation is responsible for activation of the Cdk1-inhibitory kinase Wee1 and for inactivation of the Cdk1-activating phosphatase Cdc25, it is possible that PKA has additional roles in controlling meiotic maturation, perhaps via α-endosulfine. In fact, two forms of Endos with different electrophoretic mobilities are present in Drosophila ovaries (Drummond-Barbosa, 2004), with the lower mobility form being specifically present in stage 14 oocytes. However, it remains to be determined whether these different forms of Endos are caused by phosphorylation, and, if so, what the effect of phosphorylation is on Endos activity (Von Stetina, 2008).

Finally, some of the results suggest that Endos does not regulate insulin secretion, which is different from mammalian studies that link α-endosulfine to this process. It is possible that this discrepancy results from differences in the function of α-endosulfine between species, perhaps reflecting an evolutionarily newer role of α-endosulfine in the control of insulin secretion. It is important, however, to emphasize that the role of α-endosulfine in insulin secretion has not been tested in vivo. Nevertheless, human α-endosulfine mRNA is expressed in multiples tissues, including heart, brain, lung, pancreas, kidney, liver, spleen, and skeletal muscle, and this study shows that it is also expressed in the ovary. The wide range of expression of human α-endosulfine suggests that it is likely to play multiple biological roles, perhaps including, as these studies point to, a potential role in meiotic maturation (Von Stetina, 2008).

The chromosomal passenger complex activates Polo kinase at centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. This study concerns the coordination of the activities of these two kinases in vivo. INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. This study shows that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. The results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation (Carmena, 2012).

Coordination of Polo and Aurora B activity at kinetochores is critical in early mitosis, as the two kinases play potentially antagonistic but complementary roles in regulating kinetochore-microtubule interactions. Aurora B is essential for the correction of aberrant attachments, and indeed, tethering Aurora B too close to kinetochores interferes with the formation of stable attachments. In contrast, Plk1 activity is required for initial stabilisation of microtubule attachments to kinetochores. It is suggested that interactions with INCENP may provide a mechanism to coordinate the activities of these two essential kinases during early mitosis (Carmena, 2012).

Recent studies suggest that Plk1 is activated at centrosomes when its T-loop (T210) is phosphorylated by Aurora A kinase-Bora, and that this promotes the G2/M transition upstream of Cdk1, although Polo activity is not required for mitotic entry. How Plk1 is activated at kinetochores remained an important unsolved question. The present results show that Aurora B and INCENP, which are concentrated at inner centromeres, function there to activate Polo by phosphorylating its T-loop (Carmena, 2012).

Plk1 recruitment to centromeres in late G2 has been variously proposed to be mediated by Bub1, INCENP, and BubR1. Another report implicated the self-primed interaction of Plk1 with PBIP1/CENP-U. This could potentially explain why Plk1 activity is reportedly required for its localisation to kinetochores in human cells (Carmena, 2012).

The current RNAi studies confirmed that Plk1 is partially dependent on the CPC for its centromeric localization in human cells. However, this appears not to be the case in Drosophila, where Polo is present at centromeres before NEB, at a time when INCENP is not yet concentrated at inner centromeres and before PoloT182ph, the active form of the kinase, is detected there. Indeed, no significant decrease was observed in kinetochore-associated Polo levels after INCENP RNAi in Drosophila cells (Carmena, 2012).

Although Polo targeting to kinetochores is independent of the CPC in Drosophila, its activation there does require the CPC with active Aurora B. The data suggest that INCENP binding to Polo facilitates its subsequent activation by Aurora B kinase. Indeed, INCENP and Polo interact physically in vitro and co-immunoprecipitate in mitotic cell extracts. Although most centromeric Polo kinase is concentrated in the outer kinetochore in prophase and prometaphase, active Polo (PoloT182ph) is also found in inner centromeres, where it overlaps with INCENP as confirmed by a proximity ligation assay (PLA)(Carmena, 2012).

A range of evidence presented in this study suggests that Aurora B is the upstream kinase responsible for Polo kinase activation at centromeres. Firstly, Aurora B phosphorylates Polo at Thr182 in vitro. Secondly, RNAi depletion of INCENP or Aurora B, but not Aurora A, reduces levels of active PoloT182ph at kinetochores. Thirdly, tissue culture cells and third larval instar neuroblasts treated with a specific inhibitor of Drosophila Aurora B kinase show decreased levels of PoloT182ph at kinetochores. In all of the preceding experiments, PoloT182ph levels are affected at kinetochores but not at centrosomes, where Polo is presumably activated by Aurora A. Importantly, this involvement of Aurora B in Polo activation at centromeres discovered in Drosophila is conserved for Plk1 in human cells (Carmena, 2012).

The current results suggest a model for interactions between Polo kinase and the CPC at centromeres (see Model for the interactions between the CPC and Polo kinase at the centromere/kinetochore). In Drosophila cells, Polo targets to centromeres before the CPC is recruited by Survivin binding to histone H3T3ph (Yamagishi, 2010: see Schematic depiction of the pathways that regulate CPC targeting to centromeres). At inner centromeres of chromosomes whose kinetochores are not under tension, Polo now binds to INCENP. This promotes Polo kinase activation, as Aurora B phosphorylates PoloT182. It is suggested that interactions with INCENP link the two complementary kinase activities, thereby potentially creating a microtubule attachment/detachment cycle at kinetochores. Such a cycle would not be possible without a balancing phosphatase activity, and PP2A-B56 has recently been shown to oppose both Aurora B and Plk1 activities at kinetochores to promote stable attachments (Carmena, 2012).

At metaphase, when chromosomes are bioriented and under tension, the CPC and Polo kinase exhibit only a partial overlap. A weakening of the INCENP/Polo PLA signals in metaphase suggests that Polo may be released from INCENP after its activation—possibly moving to the outer kinetochore. During metaphase, the CPC localizes in the inner centromere, stretching between sister kinetochores, whereas Polo and PoloT182ph concentrate mainly at the kinetochores. This separation may be necessary to allow Polo-mediated stabilisation of kinetochore-microtubule attachments. The coordinated activities of both kinases at kinetochores and their tension-mediated separation might facilitate a dynamic equilibrium between attached and unattached kinetochores, selectively stabilizing proper chromosome attachments (Carmena, 2012).

In summary, the results reveal that INCENP and Aurora B are responsible for Polo kinase activation at centromeres but not at centrosomes during mitosis. These experiments support the hypothesis that INCENP acts as a scaffold integrating the cross-talk between these two important mitotic kinases (Carmena, 2012).

Role of Survivin in cytokinesis revealed by a separation-of-function allele

The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. This study identified a missense allele of Drosophila Survivin (FlyBase name: Deterin) that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this 'separation-of-function' allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems (Szafer-Glusman, 2011).

The chromosomal passenger complex (CPC), composed of the Ser-Thr kinase Aurora B and three partner proteins, plays several key roles in mitosis and meiosis, including regulation of attachment of kinetochores to microtubules, the spindle checkpoint that delays anaphase onset until all chromosomes are under tension on the spindle, regulation of sister chromatid cohesion, and cytokinesis. To accomplish these different tasks, the Aurora B kinase must be exquisitely localized in space and regulated in time. During mitosis, Aurora B associates with the microtubule-binding protein Inner Centromere Protein (INCENP), Borealin/DASRA/CSC-1, and the small, multifunctional BIR-motif protein Survivin to form the CPC (Szafer-Glusman, 2011).

Dissecting the role of individual CPC components has been hampered by the extraordinary interdependence of the four subunits; depletion of any single CPC protein by RNA interference knockdown in human cells affected the structural unit, localization, and function of the CPC. The structural basis of this interdependence is evident in the crystal structure of the Survivin-Borealin-INCENP core of the CPC complex, in which Borealin and INCENP associate with the C-terminal helical domain of Survivin to form a tight three-helix bundle (Szafer-Glusman, 2011 and references therein).

Strict localization of Aurora B by the CPC ensures that this kinase, which has multiple substrates, phosphorylates the correct targets at the proper points in cell cycle progression. Concentrated on chromosomes from G2, then at inner centromeres from prometaphase until the metaphase-to-anaphase transition, the CPC is required to regulate chromosome condensation, spindle formation and dynamics, kinetochore maturation, kinetochore-microtubule interaction, correct chromosome alignment, and control of the spindle checkpoint. At anaphase onset the CPC then translocates to the central spindle (CS) midzone and equatorial cortex and is involved in CS formation. At the CS Aurora B phosphorylates the centralspindlin components, Pav/mitotic kinesin-like protein 1 (MKLP1)/Zen-4 and the RacGAP50/MgcRacGAP/Cyc-4. However, the mechanisms that target the CPC to the spindle midzone and equatorial cortex after onset of anaphase and the mechanisms by which the CPC regulates central spindle formation and cytokinesis are not understood. In addition, the requirements for CPC function for critical events in metaphase and at the metaphase-anaphase transition have complicated analysis of how the CPC is localized and functions at later stages for cytokinesis (Szafer-Glusman, 2011).

This study characterized the role of Drosophila Survivin (dSurvivin), previously termed deterin and analyzed in its antiapoptotic activity, as a regulator of cell division, identifying a missense mutation (scapolo) in the Drosophila Survivin BIR domain that allows recruitment and function of the CPC in metaphase but disrupts CPC localization and function in anaphase and telophase. The findings reveal that Survivin plays a role in targeting the CPC and centralspindlin to the central spindle and the equatorial cell cortex during anaphase. In spermatocytes, Survivin function is also required to localize Polo and localize the small GTPase RhoA to set up the contractile ring machinery at the onset of cytokinesis. In larval neuroblasts undergoing mitotic division, however, the scapolo mutant did not block initial accumulation of Rho to a band at the equatorial cortex, although it did cause failure of cytokinesis. The different requirements for Survivin function for equatorial accumulation of Rho in spermatocytes versus neuroblasts may reflect a fundamental difference in the series of steps that lead to formation of the contractile ring in these two cell types (Szafer-Glusman, 2011).

A missense mutation leading to substitution of serine for the wild-type Pro-86 of Drosophila Survivin uncouples the function of Survivin in metaphase from function during anaphase and telophase, indicating a direct requirement for Survivin and the chromosomal passenger complex in orchestrating the profound reorganization of the cortical cytoskeleton at the cell equator at the onset of cytokinesis. This 'separation-of-function' allele allowed analysis of Survivin and CPC function during cytokinesis, which is normally obscured by the better-known roles of the CPC at centromeres during metaphase, when it facilitates alignment of chromosomes to the spindle equator and mediates the spindle checkpoint. The finding that a point mutation in the BIR domain disrupts activity of Survivin during cytokinesis challenges the model that the C-terminal domain of Survivin is sufficient for cytokinesis function (Lens, 2006) and indicates that residues in the BIR domain are important for localization and activity of Survivin at the central spindle (Szafer-Glusman, 2011).

Survivin associates with kinetochores and the central spindle with different dynamics, being highly mobile in prometaphase and metaphase and strongly immobile at the anaphase central spindle. This change in dynamics may underlie the largely normal localization and function of scapolo (scpo) (a missense allele of the Drosophila Survivin) at metaphase but the fully penetrant effect on assembly of the F-actin contractile ring and cytokinesis observed in scpo mutants (Giansanti, 2004; Szafer-Glusman, 2011).

Cytokinesis depends on the assembly of an equatorial actomyosin ring regulated by local activation of the small GTPase RhoA at the cortex, in turn catalyzed by the RhoGEF Ect2/Pebble. It has been proposed that association of RhoGEF/Pebble with centralspindlin promotes local RhoA activation at the cortex. In addition, the kinase polo (PLK1) has been implicated in RhoGEF localization and Rho activation, at least in part by phosphorylation of the centralspindlin component MgcRacGAP. The current observations that the Drosophila RhoA homologue, Rho1, failed to accumulate at the equatorial cortex in scpo mutant spermatocytes implicate Survivin and the CPC in the mechanism(s) that localize and activate RhoA at the equatorial cortex in these cells. This requirement may in part act through effects on Polo kinase. Failure to localize Polo to the central spindle in scpo mutant spermatocytes could prevent localization of RhoGEF by the centralspindlin complex and the consequent activation of Rho at the cortex. In this model, failure to localize Polo may contribute to the failure to form an equatorial ring of localized Rho1 and, in consequence, the inability to form a localized ring of myosin regulatory light chain and F-actin in scpo mutant male germ cells undergoing meiotic division. This mechanism may also explain the failure to maintain pole-to-equatorial microtubules observed in scpo mutant spermatocytes. It is likely that Rho-mediated activation of the Formin Dia helps stabilize microtubule arrays at the equatorial cortex of dividing cells, as active Rho and Formin (mDia) have been shown to regulate stabilization of microtubule arrays at the cortex of migrating fibroblasts. Consistent with this model, this study found that microtubules reached the plasma membrane at the equator of scpo dividing spermatocytes, but the bundles are transient and fail to form stable arrays at the cortex (Szafer-Glusman, 2011).

A striking finding of this work is the difference in requirement for Survivin function for localization of the Polo kinase and RhoA in anaphase neuroblasts versus spermatocytes. This difference raises two possibilities: either Survivin is not part of a universal signaling mechanism that directs cytokinesis, or different semiredundant mechanisms can drive cytokinesis, similar to redundancy between astral pulling and sliding of central spindle microtubules for anaphase B, and different cell types rely more strongly on one mechanism or the other. Indeed, consistent with the latter possibility, spermatocytes and neuroblasts display different cytoskeletal architectures during cytokinesis (Giansanti, 2006). In neuroblasts, actomyosin initially accumulates in a broad cortical band, presumably because this is the region of the cell cortex that escapes repression of Rho associated with the plus ends of astral microtubule. This initial wide band gradually narrows into a tight equatorial ring as the cell progresses into telophase. Thus assembly of the contractile apparatus in neuroblasts proceeds, as proposed for Caenorhabditis elegans embryos, in 'two genetically separable steps' in which localization of contractile machinery is initially independent of the central spindle. In support of this model, this study found that Rho1 accumulates in a broad cortical band in scpo mutant neuroblasts, suggesting that the first stage can occur independent of Survivin and CPC localization to the central spindle (Szafer-Glusman, 2011).

Spermatocytes, in contrast, do not form an initial wide equatorial band of contractile ring components. Instead, from their first appearance in early anaphase, the actomyosin rings in spermatocytes are tightly focused at the cell equator). It is speculated that this restricted initial localization of contractile ring components and the apparent lack of a preceding wide equatorial band may be a consequence of a more stringent global block to Rho1 activation at the cortex in spermatocytes than in neuroblasts. It is proposed that this global block is eventually overridden by positive regulation of Rho1 by local concentration of RhoGEF, in turn facilitated by CPC-dependent events associated with and/or localized by central spindle microtubules. Rho1 activation would then occur within a narrow peak exactly at the site where pole-to-equator microtubules interact to maximize RhoGEF deposition/concentration at the cortex. Indeed, F-actin ring assembly occurs locally and cytokinesis initiates immediately after the pole-to-equator microtubules contact the cortex in Drosophila spermatocytes. It is proposed that, according to this model, the defects in Survivin lead to lack of CPC activity and abnormal centralspindlin, resulting in absence of Rho1 and Polo kinase from the equator of scpo mutant spermatocytes (Szafer-Glusman, 2011).

In neuroblasts, where a more permissive cortex allows a broad belt of Rho1 activation at the cell equator, Survivin and CPC appear to promote gradual convergence of the initial broad band into a narrow ring centered at the maximum of RhoGEF activity at the cortex. In scpo mutants, which display irregular anaphase central spindles devoid of Pav, the broad Rho1 cortical band fails to narrow, the cells fail to form a focused, narrow ring of myosin, and cell division proceeds with inefficient and incomplete constriction (Szafer-Glusman, 2011).

A key difference between neuroblasts and spermatocytes that may account, at least in part, for the differences in behavior of Rho1 and myosin complex proteins is in the relationship between Polo kinase and the CPC observed in scpo mutant mitotic versus male meiotic cells. In spermatocytes, Polo and the CPC are interdependent and Polo colocalizes with the CPC along its full journey from metaphase through anaphase and telophase. In neuroblasts, in contrast, Polo localization during cytokinesis appears to be independent of the CPC and centralspindlin, at least at early stages of cell division, but Polo appears to colocalize with Feo, the Drosophila homolog of PRC1, that required for central-spindle formation and cytokinesis. A second difference between neuroblasts and spermatocytes may be the recently described, spindle-independent backup system that can localize myosin to a broad band at the cell cortex near the future cleavage plane under control of the neuroblast cell polarity system. The broad localization of myosin to the cell cortex observed in ana/telophase neuroblasts in scpo mutants may be in part due to these redundant mechanisms (Szafer-Glusman, 2011).

Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts

During interphase in Drosophila neuroblasts, the Centrobin (Cnb)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. This study shows that daughter centrioles depleted of CNB cannot fulfill this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include gamma-Tub, Ana2, Cnn, Sas-4, Asl, DGRIP71, Polo and Sas-6. Following chemical inhibition of Polo or removal of three Polo phosphorylation sites present in Cnb, the interphase microtubule aster is lost. These results demonstrate that centriolar Cnb localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for Polo in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation (Januschke, 2013).

Since first described in germline stem cells, unequal mother-daughter centriole behaviour has become a major issue in stem cell self-renewing asymmetric division that has now been reported to occur in mice and in other cell types in Drosophila melanogaster including neuroblasts. Drosophila neuroblasts are neural precursors that generate the flys central nervous system through repeated rounds of self-renewing asymmetric divisions. Neuroblast asymmetric division is driven by the polarized localization of protein complexes at the apical and basal cortex and by the orientation of the mitotic spindle along the apico-basal axis. As a result, the apical and basal sides of the cortex are cleaved apart into the renewed neuroblast and the smaller differentiating ganglion mother cell (GMC), respectively. Dysfunction of several of the molecules involved in these processes results in tumour growth (Januschke, 2013).

Neuroblast cortical polarity and mitotic spindle alignment are tightly linked to the centrosome cycle of these cells in which centriole splitting is a very early interphase event and mother and daughter centrosomes exhibit significant differences in structure, function and fate. In Drosophila neuroblasts, the mother centriole, which has no significant microtubule organizing activity and migrates extensively during interphase, is inherited by the GMC after mitosis. In contrast, the daughter centriole, which retains high microtubule organizing activity and remains rather stable near the apical cell cortex throughout interphase, is retained by the neuroblast at mitosis. Thus, unlike most Drosophila cells where centrosomes are rather feeble microtubule organizing centres (MTOCs) during interphase, Drosophila neuroblasts posses a prominent interphase microtubule aster that is organized by the cortex-bound daughter centriole. Drosophila centrioles do not seem to have age-dependent ultrastructural attributes such as the satellites and appendages observed in vertebrates. However, Drosophila CNB, similarly to its homologues in mouse and human cells, binds only to the daughter centriole, revealing a molecular dimorphism that could contribute to daughter-centriole-specific behaviour in Drosophila larval neuroblasts (Januschke, 2013).

This study shows that in Drosophila neuroblasts, daughter centrioles without CNB behave like mother centrioles, and mother centrioles with ectopically localized CNB behave like daughter centrioles. Moreover, by co-immunoprecipitation and tandem mass-spectroscopy (MS/MS) this study shows that CNB co-precipitates with γ-Tub, Ana2, Cnn, Sas-4, Asl, Asl, DGRIP71, Polo and Sas-6. These centriolar and PCM proteins are part of a well-characterized network that drives PCM accumulation during mitotic centrosome maturation (see Gopalakrishnan, 2011). This study also shows that CNB phosphorylation by Polo is essential to maintain the interphase aster, hence revealing a requirement for Polo function during interphase (Januschke, 2013).

In neuroblasts expressing YFP-CNB-PACT (The PACT domain is a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin), both mother and daughter centrioles carry CNB and behave as daughter centrioles do in wild-type neuroblasts (see Centrobin function and graphical summary). Interestingly, ectopic activation of the interphase PCM retention program on both centrioles forces a canonical centrosome cycle in these cells whereby one centrosome-aster complex splits in two that segregate away from each other. In these cells, spindle assembly frequently occurs at an angle with respect to the apico-basal axis, but eventually rotates to align with it as in delaminating neuroblasts in the Drosophila embryo. In neuroblasts mutant for Cnb or expressing YPF-CNBT4A T9A S82A, the two centrioles behave as mother centrioles do in wild-type neuroblasts; they retain little or no PCM during interphase, but centrosomes mature at mitosis entry. In all three cases the spindle aligns with the apico-basal axis. CNB depletion, similarly to CNB ectopic localization on the two centrioles, randomizes age-dependent centriole fate. Apico-basal orientation can change from one cycle to the next in CNB-mutant cells, but not in neuroblasts that express CNB-PACT, strongly suggesting that the orientation of cortical polarity is critically dependent on daughter centriole behaviour and that whether this is provided by one or two centrioles seems to be irrelevant as long as a cortex-bound microtubule network is maintained during interphase (Januschke, 2013).

The results show that interphase PCM retention in neuroblasts requires Polo to act on CNB-decorated centrioles to mediate Cnn and PCM recruitment. Which centriole and where exactly on the centriole CNB is positioned do not seem to be essential because ectopic localization of CNB by the PACT domain works just as well. Indeed, the interaction of CNB with proteins such as Ana2 and Sas6 that are essential to build the centriolar cartwheel, and with Cnn and Polo that are key regulators of PCM assembly, strongly suggests that CNB might serve its function by providing a critical bridge between centrioles and PCM. It also suggests that the neuroblast and daughter-centriole-specific process of PCM retention by the CNB-containing centriole has co-opted at least some of the proteins that drive PCM enrichment on centrosomes during mitosis in many cell types. Nevertheless, the fact that mitotic centrosome maturation does not require CNB and is hardly affected by 20 nM of the Polo kinase inhibitor BI2536 strongly suggests that important differences apply between interphase PCM retention in Drosophila neuroblasts and the ubiquitous process of mitotic centrosome maturation (Januschke, 2013).

Interphase PCM retention by CNB-containing centrioles also requires Pins, but seems to require neither Mud, nor the Khc-73/Dlg pathway. Centriole migration to the cortex shortly after mitosis in neuroblasts mutant for pins suggests that Pins function is required at a later stage. Such a function is unlikely to require cortical Pins because CNB-decorated centrioles detached from the cortex by microtubule poisons efficiently retain PCM and, indeed, no evidence of Pins cortical localization during interphase has been reported in Drosophila larval neuroblasts. The seemingly normal mitotic centrosome maturation in >pins-mutant neuroblasts further substantiates that despite shared molecular factors, important differences apply between interphase PCM retention in Drosophila neuroblasts and mitotic centrosome maturation (Januschke, 2013).

Neither cell size asymmetry nor Mira cortical polarization seem perturbed by CNB depletion or by CNB ectopic localization in both centrioles. However, given the extraordinary complexity of cell fate determination, which is only partially understood, these observations cannot substantiate any solid conclusion on the effect that CNB dysfunction might have on cell fate determination and brain development. CNB is a ubiquitous protein that is one of the two centrioles in different cell types in Drosophila. This study also found that some CNB allelic combinations are lethal, which strongly suggests that CNB might have functions in other tissues. In vertebrates, Centrobin has been reported to bind to the tumour suppressor BRCA2, and some common genetic variants of human Centrobin have been associated with breast cancer susceptibility (Januschke, 2013 and references therein).

A recent study has revealed that in human neuroblastoma cell lines, the sister cell that retains the NuMA crescent, which is thought to have more self-renewal potential, frequently inherits the daughter centrosome. These results suggest the tantalizing possibility that the age-dependent centrosome segregation demonstrated in Drosophila neuroblasts might also occur in mammals and may have relevance in human disease (Januschke, 2013).

PLP inhibits the activity of interphase centrosomes to ensure their proper segregation in stem cells

Centrosomes determine the mitotic axis of asymmetrically dividing stem cells. Several studies have shown that the centrosomes of the Drosophila melanogaster central brain neural stem cells are themselves asymmetric, organizing varying levels of pericentriolar material and microtubules. This asymmetry produces one active and one inactive centrosome during interphase. This study identifies pericentrin-like protein (PLP or cp309) as a negative regulator of centrosome maturation and activity. PLP is enriched on the inactive interphase centrosome, where it blocks recruitment of the master regulator of centrosome maturation, Polo kinase. Furthermore, it was found that ectopic Centrobin expression influenced PLP levels on the basal centrosome, suggesting it may normally function to regulate PLP. Finally, it is concluded that, although asymmetric centrosome maturation is not required for asymmetric cell division, it is required for proper centrosome segregation to the two daughter cells (Lerit, 2013).

A model is presented in which PLP functions as a negative regulator of centrosome maturation in interphase NBs by preventing the localization of Polo to the basal centrosome. Loss of PLP leads to the activation of both centrosomes in interphase and greatly reduces their mobility, resulting in the atypical apical positioning and inheritance of both centrosomes. Temperature shift experiments illustrate that plp- mutants are sensitized to increased centrosome segregation and mitotic spindle defects. It is concluded that inhibition of basal/mother centrosome maturation during interphase is critical for proper centrosome partitioning and mitotic spindle organization in neural stem cells (Lerit, 2013).

It is unknown how PLP functions as a negative regulator, but one model may involve an indirect role for PLP. For example, PLP might normally promote early centriole separation to ensure efficient movement of the mother centriole away from the apical cortex to allow for its inactivation. Two experiments were performed to test this model. First, the timing of centriole separation was analyzed in cells exiting mitosis in WT and plp- NBs, and it was found that centriole disengagement and the earliest signs of independent centriole movement are unaffected. Therefore, centrosomes remain apically positioned in early interphase in both WT and plp- NBs. Next, whether apical positioning of centrioles is sufficient for their activation was tested. Early interphase WT NBs were analyzed that contained two apical centrioles (<30o separation) and no correlation was found between centrosome position and activity. Collectively, these observations argue that PLP does not influence early centrosome separation and that centrosome location is not critical for mother centrosome inactivation (Lerit, 2013).

Instead, the alternative hypothesis is favored that PLP directly regulates centrosome activity, which, in turn, influences centrosome position. One possible model is that PLP acts on the centrosome by physically shielding the docking of promaturation factors until mitotic entry. A biochemical modification and/or conformational change would then allow PLP to transition to a positive regulator in mitosis that scaffolds other PCM factors. Another possible direct function for PLP might be to differentially modulate the behavior of proteins at the apical and basal centrosomes. It will be critical to examine how the dynamics of other centrosome proteins are affected by the loss of PLP. Given the importance of interphase centrosome asymmetry in stem cell division, it is believed that an in depth understanding of the interplay between PCM proteins in both interphase and mitosis is a critical area of future research (Lerit, 2013).

The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts

Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates and Drosophila neuroblasts, the neural precursors of the fly's central nervous system, contain molecularly and physically asymmetric centrosomes, established through differences in pericentriolar matrix (PCM) retention. For instance, the daughter centriole maintains PCM and thus microtubule-organizing center (MTOC) activity through Polo-mediated phosphorylation of Centrobin (Cnb). The mother centriole, however, quickly downregulates PCM and moves away from the apical cortex, randomly migrating through the cytoplasm until maturation sets in at prophase. How PCM downregulation is molecularly controlled is currently unknown, but it involves Pericentrin (PCNT)-like protein (plp) to prevent premature Polo localization and thus MTOC activity. This study reports that the centriolar protein bld10, the fly ortholog of Cep135, is required to establish centrosome asymmetry in Drosophila neuroblasts through shedding of Polo from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM, generating two active, improperly positioned MTOCs. Failure to shed Polo and PCM causes spindle alignment and centrosome segregation defects, resulting in neuroblasts incorrectly retaining the older mother centrosome. Since Cep135 is implicated in primary microcephaly, it is speculated that perturbed centrosome asymmetry could contribute to this rare neurodevelopmental disease (Singh, 2014).

In a gene candidate approach to identify molecules required for centrosome asymmetry in Drosophila neuroblasts, this study identified bld10/Cep135 as a potential centrosome dematuration regulator. bld10 is a ubiquitous centriolar protein, localizing to centrioles in Drosophila larval neuroblasts and other cell types. To investigate centrosome asymmetry, live imaging experiments were performed in intact third-instar larval brains, labeling centrosomes with the centriolar markers DSas6::GFP or DSas4::GFP and mCherry::Jupiter. jupiter encodes for a microtubule binding protein, sharing properties with several structural microtubule-associated proteins (MAPs), and is ideally suited to visualize microtubule dynamics and microtubule-organizing center (MTOC) activity. In agreement with previous findings, it was found that wild-type (WT) interphase neuroblasts contained one apical MTOC only. The second MTOC appeared during prophase in close proximity to the basal cortex. By prometaphase, both MTOCs reached maximal activity and intensity. However, in bld10 mutant interphase neuroblasts (bld10c04199/Df(3L)Brd15, two centrosomes of similar size and MTOC activity were observed close together on the apical cortex. The two centrosomes progressively separated from each other until they reached their respective positions on the apical and basal cortex by prometaphase. Thus, in contrast to the wild-type, bld10 mutant neuroblasts show symmetric centrosome behavior. bld10's centrosome asymmetry defect could be rescued with bld10::GFP, and immunohistochemistry experiments confirmed the live imaging results (Singh, 2014).

The bld10c04199 allele is predicted to produce a truncated protein, retaining bld10's N terminus. A new N-terminal deletion allele (bld10ΔN was generated that showed the same centrosome asymmetry phenotype. In addition, neuroblasts were found containing monopolar and multipolar spindles, which are not observed with the bld10c04199 allele. This suggests that bld10c04199 is a separation-of-function allele, specifically disrupting centrosome asymmetry. Unless otherwise noted, all of the experiments described in the following sections were performed with the bld10c04199 allele (Singh, 2014).

The lack of centrosome asymmetry in bld10 mutant neuroblasts could be due to aberrant centriole migration. For example, the mother centriole could either fail to migrate through the cytoplasm or migrates back to the apical cortex to mature. This hypothesis was tested, measuring centriole migration as a function of time, and it was observed that centriolar migration in wild-type and bld10 mutant neuroblasts occur in two distinct phases: (1) centrioles steadily separated from each other, followed by (2) a sudden increase in intercentriolar distance, which peaked when centrioles reached a separation distance of ~4-6 μm in the wild-type and bld10 mutants. Centrioles in bld10 mutants did not require more time to reach this threshold distance and did not return to the apical cortex to mature. It is concluded that bld10's centrosome asymmetry defect is not due to aberrant centriole migration (Singh, 2014).

To get mechanistic insight into the bld10 phenotype, live imaging was used to measure the dynamic localization of three GFP-tagged pericentriolar matrix (PCM) markers: γ-tubulin (γ-Tub), Mini spindles (Msps; CKAP5 in vertebrates) and centrosomin (Cnn; CDK5Rap2 in vertebrates). Wild-type neuroblasts showed robust localization of γ-Tub, Msps, and Cnn to the apical centrosome during interphase. After centrosome splitting, all three PCM markers were downregulated from the basal centrosome (shedding phase) but reaccumulated during prophase (maturation phase. bld10 mutant neuroblasts also correctly localized γ-Tub, Msps, and Cnn to the apical interphase centrosome. However, similar to the MTOC marker Jupiter, γ-Tub, Msps, and Cnn were not downregulated from the separating centriole. Centrosome size was measured and a centrosome asymmetry index was plotted, starting at centrosome splitting until metaphase. Wild-type centrosomes developed a clear size asymmetry during the shedding phase and reduced it during the maturation phase. bld10 mutant centrosomes stayed similar in size, manifested in an asymmetry index below 1.5. Centrosome size and intensity measurements also revealed that in most wild-type neuroblasts, γ-Tub, Msps, and Cnn were removed from the basal centrosome ~15 min after centrosome splitting. Basal wild-type centrosomes were essentially devoid of γ-Tub after that time, whereas bld10 mutants contained equal amounts of this PCM marker. Changes in centrosome size were further compared, and it was found that wild-type apical centrosomes predominantly grew, whereas basal centrosomes increased (maturation phase) and decreased (shedding phase) their size to almost the same extent. bld10 mutant centrosomes were able to enlarge but showed very little size reduction, comparable to apical wild-type centrosomes. It is concluded that bld10 mutant centrosomes are able to mature but fail to downregulate the PCM markers γ-Tub, Msps, and Cnn (Singh, 2014).

The results suggest two possible mechanisms for centrosome asymmetry: (1) bld10 could prevent premature mother centrosome maturation by blocking the precocious accumulation of PCM proteins. (2) Alternatively, bld10 could promote PCM shedding right after centrosomes separate, thereby preventing the basal mother centrosome to prematurely become an MTOC. An in vivo pulse-chase labeling experiment was devised to distinguish between these two possibilities. To this end, Cnn at its endogenous locus was tagged with the photoconvertable fluorescent protein mDendra2. If bld10 blocks premature PCM accumulation, mother centrioles should quickly shed photoconverted Cnn and prematurely reaccumulate unconverted Cnn in bld10 mutants. Vice versa, if PCM shedding is compromised, it should be possible to follow the photoconverted centrosomes from the moment they separate until telophase. It was found that apical wild-type daughter centrioles retained the majority of photoconverted Cnn::mDendra2 from early interphase until prophase (possibly longer), indicating that very little Cnn protein gets exchanged. The basal mother centriole, however, lost photoconverted Cnn::mDendra2 within approximately 10-15 min after centriole separation, confirming that Cnn is shed quickly. Interestingly, bld10 centrioles retained photoconverted Cnn::mDendra2 for at least 45 min after separation. In some cases, one of the centrioles decorated with photoconverted Cnn::mDendra2 was even inherited by the newly formed GMC. It is conclude that (1) on the apical centrosome, Cnn protein turnover is absent or significantly reduced during interphase, that (2) on the basal centrosome, Cnn is shed quickly and replaced with new Cnn when maturation sets in, and that (3) bld10's centrosome asymmetry defect is not due to premature centrosome maturation. Instead, separating basal centrioles fail to shed Cnn in particular and possibly PCM proteins in general (Singh, 2014).

To elucidate the molecular mechanism underlying PCM shedding, the relationship was analyzed between bld10, Centrobin (Cnb), and Pericentrin (PCNT)-like protein (plp). Recently, it was shown that Cnb is necessary and sufficient for PCM retention on the apical daughter neuroblast centrosome. However, gain- and loss-of-function experiments with Cnb did not perturb bld10's localization. Similarly, as in the wild-type, Cnb was localized asymmetrically in bld10 mutants. plp mutants fail to downregulate γ-Tub on the mother centrosome. In plp mutant neuroblasts, the basal centrosome retained Cnn and MTOC activity during interphase. Interestingly, photoconversion experiments showed that similar to bld10, Cnn shedding from the basal centrosome was compromised in plp mutants. However, plp localization is not perturbed in bld10 mutant neuroblasts, and bld10 was normally localized in plp mutants. Knockdown of plp in bld10 mutants did not enhance bld10 PCM shedding phenotype, but due to the occurrence of additional phenotypes (fragmented or multiple centrosomes), the shedding phenotype could also be partially masked. In sum, it is concluded that bld10 is regulating centrosome asymmetry independently of Cnb and that plp is also required to shed Cnn (Singh, 2014).

Since the mitotic kinase Polo has been implicated in PCM retention during interphase, Polo localization dynamics were analyzed in wild-type and bld10 mutant neuroblasts. Recently, it was reported that Polo localizes to the apical centrosome during interphase and is only detectable at the basal centrosome during prophase, when maturation sets in. A Polo::GFP protein trap line was used and it was confirmed that Polo is stably localized to the apical interphase centrosome. Surprisingly, weak Polo was also found on the separating mother centrosome. Subsequently, Polo disappeared from the basal mother centriole within 10 min, comparable to Cnn, γ-Tub, and Msps shedding times. With a genomic Polo::GFP transgene, showing lower fluorescence intensity, Polo was found to be localized on both centrosomes in bld10 mutants. These data suggest that in wild-type neuroblasts, Polo is not just recruited onto the basal mother centrosome by prophase as previously reported, but is also subject to shedding during interphase. Polo is required for PCM retention since in bld10 mutant neuroblasts treated with the Polo inhibitor BI2536, both centrosomes lose MTOC activity. Thus, it is concluded that shedding of Polo is a requirement for the subsequent shedding of Cnn, γ-Tub, and Msps, enabling basal mother centrosome dematuration and the establishment of centrosome asymmetry (Singh, 2014).

Finally, the consequences were analyzed of disrupted centrosome asymmetry. The daughter centriole was labeled with Cnb::YFP ] and centrosome segregation was assayed. It was confirmed that wild-type neuroblasts faithfully retain the Cnb* daughter centriole, whereas the Cnb- centrosome segregates into the GMC. bld10 mutants showed correct asymmetric Cnb localization, but ~45% of bld10 mutant neuroblasts wrongly retained the mother centriole and segregated the daughter centriole into the GMC. Cnb+ centrosomes are usually bigger in wild-type and bld10 mutant neuroblasts, but centrosome segregation is independent of MTOC activity and size since bld10 mutant neuroblasts often retained the smaller centrosome. It is concluded that centrosome asymmetry is required for faithful centrosome segregation (Singh, 2014).

Since bld10 mutant neuroblasts have mispositioned MTOCs in relation to the apical-basal division axis, spindle orientation was analyzed. Immunohistochemistry experiments showed that bld10 mutant neuroblast spindles deviate from the regular orientation range, with isolated cases of extreme misalignment. Metaphase spindles, aligned orthogonally to the apical-basal polarity axis, can induce symmetric neuroblast divisions, resulting in an increase of the neural stem cell pool. However, neuroblast numbers were unchanged in bld10 mutants compared to control brains, and symmetric neuroblast divisions were not found with live imaging. Instead, time-lapse experiments showed that bld10 mutant centrosomes prematurely formed misaligned bipolar spindles. Spindle rotation during metaphase corrected this misalignment. Apical-basal polarity is a prerequisite for correct spindle orientation, but apical and basal polarity markers localized normally in bld10 mutants. Similarly, the spindle orientation regulators, Partner of Inscuteable (Pins; LGN/AGS3 in vertebrates) and the NuMA ortholog Mud, were also correctly localized. It is concluded that controlled PCM shedding and maturation is required for correct centrosome positioning but backup mechanisms exist, correcting for misaligned metaphase spindles (Singh, 2014).

Many cell types, including stem and progenitor cells, contain asymmetric centrosomes and segregate them nonrandomly, suggesting a connection between centrosome asymmetry and cell fate. How centrosome asymmetry is regulated is currently not understood, but centrosome dematuration is a critical step in establishing centrosome asymmetry. It was found that the centriolar protein bld10/Cep135, known as a centriole duplication and elongation factor, is required to establish centrosome asymmetry. On the basis of these data, it is proposed that plp and bld10 induce Polo's removal from the mother centriole, triggering the shedding of PCM proteins such as Cnn, γ-Tub, and Msps. Polo has been reported to be closely associated with centrioles, ideally positioned to phosphorylate both centriolar and PCM proteins. Thus, it is proposed that Polo-mediated phosphorylation of PCM proteins maintains a stable interaction between the centriole and surrounding PCM (Singh, 2014).

How Polo localization is regulated is currently not known, and no direct molecular interaction was detected between bld10 and Polo. Although no centriolar markers were found to be mislocalized inbld10 mutants (at the resolution level of confocal microscopy), it is possible that structural centriole defects, as detected in bld10 mutant spermatocytes and wing disc cells, could affect PCM turnover rates. However, since bld10 is not asymmetrically localized, it is difficult to conceive how such defects specifically compromise the behavior of the mother but not the daughter centrosome (Singh, 2014).

Although perturbed centrosome asymmetry does not seem to undermine neuroblast polarity, the cell cycle, or physical and molecular asymmetric cell division, the possibility cannot be excluded that centrosome asymmetry could have long-term consequences currently beyond the ability to detect. Interestingly, defects in centrosome maturation or mutations in Cep135 can cause neurodevelopmental disorders such as primary microcephaly. It will be interesting to address the question whether lack of Cep135 is causing microcephaly due to compromised centrosome asymmetry and dematuration (Singh, 2014).

Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. Previous work has shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. This report shows that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. These results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis (Carmena, 2014).

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

Drosophila Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. This study shows that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks (Kachaner, 2014).

An amino-terminal Polo kinase interaction motif acts in the regulation of centrosome formation and reveals a novel function for centrosomin (cnn) in Drosophila

The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. A Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis (Eisman, 2015).

The End of a Monolith: Deconstructing the Cnn-Polo interaction

In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. A recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at two residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the two phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic (Eisman, 2016).

Phospho-Pon binding-mediated fine-tuning of Plk1 activity

In Drosophila neuroblasts (NBs), the asymmetrical localization and segregation of the cell-fate determinant Numb are regulated by its adaptor Partner of Numb (Pon) and the cell-cycle kinase Polo. Polo phosphorylates the Pon localization domain, thus leading to its basal distribution together with Numb, albeit through an unclear mechanism. This study finds that Cdk1 phosphorylates Pon at Thr63, thus creating a docking site for the Polo-box domain (PBD) of Polo-like kinase 1 (Plk1). The crystal structure of the Plk1 PBD/phospho-Pon complex reveals that two phospho-Pon bound PBDs associate to form a dimer of dimers. Evidence is provided that phospho-Pon binding-induced PBD dimerization relieves the autoinhibition of Plk1. Moreover, the priming Cdk1 phosphorylation of Pon was shown to be important for sequential Plk1 phosphorylation. These results not only provide structural insight into how phosphoprotein binding activates Plk1 but also suggest that binding to different phosphoproteins might mediate the fine-tuning of Plk1 activity (Znu, 2016).

Cdk1 phosphorylates Drosophila Sas-4 to recruit Polo to daughter centrioles and convert them to centrosomes

Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic 'centriole conversion' is regulated, but it requires Plk1/Polo kinase. This study shows that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome (Novak, 2016).

polo: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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