partner of numb: Biological Overview | References
Gene name - partner of numb
Cytological map position-4C10-4C10
Function - scaffolding protein
Keywords - asymmetric cell division, cytoskeleton
Symbol - pon
FlyBase ID: FBgn0025739
Genetic map position - X: 4,530,363..4,533,145 [+]
Classification - novel protein not conserved outside of insects; C-terminal asymmetric localization domain
Cellular location - cytoplasmic
|Recent literature||Zhu, K., Shan, Z., Zhang, L. and Wen, W. (2016). Phospho-Pon binding-mediated fine-tuning of Plk1 activity. Structure [Epub ahead of print]. PubMed ID: 27238966
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.
The proper localization of Numb depends on its interaction with the adapter protein Partner of numb (Pon). In pon mutant embryos, the formation of Numb crescent is delayed in CNS and is disrupted in mesoderm (Lu, 1998). Pon was isolated on the basis of its physical interaction with Numb. Pon is asymmetrically localized during mitosis and colocalizes with Numb. Ectopically expressed Pon responds to the apical-basal polarity of epithelial cells and is sufficient to localize Numb basally. It is proposed that Pon is one component of a multimolecular machinery that localizes Numb by responding to polarity cues conserved in neural precursors and epithelial cells (Lu, 1998 and Lu, 1999).
In principle, the asymmetric localization of Numb/Pon can be accomplished by one or a combination of the following mechanisms: localization and local translation of their mRNAs; active transport of the proteins by motor molecules along the cytoplasmic or cortical cytoskeleton; passive diffusion (3D in the cytosol or 2D along the cortex) and trapping of the proteins by basally localized anchor molecules, or protein targeting to the membrane followed by selective degradation at one side of the cortex. The available method to detect protein localization by immunostaining of fixed embryos only provides static images of the proteins in different cells and is inadequate to distinguish among the above possibilities (Lu, 1999).
The mechanism of asymmetric Pon localization has been shown to operate at the protein level. The asymmetric localization domain of Pon has been mapped to its C-terminal region. Using a fusion between this localization domain and GFP, the entire process of Pon localization was monitored in neuroblasts of living embryos. This in vivo analysis reveals that the asymmetric localization of Pon is a dynamic, multistep process. The protein is first recruited from the cytosol to the cell cortex, a step that requires cell cycle progression into mitosis. Cortically recruited Pon then moves on the cortex and is later restricted to the basal side to form a crescent. The crescent disintegrates upon exit from mitosis. Photobleaching experiments reveal both apical and basal movements of Pon on the cortex. These movements can still occur when myosin motor activity is inhibited by drug treatment. Genetic and pharmacological analyses further reveal that the formation and anchoring of the Pon crescent at the basal cortex require actomyosin and Inscuteable (Lu, 1999).
Inscuteable coordinates mitotic spindle orientation and asymmetric protein localization in neuroblasts. To gain more insight into the mechanism by which Inscuteable directs the asymmetric localization of Pon, Pon-GFP was introduced into an insc mutant background. In insc mutant neuroblasts, cortical recruitment of Pon-GFP is normal, but the Pon-GFP crescent fails to form in 70% of the cases, and, in the remaining 30% of the neuroblasts, the crescent forms but it is randomly positioned. These observations are consistent with previous findings about protein localization in insc mutants. However, the ability to continuously monitor Pon-GFP in living embryos revealed a previously unrecognized function of Inscuteable. In wild-type embryos, the Pon-GFP crescent is stably anchored to the basal cortex once the crescent is formed. In contrast, in insc mutant embryos, the crescent is not stably anchored on the cortex, and it shifts position gradually. Depending on the orientation of the mitotic spindle and the position of the crescent at the time of cytokinesis, the protein can be segregated equally or unequally between the two daughters. Therefore, Inscuteable is required to provide positional cues that direct the formation and the anchoring and maintenance of Pon-GFP crescent at the basal cortex (Lu, 1999).
The gradual recruitment of Pon from the cytosol to the cell cortex at early stages of the cell cycle appears to be coupled to cell cycle progression. To test whether entry into mitosis is a prerequisite for this cortical recruitment, Pon-GFP was introduced into cell cycle mutant backgrounds. In string mutants, postblastoderm cells arrest at the G2 phase of the cell cycle. In neuroblasts of string mutants, the GFP signal is diffuse in the cytoplasm, with some uniform cortical staining. Even after 1 hr of recording, the cytoplasmic signal is not cleared, although the uniform cortical signal seems to increase slightly over time. In contrast, in wild-type neuroblasts, the cytoplasmic signal is cleared within 5-6 min after its appearance. Thus, cortical recruitment of Pon-GFP depends on entry into mitosis (Lu, 1999).
In pebble mutants, cytokinesis of postblastoderm cell divisions is blocked, but other cell cycle events including the asymmetric localization of Numb and Prospero still occur. The initial cortical recruitment and formation of a basal Pon-GFP crescent are normal in pebble mutant neuroblasts. However, by continuously monitoring the Pon-GFP crescent, it was observed that within 10-15 min of its formation, the crescent starts to disintegrate and the protein is dispersed uniformly on the cortex. Gradually, the cortical signal is decreased to background levels, presumably due to degradation or release from the cortex. By comparing the time interval between crescent formation and disintegration in pebble mutants (10-15 minutes) with the interval between crescent formation and the later stages of the neuroblast cell cycle in wild-type embryos, it was determined that the timing of Pon-GFP crescent disintegration in pebble mutants coincides with the end of a normal neuroblast division. This result suggests that at the exit from mitosis certain cell cycle events disassemble or inactivate the localization machinery and that this can occur in the absence of cytokinesis (Lu, 1999).
The defect in the cortical recruitment of Pon-GFP in string mutants suggests that the assembly or proper functioning of the machinery that recruits Pon to the cortex depends on cell cycle progression into mitosis. It is also possible that the cortical recruitment of Pon may depend on its posttranslational modification such as phosphorylation by the p34cdc2 kinase, which is inactive in string mutants. Further biochemical characterization of Pon protein, such as analyzing its posttranslational modification during the cell cycle, should provide more insight into the mechanistic aspects of this regulation (Lu, 1999).
The disintegration of Pon-GFP crescent at the exit from mitosis in pebble mutants implicates a role for the cell cycle machinery in disabling the protein localization machinery. The absence of cytokinesis in pebble mutants allows this step to be observed in more detail. In wild-type neuroblasts, the cleavage furrow coincides with the border of the Pon-GFP crescent at cytokinesis, therefore the GFP signal is distributed all around the GMC cell membrane as soon as the GMC is formed. This makes the crescent disintegration step not observable in wild-type embryos. However, in both wild-type GMC cells and pebble mutant neuroblasts, the uniform cortical GFP signal is gradually decreased to background levels as the cell cycle progresses, suggesting that Pon-GFP is eventually released from the cortex and becomes degraded or delocalized in both wild-type and pebble mutant embryos. In this regard, it will be interesting to test whether the anaphase-promoting complex/cyclosome ubiquitin ligase or components of the mitosis exit signaling pathway are involved in the disintegration of Pon-GFP crescent and the subsequent release of the protein from the cortex (Lu, 1999).
The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).
Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001 and references therein).
The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?
To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).
Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).
After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).
Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).
In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).
Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).
To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homolog of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore, the function of E-APC in epithelial cell division was tested (Lu, 2001).
In about 60% of E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells. Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).
To test whether E-APC functions with EB1 to orient the mitotic spindle, RNAi was performed on a closely related fly homolog of EB1 (dEB1). In dEB1(RNAi) embryos, the epithelial divisions are also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. The penetrance of dEB1(RNAi) phenotype (20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologs in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain, which is required for interaction with EB1, and no direct interaction between E-APC and EB1 could be detected in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orient spindles (Lu, 2001).
These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orient their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in an insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behavior of the adherens junction (Lu, 2001).
When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orienting axis division may have its precedent in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlight the importance of tumor suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).
Asymmetric divisions with two different division orientations follow different polarity cues for the asymmetric segregation of determinants in the sensory organ precursor (SOP) lineage. The first asymmetric division depends on frizzled function and has the mitotic spindle of the pI cell in the epithelium oriented along the anterior-posterior axis, giving rise to pIIa and pIIb, which divide in different orientations. Only the pIIb division resembles neuroblast division in daughter-size asymmetry, spindle orientation along the apical-basal axis, basal Numb localization, and requirement for inscuteable function. Because the PDZ domain protein Bazooka is required for spindle orientation and basal localization of Numb in neuroblasts, it was of interest to enquire whether Bazooka plays a similar role in the pIIb in the SOP lineage. Surprisingly, in pI and all subsequent divisions, Bazooka controls asymmetric localization of the Numb-anchoring protein Pon, but not spindle orientation. Bazooka also regulates cell proliferation in the SOP lineage; loss of bazooka function results in supernumerary cell divisions and apoptotic cell death (Roegiers, 2001).
During embryonic neuroblast divisions, Bazooka is required not only to localize Inscuteable to the apical cortex and Numb, Miranda, Prospero, and Pon to the basal cortex, but also to orient the mitotic spindle along the apical-basal axis. To determine the requirement of bazooka in the asymmetric divisions of the adult SOP lineage, the MARCM system was used to generate baz mutant clones expressing both Pon-GFP (as a reporter for Numb localization) and Tau-GFP (as a reporter for spindle orientation) under the control of scabrous-GAL4, which is strongly expressed in the SOP cell and in the SOP lineage. The movements of Pon-GFP and Tau-GFP were monitored in live tissue throughout all asymmetric divisions of the SOP lineage. In bazxi106 or bazEH171 null mutant clones, pI cells underwent mitosis at ~15 h APF as in wild type. However, in all mutant pI cells observed, Pon-GFP remained uniformly distributed and never formed an anterior crescent as seen in dividing wild-type pI cells. Nor did Pon-GFP crescents form in the subsequent divisions in the lineage. Thus, although only the pIIb resembles the embryonic neuroblast in its orientation of division and requirement for Inscuteable, Bazooka is required for the asymmetric Pon/Numb localization in the pI division, as well as all subsequent divisions (Roegiers, 2001).
Because Numb functions as an asymmetrically localized cell-fate determinant in the SOP lineage, the absence of Numb crescents in baz mutant clones could lead to cell-fate transformations in the daughters of the pI cell. Thus the anterior daughter cell of the pI in bazooka mutant clones (the pIIb cell in the wild type) is referred to as pIIbb, and the posterior daughter cell as pIIab. It is worth noting, however, that either loss-of-function or misexpression of numb causes cell-fate transformation only in a subset of sensory organs, presumably because the Notch-mediated mutual inhibition may still allow the two daughter cells to adopt different cell fates, albeit without a bias set by the Numb crescent. Transformation of pIIa to pIIb cell fate is known to alter the timing of mitosis of the transformed pIIa cell. Timing of the pIIbb, pIIab, and pIIIbb divisions is indistinguishable from wild-type pIIb, pIIa, and pIIIb cells. In addition, the pI and pIIab spindles align along the A-P axis in all mutant clones. And in eight of the nine clones examined the pIIbb spindles were oriented along the apical-basal axis as in wild type (the remaining pIIbb cell divided before the pIIab, but had its spindle oriented along the anterior-posterior axis). Because an apically localized Inscuteable is required for mitotic spindle positioning in the pIIb cell, Inscuteable localization was also examined in the pIIbb cell in bazooka mutant clones. Inscuteable is localized to an apical stalk in pIIbb, similar to the wild-type pIIb (n = 12). Thus the great majority of pIIbb and pIIab cells resemble wild-type pIIb and pIIa cells in their timing and orientation of division, as well as the expression of Inscuteable in the pIIbb. It therefore appears that these bazooka mutations do not cause detectable cell-fate transformation in most of the pIIbb and pIIab cells, although it remains possible that there are partial transformations and cell-fate changes in a subset of these cells. In light of these observations, the complete loss of Pon-GFP crescents in every mitotic pIIbb and pIIab cell examined strongly supports a model wherein Bazooka controls Pon/Numb asymmetric localization in not only pI but also pIIb and pIIa cells (Roegiers, 2001).
In the dorsal thorax (notum) of the Drosophila pupa, the pI cell divides unequally with its spindle axis aligned with the anterior-posterior (a-p) axis of the fly body. It produces two different daughter cells, pIIa and pIIb. During this division, Numb and its adaptor protein Partner of Numb (Pon) form an anterior crescent and segregate unequally into the anterior pIIb cell. In fz mutant pupae, the division of the pI cell is oriented randomly relative to the a-p axis and the Numb crescent does form, but at a random position. Thus, Fz is not required to establish planar asymmetry per se, but is necessary to orient the axis of the asymmetric cell division. This indicates that additional genes may be required for establishing, rather than orienting, planar asymmetry in the pI cell (Bellaïche, 2001 and references therein).
Fz organizes the actin cytoskeleton at the site of hair formation. Planar polarity in the pI cell is established by a mechanism that involves a remodeling of the previously established apical-basal polarity. During the pI cell division, Baz and DaPKC relocalize from the apical cortex to the posterior lateral cortex, while Dlg and Pins accumulate asymmetrically at the anterior lateral cortex. This redistribution along the a-p axis leads to the formation of two complementary planar domains at the cell cortex. This mechanism of polarity establishment is distinct from the one described in Drosophila neuroblasts. In these cells, Pins is recruited via Insc by Baz to the apical cortex, and acts in a Dlg-independent manner to maintain the Baz/DmPAR-6/DaPKC/Insc complex at the apical cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell (Bellaïche, 2001).
In the dividing pI cell, Numb and Pon colocalize at the anterior pole of the lateral cortex, marked with Fasciclin3 (Fas3), below the adherins junction (AJ), marked with DE-Cadherin (Shotgun). In epithelial cells in interphase, Baz colocalizes with Shotgun at the AJ around the apical cortex. In the pI cell, Baz accumulates at the posterior cortex during mitosis. Prior to chromosome condensation, this accumulation is seen at the level of the AJ. Then, during prophase and metaphase, Baz forms a posterior crescent below AJ and opposite to Numb. At telophase, the pIIa cell inherits a higher level of Baz than its sister cell. DaPKC shows a similar distribution to Baz in the pI cell (Bellaïche, 2001).
In neuroblasts, a key function of the Baz/DaPKC/DmPAR-6 complex is to recruit the Insc and the Pins proteins. However, in the pI cell, Insc is not expressed and Pins does not colocalize with Baz at the posterior cortex. Rather, it localizes to the anterior pole in early prophase and colocalizes with Numb at the anterior lateral cortex at metaphase (Bellaïche, 2001).
The roles of Dlg and Pins in the asymmetric localization of Numb and Pon were examined. The interphase localization of Numb at the cortex and of Pon around the nucleus does not depend on the function of the dlg or pins genes. At metaphase, however, the anterior localization of both proteins requires the activity of both dlg and pins. Thus, in pins mutant cells at prometaphase, the crescent of Numb and Pon is either not detected or weak. Nevertheless, both proteins segregate into the anterior cell at anaphase and telophase. In dlg1P20 mutant pI cells, Numb does not accumulate at the anterior cortex and Pon remains cytoplasmic at metaphase. At telophase, Numb and Pon segregate equally into both daughter cells. These results show that Dlg and Pins are required to localize Numb and Pon at the anterior cortex in the pI cell. Consistently, nonsensory cells are transformed into neurons leading to a bristle loss phenotype in adult flies. Furthermore, the genetic interaction seen between dlgsw and pins suggests that dlg and pins act in the same process to specify the fate of the pI daughter cells (Bellaïche, 2001).
Since Pins localizes asymmetrically in a Fz-independent manner, it was asked whether Pins is necessary to localize Baz at one pole of the pI cell in the absence of Fz. It was found that Baz localizes uniformly around the basal-lateral cortex in 82% of the fz;pins double mutant pI cells at metaphase. Moreover, although Numb forms a crescent at anaphase in pI cells mutant for pins or fz, no Numb crescent is seen at either metaphase or anaphase in fz;pins double mutant pI cells. Consistently, loss of fz activity enhances the pins bristle loss phenotype. These data show that Pins and Fz act in a redundant manner to exclude Baz from the anterior cortex and to establish planar asymmetry in the pI cell (Bellaïche, 2001).
These results show that Pins localizes to the anterior cortex in a Baz-independent manner, in an orientation opposite that of Baz, as does Numb. Pins cooperates with Fz to exclude Baz from the anterior cortex of the pI cell. In contrast, in neuroblasts, Pins localizes in a Baz-dependent manner to the apical pole, opposite Numb, and stabilizes the Insc/Baz/DmPAR-6/DaPKC complex. Nevertheless, Pins promotes the localization of Numb in both cell types (Bellaïche, 2001).
One important difference between pI cells and neuroblasts is the lack of insc expression in pI cells. To test the functional significance of this lack of Insc, Insc was expressed in the pI cell. Under these circumstances, Insc and Pins localize at the anterior cortex. Insc triggers the anterior relocalization of Baz, while Numb forms a posterior crescent at anaphase. The pI cell division remains planar. This contrasts with the effect of Insc in epithelial cells. In these cells, Insc localizes apically and orients the spindle along the apical-basal axis. This further indicates that the apical-basal polarity is remodeled in the pI cell. It is concluded that the ectopic expression of Insc is sufficient to reverse the planar polarity axis of the pI cell and to modify the activity of Pins relative to Baz. In the absence of Insc, the Dlg/Pins complex excludes Baz, while expression of Insc leads to the formation of a Pins/Insc/Baz complex. In both cases, Numb localization is opposite that of Baz (Bellaïche, 2001).
In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. This study used quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. It was demonstrated that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. It is proposed that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis (Mayer, 2005).
In order to study the dynamics of asymmetric protein localization, a time series of the division of an SOP cell expressing GFP-Pon and Histone2B-RFP was recorded under the control of a specific promoter. Histone2B-RFP was used to visualize DNA, thus allowing correlation of distinct steps of GFP-Pon localization with other mitotic events. In interphase, some GFP-Pon is cortical, but a large part localizes to the cytoplasm. As the cell enters mitosis, it rounds up and undergoes strong membrane blebbings, indicative of local rearrangements of the cortical cytoskeleton. Interestingly, similar blebbing events have also been observed in the first division of the C. elegans zygote. Unlike in SOP cells, however, they only occur on the anterior side of the C. elegans zygote, where Par-3/6 localize. Shortly after blebbing has started, chromosomes condense and GFP-Pon accumulates on random sites of the cell cortex. The accumulations are transient and do not necessarily predict the position of the final Pon crescent. This suggests that the process leading to Pon accumulation can take place all around the cell but is reinforced specifically in the crescent region. Some GFP-Pon was also observed at the nucleus. This signal might be due to GFP-Pon binding to the nuclear envelope or to the endoplasmic reticulum, and it disappears slowly after nuclear-envelope breakdown. At nuclear-envelope breakdown, cortical blebbing ceases, the cell cortex smoothes, and first signs of asymmetric localization of GFP-Pon into an anterior cortical crescent are observed. As the cell progressed into metaphase, the GFP-Pon signal in the crescent area becomes stronger. Surprisingly, the intensity of the cortical area opposite of the crescent is almost not changed during this process. Thus, GFP-Pon might actually be recruited to the crescent directly from the cytoplasm rather than being transported along the cell cortex. Indeed, quantification of fluorescence intensity showed that GFP-Pon recruitment at the cell cortex is accompanied by a comparable loss of cytoplasmic GFP-Pon. Note that local degradation of GFP-Pon in the cytoplasm is not responsible for this reduction because total GFP-Pon remains unchanged (Mayer, 2005).
Subsequently, the metaphase plate was oriented with respect to the crescent, and during cytokinesis, GFP-Pon segregated largely into the anterior daughter cell. It is proposed that GFP-Pon localization is a two-step process involving the establishment of a cortical area where the crescent will form and the progressive recruitment of protein to the predefined site until metaphase (Mayer, 2005).
Asymmetry of Numb and Pon could be created by lateral movement along the cell cortex or by direct recruitment from the cytoplasm to one side of the cell cortex. To quantify the exchange of Numb and Pon between the cell cortex and the cytoplasm, fluorescence recovery after photobleaching (FRAP) was used of GFP fusions to Numb and Pon. Numb-GFP can partially rescue the numb mutant phenotype, indicating that it is functional. GFP-Pon contains just the asymmetric-localization domain. Its rescue behavior is unknown, but it colocalizes with endogenous Pon throughout mitosis. When cytoplasmic GFP-Pon is photobleached, fluorescence recovers with a half-time of 0.48 s, indicating that diffusion is not limiting. Recovery of cortical GFP-Pon fluorescence occurred with single exponential kinetics and a half-time of 35 s, whereas the half-time for Numb-GFP was 27 s. Therefore, Numb and Pon showed a surprisingly dynamic association with the cell cortex (Mayer, 2005).
Either cortical recruitment of cytoplasmic GFP-Pon or lateral diffusion/transport of cortical GFP-Pon could be responsible for fluorescence recovery. To measure the exchange between cortical and cytoplasmic Pon, an area covering approximately 40% of the cytoplasm was repeatedly photobleached in an SOP cell expressing GFP-Pon. Fluorescence intensity was simultaneously recorded at the cortex. Cortical fluorescence intensity dropped to less than 5% with a half-time of 52 s. Thus, the cortical and cytoplasmic pools of GFP-Pon rapidly interchange with a mobile fraction of more than 95% (Mayer, 2005).
When the dynamic association with the cell cortex is taken into account, Pon asymmetry could be explained either by fast and continuous lateral transport or by directed recruitment to an asymmetric cortical binding site. To determine the contribution of lateral transport, FRAP rates were compared on the edge and in the center of a photobleached region within the GFP-Pon crescent. The bleached region was defined such that a region of nonbleached molecules was left behind at the edges of the crescent after photobleaching. To avoid recovery from above and below the image plane, a protocol was used in which the region of interest was bleached in several planes. The efficiency of this procedure was confirmed by 3D reconstruction after photobleaching in fixed tissue. FRAP curves from ten experiments were averaged. Their superposition shows that the two regions recover nearly identically with half-times of 32 s for a region close to nonbleached GFP-Pon and of 35 s for a region farther away. Taken together, these observations suggest a model where Pon is preferentially recruited from the cytoplasm to the site of crescent formation. It is proposed that a cortical high-affinity binding site for Pon is established during mitosis and mediates specific recruitment of Pon to one side of the cell cortex (Mayer, 2005).
To test the role of Lgl in asymmetric protein localization in SOP cells, cortical recruitment of GFP-Pon was measured in lgl1 mutant clones. In a similar experiment, Lgl has been shown to be dispensable for Pon localization, although Pon recruitment seemed to be delayed. The ratio between total cortical and total cytoplasmic fluorescence was calculated. Because GFP fluorescence intensity is proportional to GFP-Pon concentration, this ratio should give a good estimate of the fraction of GFP-Pon localized at the cell cortex. Although GFP-Pon was still asymmetric, quantitative analysis revealed that the cortical GFP-Pon fraction was slightly but significantly reduced in lgl1 mutant clones. This might be a hypomorphic phenotype caused by small residual amounts of Lgl protein present in the mutant clones. Therefore expression of deregulated aPKC (aPKC-deltaN) was used as another means to inactivate Lgl. Expression of aPKC-deltaN was shown to phenocopy lgl mutants in embryonic tissues, presumably because it phosphorylates and inactivates Lgl all around the cell. In contrast to lgl1 mutant SOP cells, a much stronger decrease of cortical GFP-Pon recruitment was observed upon aPKC-deltaN expression. Still, a slight cortical asymmetry was observed, which is thought is due to the presence of endogenous aPKC. Even at anaphase, the degree of recruitment hardly reached that of control cells in prophase. To test whether Lgl phosphorylation was responsible for this phenotype, aPKC-deltaN was coexpressed with nonphosphorylatable lgl3A. Expression of lgl3A completely rescued the cortical-recruitment defect. The observed differences are not due to increased protein levels because total cellular GFP-Pon fluorescence remains constant (Mayer, 2005).
Thus, active, nonphosphorylated Lgl is needed for cortical recruitment of GFP-Pon although lgl1 mutant clones did not show a very strong phenotype. The easiest explanation for the discrepancy between the lgl1 mutant and ectopic Lgl phosphorylation is the perdurance of residual Lgl protein in mutant tissue. This is supported by previous observations describing Numb-localization defects in temperature-sensitive alleles of lgl. It is possible that Lgl can mediate its effects even at protein concentrations below the detection limit of the antibody. Thus, Lgl may not be needed at stoichiometric levels for asymmetric protein localization in SOP cells, but it instead plays a catalytic or signaling role (Mayer, 2005).
How could Lgl recruit Pon to the cell cortex? Formally, it is possible that Pon simply binds Lgl in a phosphorylation-dependent manner. However, no direct interaction has been described and such a model would not explain why Pon is cortical even when Lgl levels are strongly reduced. Two other models are more likely: Either cortical binding sites for Numb and Pon are present all around the cell, but their affinity depends on Lgl and its phosphorylation status and therefore varies along the cell cortex (Model 1); or a limiting number of cortical binding sites are present only on one side of the cell, and Lgl is responsible for their asymmetric distribution (Model 2). To distinguish between these models, FRAP rates were measured for cortical GFP-Pon in different genetic backgrounds. The FRAP rate is a function of the rate constants for both association and dissociation of GFP-Pon with its postulated cortical binding site. In Model 1, expression of activated lgl (lgl3A) or deregulated aPKC (aPKC-?N) should alter the affinity of the binding site and therefore change the rate constants, resulting in a variation of the FRAP rate. Because the FRAP rate is independent of receptor concentration, however, it would remain constant under the same conditions in Model 2. Cortical GFP-Pon FRAP rates were measured in wild-type SOP cells, in cells expressing lgl3A, and in cells where Lgl was inactivated by expression of aPKC-deltaN. Although expression of aPKC-deltaN dramatically reduced the amount of GFP-Pon present at the cortex, it did not influence the kinetics of GFP-Pon binding to the cortical binding site. Thus, the number of Pon binding sites at the cell cortex, and not their affinity for Pon, seems to be reduced by aPKC-deltaN expression (Mayer, 2005).
To gain independent evidence for the two models, the fraction of GFP-Pon present at the cell cortex was quantitated. If Lgl regulated GFP-Pon binding site affinity, expression of lgl3A would change the entire SOP cell cortex to high affinity, and therefore it would increase the cortical GFP-Pon fraction. If Lgl regulated only the distribution of binding sites, however, the cortical fraction of GFP-Pon should remain the same. Cortical recruitment was quantified by measuring the ratio of cortical to cytoplasmic fluorescence for GFP-Pon and Numb-GFP at different time points in mitosis. Compared to wild-type cells, expression of lgl3A did not cause a significant increase in cortical recruitment. This is not because cytoplasmic GFP-Pon is limiting; increased GFP-Pon expression predominantly increased the cytoplasmic signal. Taken together, these results favor Model 2, in which Lgl acts by asymmetrically distributing a limiting number of cortical GFP-Pon binding sites. The loss of cortical fluorescence upon aPKC-deltaN expression indicates that lgl is also required for binding site formation, in addition to binding site positioning. However, this second role of lgl does not seem to be rate limiting under normal conditions because lgl3A expression does not increase the cortical GFP-Pon fraction. Although these results are most consistent with Model 2, more-complex models cannot be excluded. For example, lgl could distribute a limiting adaptor protein that links Pon to a receptor but is not the receptor itself (Mayer, 2005).
The direct cortical binding partners for Pon or Numb have not yet been identified. Thus, it is only possible to speculate on the molecular mechanisms of their postulated asymmetric distribution. Although the results are inconsistent with lateral transport of GFP-Pon, they do not exclude lateral transport of its cortical anchor. Similar to what has been proposed for asymmetric cell division in C. elegans, a possible mechanism could be local tearing and contraction of the cortical actin cytoskeleton. Lgl was shown to inhibit the cortical localization of myosin II, and it has been proposed that cortical myosin II might exclude asymmetrically segregating proteins. These data could be integrated with the model if myosin II excludes the cortical binding sites rather than influencing determinant localization directly. Alternatively, transmembrane receptors for Pon or Numb could be delivered to the position of crescent formation by vesicle transport. Such a mechanism in which transmembrane receptors are present on vesicles that dock at the membrane in an Lgl-dependent fashion would be consistent with the quantitative observations. It would also explain why Lgl is essential for crescent formation but not needed in metaphase for maintenance of asymmetric protein localization. It is remarkable that lateral diffusion of transmembrane proteins is slow enough to allow a stable asymmetric distribution, if the delivery of the protein is asymmetric, both in yeast and in SOP cells. The yeast Lgl orthologs Sro7p and Sro77p have been implicated in plasma-membrane fusion of secretory vesicles (Lehman, 1999), and Lgl has been proposed to regulate vesicular targeting to specific membrane domains. Furthermore, asymmetric protein localization in Drosophila requires myosin VI, a motor whose main function is vesicle movement, suggesting that vesicle trafficking plays some role (Mayer, 2005).
These data provide insight into the dynamic protein movements of cell-fate determinants and their associated adaptor proteins during asymmetric cell division. It is proposed that these determinants are preferentially recruited from the cytoplasm to a high-affinity binding site during late prophase. Establishment of this binding site is regulated by the phosphorylation status of Lgl. The role of Lgl is more to concentrate binding sites on one side of the cell than to act as a receptor itself or change the affinity of another Numb or Pon binding site (Mayer, 2005).
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).
Search PubMed for articles about Drosophila Partner of numb
Bellaïche, Y., et al. (2001). The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila. Cell 106: 355-366. PubMed ID: 11509184
Lehman, K., Rossi, G., Adamo, J. E. and Brennwald, P. (1999). Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9. J. Cell Biol. 146: 125-140. PubMed ID: 10402465
Lu, B., Rothenberg, M., Jan, L. Y. and Jan, Y. N. (1998). Partner of numb colocalizes with numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 95: 225-23. PubMed ID: 9790529
Lu, B., Ackerman, L., Jan, L. Y. and Jan, Y. N. (1999). Modes of protein movement that lead to the asymmetric localization of partner of Numb during Drosophila neuroblast division. Molec. Cell 4: 883-891. PubMed ID: 10635314
Lu, B., et al. (2001). Adherens junctions inhibit asymmetric division in the Drosophila epithelium. Nature 409: 522-525. PubMed ID: 11206549
Mayer, B., Emery, G., Berdnik, D., Wirtz-Peitz, F. and Knoblich, J. A. (2005). Quantitative analysis of protein dynamics during asymmetric cell division. Curr. Biol. 15(20): 1847-54. PubMed ID: 16243032
Roegiers, F., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (2001). Bazooka is required for localization of determinants and controlling proliferation in the sensory organ precursor cell lineage in Drosophila. Proc. Natl. Acad. Sci. 98: 14469-14474. PubMed ID: 11734647
Wang, H., Ouyang, Y., Somers, W. G., Chia, W. and Lu, B. (2007). Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon. Nature 449(7158): 96-100. PubMed ID: 17805297
date revised: 4 January 2008
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