atypical protein kinase C


Prospero, targeting aPKC, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Interplay between the transcription factor Zif and aPKC regulates neuroblast polarity and self-renewal

How a cell decides to self-renew or differentiate is a critical issue in stem cell and cancer biology. Atypical protein kinase C (aPKC) promotes self-renewal of Drosophila larval brain neural stem cells, neuroblasts. However, it is unclear how aPKC cortical polarity and protein levels are regulated. This study identified a zinc-finger protein, Zif, which is required for the expression and asymmetric localization of aPKC. aPKC displays ectopic cortical localization with upregulated protein levels in dividing zif mutant neuroblasts, leading to neuroblast overproliferation. Zif was shown to be a transcription factor that directly represses aPKC transcription. It was further shown that Zif is phosphorylated by aPKC both in vitro and in vivo. Phosphorylation of Zif by aPKC excludes it from the nucleus, leading to Zif inactivation in neuroblasts. Thus, reciprocal repression between Zif and aPKC act as a critical regulatory mechanism for establishing cell polarity and controlling neuroblast self-renewal (Chang, 2010).

Drosophila larval brain neural stem cells, neuroblasts (NBs), divide asymmetrically to give rise to a self-renewing daughter and a ganglion mother cell (GMC) that is committed to differentiation. The mechanisms of NB asymmetric division have been studied primarily in embryonic NBs, and are conserved in larval NBs. Asymmetric division of NBs depends on asymmetric protein localization during mitosis. Apically localized proteins include the Bazooka (Drosophila Par3)/Par6/aPKC complex, Partner of Insc (Pins), Locomotion defects (Loco), and mushroom body defect (Mud). Gβγ and Ric-8 regulate Gαi localization. Apical proteins allow asymmetric localization of basal proteins Numb, Prospero (Pros), Brat, Partner of Numb (Pon), and Miranda (Mira), which are segregated into GMCs. Basal protein localization is also mediated by Discs large, Lethal (2) giant larvae (Lgl), Myosins II and VI, and Protein Phosphatases 2 and 4. aPKC can directly phosphorylate Numb and Mira to regulate their asymmetric localization (Chang, 2010).

Drosophila larval brain NBs utilize the asymmetric division machinery to distribute 'proliferation factors' and 'differentiation factors' to different daughters. Failure in asymmetric division can result in NB overproliferation. Larval brain tissues from mutants of pins, mira, and numb when transplanted into wild-type adults, can form malignant tumors. Two distinct populations of NBs have recently been identified in larval brains. Unlike type-I NBs, type-II NBs are Asense (Ase) negative and divide to produce a NB and an intermediate neural progenitor cell (INP) which produces multiple GMCs. aPKC is a NB proliferation factor in both type-I and -II NBs, as its ectopic expression throughout the entire cell cortex by CAAX motif leads to overgrowth in larval brains. Aurora-A and Polo kinases inhibit NB overgrowth primarily by regulating Numb asymmetry, while all basal proteins have brain tumor suppressor functions that prevent NB overgrowth. Aurora-A acts in both type-I and -II NBs; it also directly phosphorylates Par-6. Dap160/intersectin interacts with and stimulates aPKC activity, which is required for maintaining NB proliferation (Chang, 2010).

This study identifies a C2H2-type zinc-finger transcription factor that has been named 'Zif,' which is required for regulating both aPKC expression and cortical polarity. In addition, a reciprocal activity of aPKC on Zif—that aPKC directly phosphorylates Zif, regulating its activity via control of its subcellular localization (Chang, 2010).

A clonal screen was performed using mosaic analysis with a repressible cell marker (MARCM) and three ethlymethane sulfonate (EMS)-induced mutants (1L15, 2L745, and 2L497) belonging to a single complementation group were isolated. Compared with wild-type clones which mostly possess only one NB, supernumerary NB-like cells labeled by Mira were observed in these mutant clones. Genetic mapping and molecular analysis indicated that 1L15, 2L745, and 2L497 each carried a nonsense mutation in CG10267, a C2H2-type Zinc-finger protein (Zif), at Q110, Q192, and K306, respectively. Zif contains five zinc fingers at the C terminus which confers DNA-binding capability. All three mutants are embryonic lethal and are likely to be loss-of-function alleles, as Zif protein was undetectable in them. 1L15 (referred to as zif mutant hereafter) was used for most experiments. The NB overgrowth defects observed in all zif mutants could be completely rescued by expressing full-length Zif (Chang, 2010).

Examined next was whether Zif functions similarly to inhibit NB overgrowth within type-I and -II larval NB lineages. zif MARCM clones were generated by labeling NBs with Deadpan (Dpn)/Ase and differentiated neurons with Embryonic Lethal Abnormal Vision (Elav). Wild-type type-I NB clones contain one Dpn-positive/Ase-positive (Dpn+Ase+) NB with a large number of ELAV+ neurons. However, in zif mutant type-I NB clones, ectopic NBs were evident by the presence of multiple Dpn+Ase+ cells. This NB overgrowth occurs at the expense of neuron formation, as very few Elav+ neurons were observed in these clones. In type-II NB clones, zif mutants also had an increased number of Dpn+Ase- NBs, compared to one Dpn+Ase- NB in a wild-type type-II clone. zif mutant type-II clones contained 24.1 ± 8.1 INPs, similar to wild-type clones, indicating that Zif is not obviously required for INP self-renewal. Therefore, Zif functions in both type-I and -II NB lineages to inhibit NB overgrowth (Chang, 2010).

Zif protein, as detected by antibodies, is nuclear in interphase NBs and cytoplasmic throughout mitosis. This localization pattern of endogenous Zif is recapitulated by an inducible construct of Venus-tagged full-length Zif (UAS-Venus::zifWT-FL) expressed using Insc-Gal4. Zif is expressed in NBs, GMCs, and neurons, suggesting a ubiquitous expression of Zif in most cell types in larval brains (Chang, 2010).

Whether Zif regulates asymmetric protein localization in NBs was investigated. In contrast to wild-type prometa/metaphase NBs in which 84% had a robust localization of aPKC to the apical cortex (12% with weaker crescent of aPKC), 78% of the zif mutant NBs exhibited aPKC delocalization throughout the cortex. The remaining 22% of the zif prometa/metaphase NBs analyzed also exhibited disrupted aPKC localization, either appearing as punctate structures on the cortex or diffused crescents. Consistent with the observed aPKC delocalization, Baz is also delocalized or absent in 56% of prometa/metaphase zif mutant NBs. Zif is also required for asymmetric localization of Numb, Mira, Pros, and Pon. Numb is cortically localized or absent in zif mutant prometa/metaphase NBs. Pon, Mira, and Pros are also delocalized in zif prometa/metaphase NBs. Thus, Zif regulates asymmetric localization of apical and basal complex proteins during NB asymmetric divisions (Chang, 2010).

The NB overgrowth phenotype in zif mutant clones was significantly suppressed in aPKC06403/+ heterozygous background. aPKC asymmetric localization during prophase/metaphase is also mostly restored. zif mutant NB overproliferation was also suppressed by overexpression of Lgl3A. These data indicate that Zif inhibits NB overgrowth primarily by suppressing aPKC function (Chang, 2010).

As a putative transcription factor, it was asked if Zif regulates the expression of asymmetric cell division genes. Examination of the mRNA transcript levels of most known asymmetric cell division genes by reverse transcriptase (RT)-PCR in zif/Df(3R)WIN11 hemizygous embryos versus wild-type showed that aPKC transcripts were dramatically increased in zif/Df(3R)WIN11 mutant embryos compared to wild-type. The levels of transcript of all other genes tested remained similar between zif/Df(3R)WIN11 and wild-type samples. Quantitative real-time PCR was performed using the same samples, and it was found that normalized transcript levels for aPKC in zif mutant were upregulated to 363% compared to wild-type (100%), while transcript levels of Pros and Dap160 in zif mutant remained comparable to wild-type. Consistently, aPKC protein levels were dramatically increased in zifRNAi larval brains in which zif levels were further reduced by zif+/−. Knocking down zif levels in S2 cells also leads to an increase in aPKC levels; further supporting that Zif is able to repress aPKC levels in dividing cells. Knocking down of Zif in S2 cells did not change total Numb protein level but observed a dramatic increase was seen in phosphorylation of Numb by aPKC recognized by an anti-ps7Numb antibody, indicating that the excess aPKC protein in zif mutants was functionally active (Chang, 2010).

Chromatin immunoprecipitation (ChIP) was performed in S2 cells to investigate whether Zif can directly bind to aPKC promoter. At the aPKC regulatory region, a 500 bp DNA region was identified where Zif directly binds in the ChIP assay. The Zif-binding region was further narrowed down to a 200 bp DNA fragment (−652 to −424 bp upstream of the transcriptional start site, which is referred to as the mini-aPKC proximal promoter (aPKC pro). Next, a luciferase reporter assay was carried out to test the effect of Zif on aPKC pro. Zif was capable of suppressing the luciferase reporter activity under the control of aPKCpro in a dose-dependent manner, while no consistent effect was observed on the actin promoter. Taken together, Zif directly represses aPKC transcription by binding to an upstream proximal promoter region of aPKC (Chang, 2010).

Endogenous Zif was found to exist in two states differing in their net charges in two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The proportion of Zif that has more acidic residues (more negatively charged) was abolished by knockdown of aPKC in S2 cells, indicating that the putative posttranslational modification of Zif is aPKC dependent. Using group-based prediction system, two putative consensus phosphorylation sites for aPKC [S-X-(R/L)1-3] were identified at serine residues 197 and 292 of Zif. Zif is a good substrate of aPKC as shown by an in vitro kinase assay using both radiometric and nonradioactive approaches. Mutating either S197 or S292 to alanine alone does not consistently abrogate aPKC phosphorylation, whereas mutations of both completely abolished phosphorylation by aPKC. An antibody was generated that specifically recognizes phosphorylation of S292 in Zif (pS292Zif) by aPKC in the kinase assay. The phosphorylated Zif is completely absent upon aPKC knockdown in S2 cells. In addition, phosphorylated Zif is also barely detectable in homozygous aPKCk06403 larval brain extracts. In contrast, phosphorylation of Zif at S292 was unaffected in aur8839 larval brains. Taken together, these data strongly suggest that aPKC phosphorylates Zif both in vitro and in vivo (Chang, 2010).

To analyze the functional role of aPKC-directed phosphorylation of Zif, transgenic flies were generated expressing mutated Zif constructs fused to Venus reporter-phosphomimetic form of Zif by mutating S197 and S292 to aspartic acid residues (S197,292D) or a nonphosphorylatable form of Zif by mutating both serine residues to alanine residues (S197,292A). The strong nuclear localization of Venus-ZifS197, 292A is reminiscent of wild-type Venus-Zif in NBs. In contrast, Venus-ZifS197,292D which still possesses the putative nuclear localization sequence was completely excluded from the nucleus of interphase NBs. Localization of overexpressed Venus-Zif mutant proteins was verified by anti-Zif antibody. Therefore, phosphorylation of Zif by aPKC results in Zif exclusion from the nucleus in the NBs. Furthermore, the nonphosphorylatable form of Zif (ZifS197, 292A) was capable of almost complete rescue of the NB overgrowth or Mira delocalization phenotypes (76% of metaphase NBs had asymmetric localized Mira) in zif mutant MARCM clones. In contrast, expressing the phosphomimetic form of ZifS197, 292D could not significantly rescue the zif mutant overgrowth (71% of clones contained multiple NBs) or Mira mislocalization phenotypes. These data suggest that the nonphosphorylated form of Zif is active and can inhibit excess NB self-renewal (Chang, 2010).

This study has shown that Drosophila Zif is a new player regulating asymmetric cell division and the balance between self-renewal and differentiation. Several Zif-related proteins in mammals, including mouse zinc finger protein 160 and human zinc finger protein 287, both of which share 19% amino acid identities with Drosophila Zif and contain a serine that is conserved with S292 of Zif. These mammalian Zif-related proteins remain uncharacterized and their function during stem cell homeostasis will be of great interest (Chang, 2010).

Zif was shown to have a critical role in directly repressing aPKC expression to inhibit NB overgrowth. In zif embryos, aPKC expression in epithelium appears to be elevated compared to wild-type, indicating that the regulation of aPKC by Zif may occur in other tissues besides NBs. Overexpression of aPKC alone in NBs is insufficient to cause asymmetric division defect. It is currently unclear how Zif controls asymmetric localization of aPKC, which is likely to be independent of its regulation of aPKC transcription. It is possible that Zif regulates unidentified proteins that in turn control aPKC cortical polarity. It will be of great interest to identify such proteins and elucidate their functions in asymmetric division of NBs. In zif mutants, the delocalization of both aPKC and Numb to the entire cortex in metaphase NBs is similar to that caused by ectopic expression of aPKC-CAAX. This is inconsistent with a previous model where aPKC excludes Numb from the cortex in sensory organ precursor cells. The results may suggest that cortical aPKC does not always exclude Numb from the cortex in vivo (Chang, 2010).

Zinc-finger proteins that contain the C2H2 motif, a DNA-binding motif, are putative transcription factors. Mammalian C2H2-type zinc-finger transcription factors are abundant. However, very little is known about posttranslational modification of zinc-finger proteins, which is essential for regulating the functions of these transcription factors during development. Phosphorylation of a threonine residue in the linker region (interfinger spacer) of C2H2 zinc-finger proteins can inactivate their function during mitosis. The current data suggest that aPKC phosphorylates Zif on S197 and S292, resulting in Zif exclusion from the nucleus. It will be interesting to assess whether these findings represent a conserved common mechanism by which C2H2 zinc-finger transcription factors are inactivated (Chang, 2010).

Given that Zif is a nuclear transcription factor and aPKC is predominantly localized to the cortex and cytoplasm, it is conceivable that aPKC could have access to cytoplasmic Zif during mitosis when Zif is localized in the cytoplasm after nuclear membrane breakdown. Upon phosphorylation, Zif would be retained in the cytoplasm at the interphase of the following cell cycle. Consistent with this hypothesis, an increase was detected in aPKC-phosphorylated Zif in Nocodazole-treated S2 cells that are synchronously released into mitosis where Zif is localized to the cytoplasm. In addition, it was observed that by mutating the nuclear localization signal (NLS) in Zif, there was an increase in the cytoplasmic localization of Zif in interphase S2 cells. This suggests that nuclear-cytosol shuttling of Zif may allow phosphorylation of Zif by aPKC to still take place during interphase. In summary, these findings suggest that mutual inhibition between Zif and aPKC is critical for ensuring attainment of the appropriate levels and polarity of aPKC optimal for the proper control of asymmetric division and self-renewal of NBs (Chang, 2010).

Memory enhancement and formation by atypical PKM activity in Drosophila

Synaptic stimulation activates signal transduction pathways, producing persistently active protein kinases. PKMzeta is a truncated, persistently active isoform of atypical protein kinase C-zeta (aPKCzeta), which lacks the N-terminal pseudosubstrate regulatory domain. Using a Pavlovian olfactory learning task in Drosophila, it was found that induction of the mouse aPKMzeta (MaPKMzeta) transgene enhances memory. The enhancement requires persistent kinase activity and is temporally specific, with optimal induction at 30 minutes after training. Induction also enhances memory after massed training and corrects the memory defect of radish mutants, but does not improve memory produced by spaced training. The 'M' isoform of the Drosophila homolog of MaPKCzeta (DaPKM) is present and active in fly heads. Chelerythrine, an inhibitor of PKMzeta, and the induction of a dominant-negative MaPKMzeta transgene inhibits memory without affecting learning. Finally, induction of DaPKM after training also enhances memory. These results show that atypical PKM is sufficient to enhance memory in Drosophila and suggest that it is necessary for normal memory maintenance (Drier, 2002).

The study of PKC in memory formation has a long history. However, most previous work was done before the current appreciation of the complexity of the PKC gene family. The PKC family can be divided into three classes based on their cofactor requirements. Whereas all PKC proteins require phosphatidylserine for activation, the 'conventional' (cPKC) isotypes require diacylglycerol (DAG) and Ca2+ for full activity; 'novel' (nPKC) isotypes are Ca2+ independent but still require DAG, and the 'atypical' (aPKC) isotypes are both DAG and Ca2+ independent. Structurally, these kinases can be divided into an N-terminal regulatory domain, which contains a pseudosubstrate region as well as the binding sites for the required cofactors, and the C-terminal catalytic domain. Removal of the N-terminal regulatory domain produces a persistently active kinase, referred to as PKM. Persistently active kinases have received attention as components of memory mechanisms (Drier, 2002).

The roles of PKC in hippocampal models of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) have been studied extensively. PKC/M activities may have several roles in the mechanisms that initiate and sustain LTP. However, Western blot analyses with antibodies specific for each of the rat PKC isoforms demonstrate that the only one whose levels specifically increase and remain elevated during the maintenance phase of LTP is PKMzeta, which is the truncated form of the atypical isozyme PKCzeta. Expression analyses also show that the maintenance of LTD is associated with decreasing levels of PKMzeta. Most interestingly, LTP maintenance is abolished by sustained application of low concentrations of the PKC inhibitor chelerythrine, whereas perfusion of PKMzeta into CA1 pyramidal cells produces an increase in AMPA receptor-mediated synaptic transmission (Drier, 2002 and references therein).

Experiments in honeybees also indicate a role for PKC in memory formation. Biochemical analyses of extracts made from the antennal lobes of associatively trained bees show a sustained increase in cytosolic, Ca2+-independent PKC activity. This persistent increase correlates with long-lasting bee memory in four ways: it requires multiple training trials; it persists for up to three days; it is insensitive to a drug that blocks cPKC activity, and it is blocked by protein-synthesis inhibitors. Together with the LTP data, these studies point to an important role for a nonconventional PKC activity in the maintenance of memory (Drier, 2002).

In Drosophila, the best characterized assay for associative learning and memory is an odor-avoidance behavioral task. This classical (Pavlovian) conditioning involves exposing the flies to two odors (the conditioned stimuli, or CS), one at a time, in succession. During one of these odor exposures (the CS+), the flies are simultaneously subjected to electric shock (the unconditioned stimulus, or US), whereas exposure to the other odor (the CS-) lacks this negative reinforcement. After training, the flies are placed at a 'choice point', where the odors come from opposite directions, and they decide which odor to avoid. By convention, learning is defined as the fly's performance when testing occurs immediately after training. A single training trial produces strong learning: a typical response is that >90% of the flies avoid the CS+. Performance of wild-type flies from this single-cycle training decays over a roughly 24-hour period until flies once again distribute evenly between the two odors. Flies can also form long-lasting associative olfactory memories, but normally this requires repetitive training regimens (Drier, 2002).

This task was used in Drosophila to examine the role of atypical PKM in memory formation. Induction of the mouse aPKMzeta (MaPKMzeta) transgene enhances memory, and corrects the memory defect of radish mutants. There is a single atypical PKC in Drosophila, and the truncated 'M' isoform, DaPKM, is preferentially expressed and active in fly heads. Both pharmacological and dominant-negative genetic intervention of DaPKC/M activity disrupts normal memory. Finally, induction of the predicted DaPKM also enhances memory, further suggesting a general role of aPKM in memory processes (Drier, 2002).

To investigate the role of PKC in learning and memory in Drosophila, transgenic lines of flies were made bearing heat shock-inducible, murine atypical PKC (MaPKC) isoforms. Considering that LTP experiments indicate that MaPKMzeta levels increase after the presentation of the stimuli required for long-lasting potentiation, whether inducing MaPKMzeta after training affected olfactory memory was tesed. Induction by mild heat shock (32°C) after training strongly enhances 24-hour memory. This enhancement is not due to transgene-independent heat-shock effects, because the wild-type flies show no enhanced memory when exposed to heat shock. The transgenic flies were made in this wild-type strain, so the enhancement is not due to differences in genetic background. Finally, the memory enhancement does not result from an insertional mutation caused by the transgene, because two independent lines (MaPKMzeta-14 and MaPKMzeta-43) have similar effects (Drier, 2002).

Whether 24-hour memory could be enhanced after single-cycle training was tested by inducing MaPKMzeta with a strong heat shock (37°C) 3 hours before training, but this regimen has no effect. Because transgene induction after behavioral training enhances memory, whereas induction before training does not, the temporal specificity of this MaPKMzeta-dependent effect was examined. Optimal enhancement occurs when heat-shock induction begins 30 minutes after training ends, and the effect is absent if heat shock occurs before, or is delayed until 2 hours after training (Drier, 2002).

The memory enhancement is not observed when a kinase-inactive (KI) mutant of MaPKMzeta is induced either before or after training. The enhancement is also not observed when full-length (FL)-MaPKCzeta is induced before or after training. The failure of either the KI-MaPKMzeta or the FL-MaPKCzeta transgene to enhance memory is not due to lack of expression, because both are expressed at levels comparable to the MaPKMzeta protein. Together, these results indicate that the memory enhancement requires a persistently active aPKM isoform (Drier, 2002).

Inducible increases in MaPKMzeta protein levels and kinase activity have been detected in extracts made from Drosophila heads. Western blot analyses shows that both the mild and strong heat-shock regimens induce the MaPKMzeta and MaPKCzeta isoforms, and that these proteins persisted for ~18 hours after heat shock. The induced MaPKMzeta protein is active, since enhancement of Ca2+/DAG-independent PKC activity is observed in fly head extracts from induced but not from uninduced transgenic flies (Drier, 2002).

Drosophila can form associative olfactory memories lasting 24 hours and longer, but this normally requires repetitive training. Multiple-trial training regimens have been established that produce both anesthesia-resistant memory (ARM) and long-term memory (LTM). ARM can be produced by 10 cycles of 'massed' training with no rest intervals between the individual training trials, and lasts 2-3 days. LTM results from repetitive training that contains rest intervals (15 min each), and 10 cycles of this 'spaced' training generates LTM that lasts at least 7 days. To test whether MaPKMzeta can enhance ARM or LTM, flies were subjected to massed or spaced training regimens; the transgene was induced for 30 minutes after training, and then 4-day memory was measured (Drier, 2002).

MaPKMzeta induction substantially increases 4-day memory after massed training but does not improve 4-day memory after spaced training. These data indicate that MaPKMzeta induction enhances massed training-induced, but not spaced training-induced memory (Drier, 2002).

Previous work indicates that consolidated memory in Drosophila consists of two biochemically separable components: ARM and LTM. ARM is produced by either massed or spaced training, and it is insensitive to cycloheximide treatment. LTM is produced by spaced training and is blocked by cycloheximide treatment; thus it is considered to require acute protein synthesis. A previously identified Drosophila memory mutant, radish, is deficient in ARM, since this mutation blocks memory produced by massed training. Spaced training of radish mutants does produce memory, but this memory can be completely blocked by treating the mutants with cycloheximide. These results led to a two-pathway model of consolidated memory, one dependent on the Radish gene product (ARM) and the other dependent on activity-induced, acute protein synthesis (LTM) (Drier, 2002).

Because MaPKMzeta induction enhances memory after massed but not after spaced training, the dependence of this effect on radish was tested. The radish gene is on the X chromosome in Drosophila, and homozygous radish mutant females were crossed to males homozygous for an autosomal copy of the heat shock-inducible MaPKMzeta transgene. The radish mutant is recessive, thus the heterozygous female progeny of this mating will have normal memory after massed training, whereas the hemizygous males will display the radish memory deficit in the absence of induction. The progeny were subjected to massed training, followed by the standard MaPKMzeta induction after training, and then tested at 24 hours. Males and females were trained and tested en masse, and then separated and counted. The radish mutation did not block the memory effect of MaPKMzeta induction. The memory defect of radish males was apparent in the absence of heat-shock induction (HS-), but memory was clearly present in induced males (HS+). A lesser, but significant induction-dependent memory enhancement of the heterozygous radish females by MaPKMzeta was also observed (Drier, 2002).

There is a single atypical PKC (DaPKC) gene in the Drosophila genome, and it is highly homologous to the MaPKCzeta gene that was used. (The kinase domain shows 76% identity and 87% similarity). A Western blot of extracts made from wild-type fly heads and bodies shows that the antiserum used to detect MaPKMzeta and MaPKCzeta recognizes two bands in fly extracts, the smaller of which is enriched in head extracts. This antiserum is directed against the C-terminal 16 amino acids of MaPKC/Mzeta, which shares substantial homology with DaPKC. Antiserum from mice immunized with peptides derived from DaPKC recognizes these same bands. The molecular weights of these two bands indicate that they are probably the DaPKC (~73 kDa) and DaPKM (~55 kDa) isoforms (Drier, 2002).

The N-terminal sequence of the lower molecular weight band has not been established; however, it likely represents an endogenous DaPKM isoform. The immunoreactivity is competitively reduced by a peptide from the corresponding region of DaPKC, but not one outside of this epitope. In agreement with the Western blot data, fly heads contains more Ca2+ and DAG-independent PKC activity than does bodies. The presence of the putative DaPKM correlates strongly with this enriched activity, suggesting that most, if not all, of the endogenous atypical kinase activity measured in head extracts is due to this DaPKM isoform. These data indicate that flies possess both 'C' and 'M' forms of an atypical PKC that is highly homologous to MaPKC/M, and that the DaPKM is enriched in heads (Drier, 2002).

A P-element insertional mutant in DaPKC has been described; however, it is an embryonic lethal and thus is not suitable for examining a possible role in adult learning and memory formation. To assess whether this gene's product is necessary for memory formation, two approaches were taken. First, the effects on memory of feeding flies the PKC inhibitor chelerythrine were monitored. This drug is reported to selectively inhibit PKMzeta at low concentrations; however, its specificity is controversial, and it inhibits other PKC isotypes at higher concentrations. Memory effects produced by inducing the kinase-inactive KI-MaPKMzeta protein were tested: this form of the protein displays 'dominant-negative' activity that is likely to be specific to the atypical PKCs, leaving cPKC and nPKC responses intact (Drier, 2002).

Feeding flies chelerythrine inhibits 24-hour memory formation in a dose-dependent manner, and induction of the KI-MaPKMzeta inhibits 24-hour memory after massed training. The inhibitory effects of both chelerythrine and the KI-MaPKMzeta are not likely due to effects on olfactory acuity or shock reactivity because learning is unaffected by either treatment (Drier, 2002).

The memory enhancement produced by MaPKMzeta could have been due to properties unique to this mammalian protein. The expression data showing that DaPKM is expressed and active in Drosophila heads, when combined with the chelerythrine and dominant-negative data, suggests that DaPKM is involved in normal memory processes in Drosophila. The extensive structural homology between MaPKMzeta and DaPKM also argues against functional uniqueness. The hypothesis of functional homology makes a strong prediction: induction of DaPKM after training should also enhance memory (Drier, 2002).

Based on the approximate molecular weight of the DaPKM, the DaPKC gene was truncated within the hinge region separating the regulatory from the catalytic domains such that the putative DaPKM gene begins at methionine 223. Induction of the DaPKM transgene after training enhances 24-hour memory after single-cycle training. One of these lines was then used to show that 4-day memory after massed training is also enhanced. As with the MaPKMzeta transgenes, the DaPKM lines shows rapid heat-shock induction. These results confirm those obtained with MaPKMzeta, and thus indicate that aPKM is fundamental in the mechanisms underlying memory across species (Drier, 2002).

These results provide strong evidence that atypical PKM activity is sufficient to enhance memory in Drosophila. Ideally, necessity should have been tested by assessing potential memory deficits of flies bearing null mutations in the DaPKC/M gene. However, the lethality of such mutants precluded these analyses, and no special alleles exist that might have preserved the gene's vital function while disrupting its role in memory. In an attempt to circumvent these problems, both pharmacological and dominant-negative interventions were used. Chelerythrine inhibits normal memory in a dose-dependent manner, and induction of a predicted dominant-negative atypical PKM produces the same memory deficit (Drier, 2002).

It was found that heat-shock induction of MaPKMzeta does not enhance long-term memory, because it does not improve memory after spaced training. One explanation for this is that spaced training induces endogenous maintenance mechanisms, and thus occludes the effect of inducing the MaPKMzeta transgene. Thus, memory after single-cycle or massed training may be prolonged by transgene induction because these training regimens do not normally induce prolonged atypical PKM activity. Work in honeybees shows that single-cycle training produces neither persistent PKC activity nor long-lasting memory, but multiple-cycle training produces both. The memory enhancement observed when inducing MaPKMzeta may simply bypass the endogenous requirements (normally provided by spaced training) for prolonged activation of aPKM (Drier, 2002).

The MaPKMzeta-induced enhancement of massed, but not spaced training prompted an examination of the involvement of the radish gene product in this process. If radish were required for the enhancement, the radish mutation would have blocked the MaPKMzeta-induced effect, and this was clearly not the case. Although MaPKMzeta induction phenotypically rescues the memory defect of radish, it does not do so because radish encodes for the Drosophila aPKM. DaPKM is on the second chromosome and radish is on the X, and no Drosophila PKC gene maps to the genetically defined radish locus. There are two principal possibilities explaining how MaPKMzeta-induced memory enhancement bypasses the defect of radish mutants: (1) MaPKMzeta is downstream of radish or (2) MaPKMzeta activates a pathway that is parallel to and independent of radish. The first interpretation is favored because memory after massed training can be either enhanced or disrupted and the radish phenotype can be partially rescued (Drier, 2002).

The temporal specificity of the MaPKMzeta-dependent memory enhancement implies restrictions on its biochemical mechanism(s); enhancement requires that prior activity-dependent mechanisms be in place, and MaPKMzeta has a narrow post-training interval in which to act. If these kinetic restrictions do exist, the rapid induction achievable with the heat-shock promoter is essential for the detection of memory enhancement in these experiments (Drier, 2002).

There are two general interpretations of these data: PKMzeta acts to increase either (1) the magnitude or (2) the duration of the synaptic potentiation that underlies the behavior. In the first model, PKMzeta enhances the synaptic machinery induced by training, making a 'stronger' synaptic connection that decays more slowly. In the second model, PKMzeta acts solely to maintain the synapses previously modified by experience, with no effect on the induction of the potentiation. If one considers the behavioral measurements of learning (testing done immediately after training) and memory (testing done after a longer time) with induction and maintenance, respectively, the chelerythrine and dominant-negative data argue for a role in maintenance. Neither of these treatments affect learning, but each inhibits memory. No enhancement of learning was detected by prior induction of PKMzeta, nor was there an improvement of 3-hour memory if PKMzeta was induced 30 minutes after training. Although the magnitude and duration models may be artificially exclusive, taken together these data are most consistent with a role of PKMzeta in the maintenance of experience-dependent synaptic plasticity (Drier, 2002).

The stability of a synapse varies in response to different regimens of stimuli. Long-lasting changes normally require multiple stimuli and depend on new protein synthesis. Recent experiments support the existence of a synaptic marking system that enables neurons to tag recently active synapses, thus maintaining synaptic specificity during the cell-wide process of protein synthesis-dependent long-term memory formation. A synapse that would normally be stable for only a short period of time can be potentiated for a much longer period of time. However, to do so it must be activated within 2-4 hours of stimulation that produces long-term changes at a second and separate synapse within the same neuron. Although there is no direct evidence for a role of PKMzeta in this process, the similarity between the temporal windows for the proposed synaptic tag and the memory enhancement observed suggests a mechanistic relationship between them (Drier, 2002).

DaPKC is part of a multiprotein complex important for both cell polarity and the asymmetrical cell divisions of early Drosophila neurogenesis. These processes show strong structural and functional parallels with the first asymmetrical cell division of Caenorhabditis elegans embryogenesis. The Drosophila homologs of C. elegans proteins important for this process, Par-3 (Bazooka) and Par-6 (DmPar-6), interact with each other and with DaPKC to direct a specific and interdependent subcellular localization of the complex. During early Drosophila embryogenesis, Bazooka, DmPar-6, and DaPKC are localized to the zonula adherens, a cell junction structure. Mutation in any one of these genes disrupts the ability of the remaining two proteins to localize to this structure properly, and this disrupts cell polarity. This mutual dependence for localization is also apparent during neurogenesis, and causes the inappropriate segregation of cell determinants. This multiprotein complex is critical in mammalian cell polarity and in organizing junctions between epithelial cells. The mouse homologs of Bazooka and Par-6 are expressed in various regions of the CNS, and their subcellular localization within CA1 hippocampal neurons is consistent with a role in synaptic plasticity. Bazooka and DmPar-6 are expressed in Drosophila heads, as are DaPKC and DaPKM. It remains unclear how DaPKM activity is regulated during memory mechanisms; however, the subcellular localization affected by the Bazooka-DmPar-6-DaPKC complex provides hypotheses with attractive physical properties (Drier, 2002).

Atypical PKM is sufficient to enhance memory in Drosophila, and the chelerythrine and dominant-negative data suggest that it is also necessary for normal memory. Strikingly corroborative results have also been obtained for the role of PKMzeta in the maintenance phase of LTP. Injection of MaPKMzeta into CA1 pyramidal cells is sufficient to potentiate evoked excitatory postsynaptic currents. The potentiation occludes LTP and is reversed by chelerythrine. The introduction of the KI-MaPKMzeta into a CA1 cell abolishes its ability to support LTP. The non-NMDA receptor antagonist CNQX blocks this potentiation, indicating that it occurs via AMPA receptors. When these physiological results, obtained in rat hippocampal slice preparations, are combined with the Drosophila behavioral data, they point to a central role of atypical PKM in the mechanism of memory maintenance. Understanding the regulation of atypical PKMzeta, as well as what it in turn regulates, may be critical to unraveling this process (Drier, 2002 and references therein).

Quantitative analysis of protein dynamics during asymmetric cell division

In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Partner of numb (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, 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).

Aurora-A acts upstream of aPKC as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts

The choice of self-renewal versus differentiation is a fundamental issue in stem cell and cancer biology. Neural progenitors of the Drosophila post-embryonic brain, larval neuroblasts (NBs), divide asymmetrically in a stem cell-like fashion to generate a self-renewing NB and a ganglion mother cell (GMC), which divides terminally to produce two differentiating neuronal/glial daughters. Aurora-A (AurA) acts as a tumor suppressor by suppressing NB self-renewal and promoting neuronal differentiation. In aurA loss-of-function mutants, supernumerary NBs are produced at the expense of neurons. AurA suppresses tumor formation by asymmetrically localizing atypical protein kinase C (aPKC), an NB proliferation factor. Numb, which also acts as a tumor suppressor in larval brains, is a major downstream target of AurA and aPKC. Notch activity is up-regulated in aurA and numb larval brains, and Notch signaling is necessary and sufficient to promote NB self-renewal and suppress differentiation in larval brains. These data suggest that AurA, aPKC, Numb, and Notch function in a pathway that involved a series of negative genetic interactions. This study has identified a novel mechanism for controlling the balance between self-renewal and neuronal differentiation during the asymmetric division of Drosophila larval NBs (Wang, 2006).

When aurA function is compromised, mutant NBs acquire some features of cancer stem cells. They divide to generate a large number of daughter cells capable of self-renewal. This excessive self-renewal occurs at the expense of neuronal differentiation, suggesting that the normally asymmetric NB divisions have been altered such that the mutant NBs can divide symmetrically to generate two NB-like daughters. Cell cycle regulator CycE and cell growth factor dMyc are expressed in most of these tumor-like cells. Up-regulation of CycE is required for aurA overgrowth phenotype. AurA also regulates proper orientation of the mitotic spindle probably by controlling asymmetric localization of Mud. Both proteins are localized to centrosomes and are required for centrosome function. Centrosome abnormality and chromosome segregation defects in aurA could lead to aneuploidy, and many cancer cells exhibit centrosome defects and chromosome instability. Mammalian AurA when overexpressed can be oncogenic. However, future studies on its possible role as a tumor suppressor will be particularly interesting (Wang, 2006).

The data suggest that aurA negatively regulates aPKC function to regulate NB self-renewal. aPKC appears to act as a NB proliferation factor since overexpression of a modified membrane-targeted version, aPKC-CAAX, which exhibits ectopic cortical localization throughout the NB cortex, leads to overproliferation and tumor formation, similar to loss of aurA. AurA is required for the asymmetric localization of aPKC and restrict aPKC to the cortical region associated with the future NB daughter and loss of aurA results in delocalization of aPKC to the entire cortex. Consistent with and supporting this notion, loss of aPKC can suppress, albeit partially, the aurA mutant overgrowth phenotype (Wang, 2006).

In contrast to the well-studied role of Numb as a cell fate determinant during asymmetric divisions of embryonic GMCs, SOPs, or muscle progenitors, a role for Numb during NB asymmetric divisions has not been described. This study shows that Numb also acts as a tumor suppressor in Drosophila larval brains, and that Numb is a key downstream target of AurA and aPKC in the regulation of NB self-renewal. In both aurA mutant NBs or NBs overexpressing aPKC-CAAX, the asymmetric localization of Numb is compromised and the resultant overgrowth phenotype is consistent with that of numb loss-of-function. numb and aurA mutant NBs also share several common features including excessive self-renewal at the expense of neuronal differentiation as well as the membrane enrichment of Spdo, a positive regulator of Notch signaling. These data suggest that AurA positively regulates Numb function. Genetic analysis is consistent with the notion that this is achieved through the negative regulation of aPKC that in turn negatively regulates Numb (Wang, 2006).

Numb is known to be a negative regulator of Notch signaling. The current findings indicate that Notch is necessary and sufficient for promoting larval NB proliferation and suppressing neuronal differentiation. Genetic epistasis studies suggest that an AurA-aPKC-Numb-Notch genetic hierarchy acts to regulate self-renewal of Drosophila neural progenitor cells. During a wild-type larval NB asymmetric division, aurA acts to negatively regulate aPKC and restrict its localization to the cortical region associated with the future NB daughter; aPKC negatively regulates Numb and ensures that its localization/activity is restricted to the future GMC where Numb acts to antagonize Notch. The net effect is that Notch is asymmetrically activated in the NB daughter where it acts to promote self-renewal and suppress differentiation. Although these data suggest that aurA acts through the aPKC/Numb/Notch pathway, given the partial suppression seen in the double mutants aPKC;aurA and Notchts-1;aurA, the possibility that additional mechanisms may be involved cannot be excluded (Wang, 2006).

Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation

Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. This study identified three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. It is concluded that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal (Lee, 2006b).

Mutations in aurA lead to a massive increase in larval brain neuroblasts. The major cause of this phenotype appears to be misregulation of neuroblast cortical polarity. One cortical polarity defect is increased basal localization of aPKC, which is sufficient to induce ectopic neuroblasts. Consistent with this hypothesis, aPKC aurA double mutants show strong suppression of the aurA supernumerary neuroblast phenotype, consistent with aPKC functioning downstream from AurA. While it is possible that loss of aPKC suppresses the phenotype in a nonspecific way (e.g., by arresting neuroblast cell proliferation or inducing neuroblast apoptosis), ni similarly strong suppression of the brat supernumerary neuroblast phenotype was observed in aPKC brat double mutants. This shows that aPKC functions more specifically in the AurA pathway than in the Brat pathway (Lee, 2006b).

The only other detectable cortical polarity defect seen in aurA mutant neuroblasts is a delocalization of Numb from the basal cortex. A similar Numb defect is seen during asymmetric cell division of pupal SOPs in aurA mutants, perhaps reflecting a specific and direct regulation of Numb by AurA, although Numb is not phosphorylated by AurA in vitro. The importance of the Numb delocalization phenotype is revealed by the ability of Numb overexpression in neuroblasts to rescue most of the aurA mutant phenotype (all except the component due to spindle orientation defects). Thus, Numb acts downstream from AurA to inhibit neuroblast self-renewal. Numb joins Mira/Pros/Brat as proteins that are partitioned into the GMC during neuroblast asymmetric cell division, where they function to inhibit neuroblast self-renewal (Lee, 2006b).

Where does AurA function to inhibit neuroblast self-renewal? AurA appears to be required in the neuroblast lineage, and not in surrounding glial cells or nonneuronal tissues of the larva, because neuroblast-specific expression of either AurA or the downstream component Numb can rescue most of the aurA supernumerary neuroblast phenotype. This shows that AurA is not required outside the neuroblast lineage to inhibit neuroblast self-renewal. Within the neuroblast, AurA appears to function in the cytoplasm and not at the centrosome, because cnn mutants lack all detectable AurA centrosomal localization yet do not match the aurA supernumerary neuroblast phenotype. It is concluded that AurA acts in the neuroblast cytoplasm to promote aPKC/Numb cortical polarity and spindle-to-cortex alignment (Lee, 2006b).

How does Numb inhibit neuroblast self-renewal in the GMC? Numb is a well-characterized inhibitor of Notch signaling that is segregated into the GMC, and Notch signaling is active in larval neuroblasts but not in GMCs. Thus the most obvious model is that Numb blocks Notch receptor signaling in the GMC. However, Notch mutant clones generated in larval neuroblasts do not affect neuroblast survival or clone size. Similarly, no change has been seen in neuroblast number in two different Notch-ts mutants (although the expected small wing imaginal disc phenotype was observed. In addition, no supernumerary neuroblasts were observed in larval neuroblast clones overexpressing the constitutively active Notch intracellular domain, although the same Notch intracellular domain generates the expected sibling neuron phenotype when expressed in the embryonic CNS. Thus, Notch is an excellent candidate for promoting neuroblast self-renewal, but additional experiments will be needed to test this model more rigorously. In this context, it is interesting to note that Notch promotes stem cell self-renewal in mammals (Lee, 2006b).

aurA mutant neuroblasts have essentially random orientation of the mitotic spindle relative to the apical/basal cortical polarity axis, resulting in a some neuroblasts dividing symmetrically (in size and cortical polarity markers). This phenotype may arise due to lack of astral microtubule interactions with the neuroblast cortex; aurA mutant neuroblasts have reduced astral microtubule length. Alternatively, AurA may affect spindle orientation by phosphorylating proteins required for spindle orientation, such as Cnn, Pins, or Mud. For example, Mud has a consensus AurA/Ipl1 phosphorylation site within its microtubule-binding domain, and it will be interesting to determine if this site needs to be phosphorylated for Mud to bind microtubules. Spindle orientation defects only generate part of the supernumerary neuroblast phenotype in aurA mutant brains, however, because overexpression of Numb can rescue most of the phenotype without rescuing spindle alignment, and cnn or mud mutants have nearly random spindle alignment but only a modest increase in neuroblast number. Thus, it is proposed that spindle orientation defects and cortical polarity defects combine to generate the dramatic supernumerary neuroblast phenotype seen in aurA mutants (Lee, 2006b).

Mammalian aurA has been termed an oncogene due to its overexpression in several cancers, its ability to promote proliferation in certain cell lines, and the fact that reduced levels lead to multiple centrosomes, mitotic delay, and apoptosis. However, an in vivo aurA mutant phenotype has not yet been reported. In contrast, aurA loss-of-function mutations result in a neuroblast 'brain tumor' phenotype, including prolonged neuroblast proliferation during pupal stages when wild-type neuroblasts have stopped proliferating. aurA mutants do not, however, have the imaginal disc epithelial overgrowth seen in other Drosophila tumor suppressor mutants, and aurA mutant neuroblasts have a delay in cell cycle progression. It is proposed that the aurA supernumerary neuroblast phenotype is not due to loss of growth control or a faster cell cycle time, but rather due to a cell fate transformation from a differentiating cell type (GMC) to a proliferating cell type (neuroblast) (Lee, 2006b).

It is concluded that AurA restrains neuroblast numbers using two pathways: first by promoting Numb localization into the GMC, and second by promoting alignment of the mitotic spindle with the cortical polarity axis. Absence of the first pathway leads to increased neuroblasts at the expense of GMCs, whereas absence of the second pathway leads to increased neuroblasts due to symmetric cell division. It will be interesting to determine whether mammalian AurA uses one or both pathways to regulate stem cell asymmetric division and self-renewal (Lee, 2006b).

Protein phosphatase 2A negatively regulates aPKC signaling by modulating phosphorylation of Par-6 in Drosophila neuroblast asymmetric divisions

Drosophila neural stem cells or neuroblasts undergo typical asymmetric cell division. An evolutionally conserved protein complex, comprising atypical protein kinase C (aPKC), Bazooka (Par-3) and Par-6, organizes cell polarity to direct these asymmetric divisions. Aurora-A (AurA) is a key molecule that links the divisions to the cell cycle. Upon its activation in metaphase, AurA phosphorylates Par-6 and activates aPKC signaling, triggering the asymmetric organization of neuroblasts. Little is known, however, about how such a positive regulatory cue is counteracted to coordinate aPKC signaling with other cellular processes. During a mutational screen using the Drosophila compound eye, microtubule star (mts), which encodes a catalytic subunit of protein phosphatase 2A (PP2A), was identified as a negative regulator for aPKC signaling. Impairment of mts function causes defects in neuroblast divisions, as observed in lethal (2) giant larvae (lgl) mutants. mts genetically interacts with par-6 and lgl in a cooperative manner in asymmetric neuroblast division. Furthermore, Mts tightly associates with Par-6 and dephosphorylates AurA-phosphorylated Par-6. This genetic and biochemical evidence indicates that PP2A suppresses aPKC signaling by promoting Par-6 dephosphorylation in neuroblasts, which uncovers a novel balancing mechanism for aPKC signaling in the regulation of asymmetric cell division (Ogawa, 2009).

Polarity is a fundamental characteristic of cells and underlies a variety of cellular processes involved in the development and homeostasis of living organisms. In epithelial cells, which consist of the apical and basolateral membrane domains, cell polarity creates distinct subcellular compartments to arrange the cells into a well-ordered structure. In asymmetric cell division, cell polarity is coupled with mitosis. Cell polarity creates two subcellular domains with distinct characteristics in the mitotic mother cell and coordinates the mitotic spindle with the polarity axis to allow the two daughter cells to be distinct. Because these cell polarity events are tightly linked to other elementary processes such as the cell cycle and mitotic events, cell polarity is finely controlled to coordinate with those cellular processes (Ogawa, 2009).

Drosophila neural-stem-like cells, or neuroblasts, undergo typical asymmetric divisions, providing an excellent model for the study of how cell polarity is controlled. Neuroblasts repeatedly divide into a large, self-renewing daughter (the neuroblast itself) and a smaller, differentiating daughter [the ganglion mother cell (GMC)]. Cell fate determinants, such as Prospero, Brain tumor (Brat) and Numb, are segregated to the GMC. The localization of these determinants and the coordination with mitotic spindle orientation are controlled by the apically localized protein complexes -- the aPKC-Par complex and the Pins complex -- which are mutually linked by Inscuteable (Insc). The aPKC-Par complex consists of atypical protein kinase C (aPKC), Bazooka (Baz) and Par-6 and is primarily involved in organizing cell polarity and the asymmetric distribution of the cell fate determinants along the axis of polarity. The Pins complex, which consists of Partner of Inscuteable (Pins), Locomotion defects (Loco) and Gαi, determines the orientation of the mitotic spindle relative to the cell polarity axis (Ogawa, 2009).

aPKC is a key enzyme involved in establishment of neuroblast polarity and definition of the apical cortex. A tumor suppressor protein, Lethal (2) giant larvae (Lgl), is thought to antagonize aPKC as an inhibitory substrate. Although aPKC binds to non-phosphorylated Lgl, Lgl that is phosphorylated by aPKC dissociates from it and is released from the cell cortex. In the absence of aPKC, the entire cortex becomes basal, and Miranda, an adaptor protein for Prospero and Brat, distributes uniformly throughout the cortex. However, loss of Lgl results in uniform activation of aPKC in the cortex just as if the entire cortex were apical. Consequently, Miranda misdistributes into the cytoplasm and concentrates on mitotic spindles (Ogawa, 2009).

The apical complex and the basal determinants dynamically change their localization as the cell cycle progresses. The apical complex accumulates at the apical cortex during late interphase, retains its apical localization during metaphase, and then initiates expansion through the cortex in anaphase. Miranda and its cargos are temporally found in the apical cortex in late interphase and, after spreading into the cytoplasm at the onset of mitosis, form the basal crescent that is complementary to the localization of the apical complex during metaphase. At late anaphase onwards, they are restricted to the GMC compartment, which is separated by the contractile ring from the neuroblast compartment (Ogawa, 2009).

It was recently shown that the mitotic kinase Aurora-A (AurA) has an important role in linking the cell cycle to the asymmetric cell division of neuroblasts and sensory organ precursors (SOPs) by phosphorylating Par-6. When AurA is inactive, aPKC binds to unphosphorylated Par-6 and Lgl and remains inactive. Phosphorylation of Par-6 by AurA blocks the interaction of Par-6 with aPKC, which in turn leads to activation of aPKC. Activated aPKC then phosphorylates Lgl to replace it with Baz. The Par complex that has recruited Baz is able to phosphorylate Numb, leading to an exclusion of Numb from the apical domain. Because AurA becomes active in the mitotic phase, it is able to synchronize aPKC activation with the entry into mitosis. Given the role of AurA as a positive regulator of aPKC signaling, it is also likely that dephosphorylation negatively regulates this signaling pathway (Ogawa, 2009).

The serine/threonine phosphatases are grouped into four major classes based on their sensitivity to inhibitors and requirement for divalent cations: protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B) and protein phosphatase 2C (PP2C). PP2A holoenzyme functions as a heterotrimeric complex comprising a catalytic C subunit [Microtubule star (Mts) in Drosophila], a scaffolding A subunit (PP2A-29B in Drosophila) and a regulatory B-subunit [Twins (Tws), Widerborst (Wdb) and PP2A-B' in Drosophila]. The A-subunit can serve as a linker between the C- and B-subunits, and the B-subunit can influence the enzymatic activity and substrate specificity of the holoenzyme. In a genetic screen using the Drosophila compound eye, mts was identified as an enhancer of the aPKC-induced eye phenotype. The genetic and biochemical evidence indicating that Mts suppresses aPKC activity by enhancing dephosphorylation of Par-6 in neuroblasts uncovered an antagonistic role of PP2A in the aPKC signaling pathway (Ogawa, 2009).

The Drosophila compound eye is composed of repetitive ommatidia that contain epithelial retinal cells. Because of the crystal-like arrangement of ommatidia in the compound eye, it is sensitive to defects in epithelial polarity and therefore ideal for use in mutational screens for components involved in epithelial polarity. A modifier screen was undertaken under a sensitized background to look for mutations that affected epithelial polarity. When the membrane-tethered aPKC (aPKCCAAX) is expressed by GMR-GAL4, it becomes expressed in all differentiated retinal cells, the apicobasal polarity of the retinal cells is severely impaired, and the compound eye becomes small and rough. Kinase activity of aPKCCAAX is essential for inducing this eye phenotype, because the kinase-dead version, in which Lys293 is mutated to Trp (K293W), does not alter the eye morphology. Using this system, mutants were screened that modify the aPKCCAAX-induced eye phenotype among lethal mutants available from stock centers, and mts was identified as a phenotypic enhancer. When the GMR-GAL4/UAS-aPKCCAAX fly was crossed to the mts02496 or mtsXE-2258 mutant fly, a smaller and rougher eye was observed, suggesting that Mts acts as an antagonist for the aPKC signaling pathway (Ogawa, 2009).

The mts gene is expressed ubiquitously during embryogenesis and its protein product localizes to the cytoplasm in neuroblasts as well as in epithelial cells. Because a large amount of mts mRNA is maternally supplied, zygotic mts mutant embryos do not show significant defects with regard to cell polarity, and germline clones do not produce an egg. Therefore loss-of-function phenotypes were examined in neuroblasts by overexpressing a dominant-negative mutant of Mts (dnMts) (Hannus, 2002), which lacks the N-terminal region of the phosphatase domain. In wild-type neuroblasts, the protein complex containing aPKC, Par-6 and Baz localizes to the apical cortex and directs Miranda to the basal cortex at metaphase. In the dnMts-expressing embryos, the apical complex localizes to the apical cortex, and its distribution is broader than normal. By contrast, localization of Miranda is severely affected, and it is distributed less asymmetrically along the cell cortex and into the cytoplasm, where it is concentrated on the mitotic spindles. This phenotype resembles that of lgl, raising the possibility that Mts functions in the same pathway as Lgl (Ogawa, 2009).

This study shows that PP2A functions as a negative regulator of the aPKC signaling pathway in Drosophila neuroblasts. Although several studies have suggested that PP2A negatively regulates aPKC signaling in mammalian culture cells, the critical target(s) of PP2A is unknown in these studies. The substrates of aPKC, which include Lgl and aPKC itself, can also be substrates for PP2A. However, none of them has been molecularly identified as a target to be dephosphorylated by PP2A. This study has identified Par-6 as a direct target of PP2A, which has its catalytic subunit encoded by the mts gene. Par-6 is known to be phosphorylated by AurA to trigger aPKC activation when neuroblasts and SOPs enter mitosis. Biochemical and genetic evidence reveals that Mts dephosphorylates Par-6 to suppress the aPKC pathway, suggesting an antagonistic role for Mts against AurA in the regulation of cell polarity that is governed by aPKC signaling (Ogawa, 2009).

Co-immunoprecipitation assays of overexpressed Par-6, aPKC or Lgl with Mts in S2 cells indicated that all these molecules can form a complex either directly or indirectly. Among these, the association of Mts with aPKC and Lgl is relatively weaker than the association with Par-6, although a previous study suggested that PP2A associates with aPKC to suppress its kinase activity in mammalian cultured cells (Nunbhakdi-Craig, 2002). In the current study results indicate that, in S2 cells, Par-6 most efficiently forms a complex with Mts. Consistently, the in vitro dephosphorylation assay showed that PP2A effectively dephosphorylates AurA-phosphorylated Par-6 but not the auto-phosphorylated PKCzeta or the PKCzeta-phosphorylated Lgl. It is inferred from these results that Par-6 is a direct target of Mts. Substrate specificity of PP2A is greatly influenced by the B-subunit incorporated into the holoenzyme. Thus, differences in the affinity with Mts among the three tested molecules in co-immunoprecipitation assays might, therefore, partly reflect the B-subunit(s) that is expressed in S2 cells endogenously. The Drosophila genome contains three genes for the B-subunit: tws, wdb and PP2A-B', all of which are ubiquitously expressed during embryogenesis, as is mts (Ogawa, 2009).

At present, it is not clear which of these three is used for targeting Par-6. Among them, tws mutants often show bristle duplications that are due to defective cell fate decisions of the SOP, as lgl4/lglts3 flies show. Since this lgl4/lglts3 phenotype is enhanced by mts, mts is also likely to be involved in the same pathway. Furthermore, a recent study demonstrated that Tws, together with Mts, is included in the aPKC complex to regulate the asymmetric cell division of larval neuroblasts (Chabu, 2009). These results suggest that Mts uses Tws to target Par-6 in the asymmetric cell divisions of SOPs as well as of neuroblasts (Ogawa, 2009).

Par-6 is an essential cofactor for aPKC activity, and it is known to keep aPKC inactive in the absence of AurA-dependent phosphorylation of Par-6 in neuroblasts. The complete deprivation of Par-6 results in the uniform distribution of Miranda into the cell cortex, which is reminiscent of the aPKC mutant phenotype. Thus, Par-6 is required to produce functional aPKC, and its kinase activity becomes active only when Par-6 is phosphorylated by AurA. par-6 heterozygotes (par-6δ226/+) do not show any defect in Miranda localization during the embryonic stage, which indicates that one copy of par-6 in addition to the maternal supply is sufficient to support normal aPKC function. When mts is further inactivated under this condition (par-6δ226/+, mtsXE-2258/mts02496), some neuroblasts exhibit an lgl-like phenotype in Miranda localization, which is indicative of aPKC hyperactivation and unlike the par-6 loss-of-function mutant. This result suggests that Par-6, because of its hyper-phosphorylation, becomes unable to restrict aPKC activity within the normal range. Thus, a probable normal function of Mts is to promote the inhibitory function of Par-6 on aPKC without affecting its function as an essential subunit of the aPKC complex (Ogawa, 2009).

Whereas AurA seems to be active only during the mitotic phase in cell-cycling cells, mitotically inactive or interphase epithelial cells exhibit concrete apicobasal polarity. How do those cells activate aPKC signaling even though AurA is inactive? This apparent paradox raises several possibilities. Par-6 phosphorylation is required for aPKC activation in epithelial cells but might be mediated by kinase(s) other than AurA. Alternatively, aPKC might be activated by mechanisms other than the phosphorylation of Par-6. Indeed, it has been reported that the active form of Cdc42 binds to the CRIB domain of Par-6 to relieve its inhibitory effect on aPKC, leading to the activation of aPKC (Ogawa, 2009).

Although obvious defects are not detected in the epithelial cells of zygotic mts mutant embryos, Oogenesis clones of mts show dramatic defects in their epithelial polarity. This follicle cell phenotype is different from that caused by hyperactivation of aPKC, as observed in the lgl mutant, suggesting that the action of Mts is mechanistically different in the maintenance of follicle cell polarity from that observed in neuroblasts. In photoreceptor cells, Mts operates antagonistically against Par-1 kinase, which restricts Baz to the adherens junctions. It is also known that Par-1 phosphorylates Baz directly to inhibit its incorporation into the apical aPKC complex, thereby restricting Baz to the apical domain in follicle cells. These data raise the possibility that Mts antagonizes Par-1 in Baz localization in follicle cells by inhibiting Par-1-dependent Baz phosphorylation. If this is the case, Mts positively regulates the aPKC pathway in follicle cells and photoreceptor cells, unlike the situation in neuroblasts. To date, there is no report for Par-1 function in Drosophila neuroblasts. Further study is necessary to test whether a similar antagonism between Mts and Par-1 has a role in the regulation of neuroblast polarity (Ogawa, 2009).

AurA-mediated Par-6 phosphorylation is a key step in initiating the asymmetric segregation of the cell fate determinants in the neuroblast cell cycle. Once Par-6 is phosphorylated, aPKC will be continuously activated during the mitotic phase. The apical domain would overwhelm the entire cortex unless an antagonistic reaction occurred. PP2A will be able to balance AurA in Par-6 phosphorylation during mitosis. Thus, PP2A, together with the antagonistic ligand Lgl, might have a role in maintaining aPKC activity at an appropriate level to create both apical and basal domains in the cortex during mitosis. Although both Mts and Lgl negatively regulate aPKC signaling, Mts operates on aPKC activity by directly regulating the cell-signaling cascade, whereas Lgl does so through the direct physical association as a substrate. Therefore, they are different in their mechanisms of action (Ogawa, 2009).

When neuroblasts complete cell cleavage, the basal membrane is largely segregated into the GMC, and the entire cell cortex of neuroblasts appears to become apical. It is therefore necessary to repolarize in order to make the apical and basal domains in the cell cortex for the onset of the next cell cycle. To do so, Par-6 phosphorylation must be removed before entering the next cell cycle, to reset the configuration of the apical complex. A model is proposed in which Mts actively dephosphorylates Par-6 to reset the membrane polarity after the completion of each division cycle. In this context, it will be important to examine whether Mts function depends on the cell-cycle stage in neuroblasts (Ogawa, 2009).

In eukaryotes, serine/threonine phosphatases are categorized into four major groups: PP1, PP2A, PP2B and PP2C. Recent studies have shown that PP1α affects Par-3 activity through the regulation of a phosphorylation-dependent interaction of Par-3 with 14-3-3 or PKCzeta. This study also identified a Pp1-87B mutation as an enhancer of the aPKCCAAX-induced eye phenotype in the genetic screen and found defects in localization of Miranda as well as in epithelial cell polarity in the Pp1-87B mutant. Furthermore, it has been reported that protein phosphatase 4 (PP4), which is a PP2A family member, regulates Miranda localization in Drosophila neuroblasts, although the direct substrate of PP4 is not yet clear. Thus, other classes of phosphatases in addition to PP2A are involved in the regulation of cell polarity in various cellular contexts. Further delineation of phosphatase functions and the crosstalk between phosphatases should help lead to an understanding of the global control of cellular processes regulated by cell polarity (Ogawa, 2009).

Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Thus study reports that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. It is concluded that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis (Chabu, 2009).

Drosophila aPKC regulates neuroblast cell polarity and neuroblast self-renewal, however understanding of how aPKC is regulated is far from complete. Several kinases regulate neuroblast cell polarity and cell fate, but the identity of opposing phosphatases have remained elusive. This study identified Twins as part of a protein complex containing aPKC. Twins is a regulatory subunit of PP2A, and this study also shows that the catalytic subunit of PP2A, Mts, is immunoprecipitated by aPKC. Furthermore, mts and twins mutants have similar defects in neuroblast cell polarity and expansion in neuroblast numbers. This strongly suggests that the Twins/PP2A complex regulates neuroblast polarity and self-renewal (Chabu, 2009).

The primary defect in twins mutant neuroblasts is an expansion of aPKC from the apical cortex to the basal cortex, and this ectopic aPKC is active based on its ability to exclude Miranda from the basal cortex. Twins/PP2A may promote apical Baz localization, similar to the role of PP2A in promoting Baz/Par-3 apical localization in epithelia; a reduced level of apical Baz in neuroblasts may lead to failure to localize all cortical aPKC at the apical cortex and hence ectopic basal aPKC. Alternatively, PP2A may keep active aPKC from the basal cortex by directly dephosphorylating aPKC at its N-terminus, consistent with the role of mammalian PP2A in dephosphorylating aPKCλ/ζ (Nunbhakdi-Craig, 2002). In support of this model, overexpression of aPKC lacking its N-terminus (aPKCΔN) displaces Miranda from the basal cortex into the cytoplasm, similar to twins mutant neuroblasts (Chabu, 2009).

How does Twins regulate neuroblast self-renewal? Ectopic active aPKC causes formation of supernumerary neuroblasts, as does reduced levels of the basal cortical protein Miranda. twins mutant neuroblasts have both ectopic basal cortical aPKC and a loss of basal cortical Miranda. It is likely that the primary defect causing supernumerary neuroblasts is ectopic aPKC, because reducing aPKC levels in twins mutants can rescue both basal Miranda targeting and the formation of supernumerary neuroblasts. This is in contrast to the role of another phosphatase, PP4, in regulating Miranda localization independent of aPKC (Sousa-Nunes, 2009; Chabu, 2009 and references therein).

It has been shown that Dap160, a protein related to mammalian Intersectin, is apically localized and required to anchor aPKC at the apical cortex (Chabu, 2008). This study has shown that Twins is also required for tight apical localization of aPKC. A major difference, however, is that Dap160 directly stimulates the activity of aPKC, so that in dap160 mutant neuroblasts the ectopic basal aPKC is inactive and unable to exclude Miranda from the cortex. In contrast, twins mutants have ectopic basal aPKC that remains active and thus can drive Miranda off the cortex. This supports the conclusion, from biochemical experiments, that Twins does not stimulate aPKC activity. However, the possibility that another regulatory subunit can target PP2A to aPKC in the absence of Twins cannot be excluded (Chabu, 2009).

Neuroectoderm cells of the optic lobe undergo a progressive differentiation to adopt a neuroblast fate. twins mutant optic lobes show a dramatic increase in optic lobe neuroblast numbers, suggesting that Twins normally functions to inhibit precocious neuroblast fate in the optic lobe neuroectoderm cells. How does Twins normally suppress precocious neuroectodermal-to-neuroblast differentiation? This study has show that at least one pathway utilizes aPKC to regulate neuroectoderm differentiation; twins mutant optic lobe with reduced active aPKC has a less severe phenotype compared to their twins mutant counter parts. Another pathway that has been implicated in the differentiation of neuroectoderm cells to neuroblast is the Janus Kinase/Signal transducer and activation of transcription (JAK/STAT) pathway. JAK/STAT signaling functions in neuroectoderm cells inhibits expression of proneural genes, thereby blocking precocious neuroblast differentiation. Twins/PP2A could act positively at any point in the JAK/STAT–proneural pathway, or in an independent pathway in promoting the neuroectodermal-to-neuroblast transition in the optic lobe (Chabu, 2009).

Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors

When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. This study shows that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. It was also shown that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. These observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration (Sun, 2011).

Many tissues have the capacity respond to the removal or death of cells by increasing proliferation of the remaining cells. In Drosophila, this phenomenon has been characterized both in the context of imaginal disc regeneration and compensatory cell proliferation. These studies implicate the Hippo signaling pathway as a key player in these proliferative responses to tissue damage. After genetically ablating the wing primordia by inducing apoptosis, it was observed that Yki becomes activated to high levels in surrounding cells, based on its nuclear abundance and induction of a downstream target of Yki transcriptional activity. Moreover, high level Yki activation is crucial for wing disc regeneration, as even modest reduction of Yki levels, to a degree that has only minor effects on normal wing development, severely impaired wing disc regeneration. While it was known that Yki is required for wing growth during development, the current observations establish that Yki is also required for wing growth during regeneration, and moreover that regeneration requires higher levels of Yki activation than during normal development (Sun, 2011).

These studies identify Jnk activation as a promoter of Yki activity in the wing disc. Most aspects of imaginal disc development, including imaginal disc growth, normally do not require Jnk signaling. By contrast, Jnk signaling is both necessary and sufficient for Yki activation in response to wing damage. Jnk signaling has previously been linked to compensatory cell proliferation and regeneration in imaginal discs, and it is now possible to ascribe at least part of that requirement to activation of Yki. However, Jnk signaling also promotes the expression of other mitogens, including Wg, which were linked to regeneration and proliferative responses to apoptosis. Wg and Yki are not required for each other's expression, suggesting that they are regulated and act in parallel to influence cell proliferation after tissue damage. The mechanism by which Jnk activation induces Yki activation is not yet known. The observation that it could be suppressed by over-expression of Wts or Hpo suggests that it might impinge on Hippo signaling at or upstream of Hpo and Wts, but the possibility that Jnk-dependent Yki regulation occurs in parallel to these Hippo pathway components cannot be excluded. The high level of nuclear Yki localization is striking by contrast with the more modest effects of upstream tumor suppressors in the Hippo pathway, which suggests that Jnk might regulate Yki through a distinct mechanism, or simultaneously affect multiple upstream regulators (Sun, 2011).

Strong Yki activation was detected within the wing and haltere discs in response to Jnk activation, but weaker or non-existent effects in leg or eye discs. Jnk activation has previously been linked to oncogenic effects of neoplastic tumor suppressors in eye discs, and it is possible that Yki activation might be induced in eye discs if a distinct Jnk activation regime were employed. Nonetheless, since identical conditions were employed in both wing and eye discs, isolating them from the same animals, these studies emphasize the importance of context-dependence for Yki activation by Jnk. A link between Jnk activation and Yki activation is not limited to the wing however, as a connection between these pathways was recently discovered in the adult intestine, where damage to intestinal epithelial cells, and activation of Jnk, can activate also Yki (Sun, 2011).

There was a general correspondence between activation of Jnk and activation of Yki under multiple experimental conditions, including expression of Rpr, direct activation of Jnk signaling by Egr or Hep.CA (an activated form of the Jnk kinase Hemipterous), and depletion of lgl. Some experiments, most notably direct activation of Jnk by Hep.CA, revealed a non-autonomous effect on Yki, which could imply that the influence of Jnk on Yki activity is indirect. Although the basis for this non-autonomous effect is not yet known, the hypothesis that it is actually also mediated through Jnk signaling is favored, since it has been reported that Jnk activation can propagate from cell to cell in the wing disc. Consistent with this possibility, a non-autonomous activation of Jnk adjacent to lgl depleted cells was seen to be blocked by depletion of bsk solely within the lgl RNAi cells. Conversely, alternative signals previously implicated in compensatory cell proliferation do not appear to be good candidates for mediating Yki activation, since it was found that Wg is not required for Yki activation in regenerating discs, and prior studies did not detect a direct influence of Dpp pathway activity on Yki activation (Sun, 2011).

Activation of Yki adjacent to Egr- or Rpr-expressing cells was also reduced by over-expression of Wts. This might reflect an influence of Yki on signaling from these cells, but because expression of Wts inhibits Yki activity, and activated Yki promotes expression of an inhibitor of apoptosis (Diap1), it is also possible that this effect could be explained simply by Wts over-expression resulting in reduction or more rapid elimination of Egr- or Rpr-expressing cells; the reduced survival of these cells would then limit their ability to signal to neighbors (Sun, 2011).

Although Jnk has been implicated in compensatory cell proliferation and regeneration, it is better known for its ability to promote apoptosis. The dual, opposing roles of Jnk signaling as a promoter of apoptosis and a promoter of cell proliferation raise the question of how one of these distinct downstream outcomes becomes favored in cells with Jnk activation (see Diverse inputs and outputs of Jnk signaling). Given the links between Jnk activation and human diseases, including cancer, defining mechanisms that influence this is an important question, and the identification of the role of Yki activation in Jnk-mediated proliferation and wing regeneration should facilitate future investigations into how the balance between proliferation or apoptosis downstream of Jnk is regulated (Sun, 2011).

Hippo signaling is regulated by proteins that exhibit discrete localization at the subapical membrane, e.g., Fat, Ex, and Merlin. The observation that disruption of apical-basal polarity is associated with disruption of Hippo signaling underscores the importance of this localization to normal pathway regulation. These observations establish that Hippo signaling is inhibited by neoplastic tumor suppressor mutations, resulting in Yki activation, and that this activation of Yki is required for the tumorous overgrowths associated with these mutations (Sun, 2011).

Although these results agree with these recent studies in linking lgl to Hippo signaling (Grzeschik, 2010), there are some notable differences. A previous study examined lgl mutant clones in the eye imaginal disc, under conditions where cells retained apical-basal polarity, whereas this study examined wing imaginal discs, where apical-basal polarity was lost. Intriguingly this study found that conditions associated with activation of Yki by Jnk in the wing disc were not sufficient to activate Yki in the eye disc. This observation, together with the discovery that loss of polarity in lgl depleted wing cells requires Jnk activation, suggests as a possible explanation for why lgl null mutant clones retain apical-basal polarity in eye discs, that eye disc cells have a distinct, and apparently reduced, sensitivity to Jnk activation as compared to wing disc cells (Sun, 2011).

This study also identified distinct processes linked to Yki activation in the absence of lgl. A previous study reported an effect of lgl on Hpo protein localization (Grzeschik, 2010). In wing discs, the discrete apical localization of Hpo was observed in studies of eye discs. Thus, the proposed mechanism, involving activation of Yki via mis-localization of Hpo and dRassf, might not be relevant to the wing. By contrast, this study identified an essential role for Jnk signaling in regulating Yki activation in lgl-depleted cells in the wing. Because this study did not detect an effect of direct Jnk activation on Yki in eye discs, it is possible that Lgl can act through multiple pathways to influence Yki, including a Jnk-dependent pathway that is crucial in the wing disc, and a Jnk-independent pathway that is crucial in the eye disc. Grzeschik (2010) also linked the influence of lgl in the eye disc to its antagonistic relationship with aPKC. The observation that the influence of aPKC in the wing depends on Jnk activation is consistent with an Lgl-aPKC link, and identifies a role for Jnk activation in the oncogenic effects of aPKC (Sun, 2011).

The observation that the loss of polarity in lgl RNAi discs is dependent upon Jnk signaling was unexpected, but a related observation was recently reported by Zhu; 2010). These results suggest that the established role of the Lgl-Dlg-Scrib complex in maintaining epithelial polarity depends in part on repressing Jnk activity. However, since Jnk activation on its own was not sufficient to disrupt polarity, multiple polarity complexes might need to be disturbed in order for wing cells to lose apical-basal polarity, including both Lgl and additional, Jnk-regulated polarity complexes (Sun, 2011).

The discovery of the role of Jnk signaling in Yki activation provides a common molecular mechanism for the overgrowths observed in conjunction with mutations of neoplastic tumor suppressors, and those associated with compensatory cell proliferation, because in both cases a proliferative response is mediated through Jnk-dependent activation of Yki. Although the molecular basis for the linkage of these two pathways is not understood yet, it operates in multiple Drosophila organs, and thus appears to establish a novel regulatory input into Hippo signaling that is of particular importance in abnormal or damaged tissues. Moreover, Jnk activation has also been observed in conjunction with regeneration of disc fragments after surgical wounding, and thus its participation in regeneration is not limited to paradigms involving induction of apoptosis. It is also noteworthy that under conditions of widespread lgl depletion (i.e., lgl mutant or lgl RNAi), and consequent Jnk activation, the balance between induction of apoptosis and induction of cell proliferation is shifted towards a proliferative response. By contrast, in the wing disc clones of cells mutant for lgl fail to survive, unless oncogenic co-factors are co-expressed. The loss of lgl mutant clones in wing discs was recently attributed to cell competition. Together, these observations suggest that the choice between proliferative versus apoptotic responses to Jnk activation can be influenced by the Jnk activation status of neighboring cells (Sun, 2011).

Anisotropy of Crumbs and aPKC drives myosin cable assembly during tube formation

The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, this study found that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. This study shows that Crumbs directs aPKC anisotropy which negatively regulate myosin II, probably through Rok. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized (Roper, 2012).

Myosin II has emerged as a key player in morphogenesis because of its ability to form contractile structures together with F-actin that can directly alter the shapes of cells. Different pools of myosin II within epithelial cells undergoing morphogenesis have been observed, namely apical junctional myosin, apical medial myosin, and in addition myosin organized into supracellular structures termed myosin cables or purse-strings. All three myosin II pools have been shown to be important for epithelial morphogenesis, but how much the activities of the pools depend on each other and how their specific assembly is regulated is much less clear (Roper, 2012).

Using the formation of the invagination of the salivary glands in the fly embryo as a model allowed me to analyze a morphogenetic process in which all three different pools of myosin are present. Upon specification of the gland placode, myosin II levels are drastically upregulated in the secretory cells of the placode, and myosin accumulates at cortical regions and medially within the apical 'dome' of each cell. In addition, a supracellular myosin cable surrounding the placode is formed in a process by which parts of existing structures (remnants of parasegmental cables) are joined together with a newly specified dorsal section of the cable (Roper, 2012).

Compared to mesoderm invagination in the fly, a well-studied process that depends on both apical medial and cortical myosin assemblies, the invagination of the tubes of the salivary gland topologically rather resembles wound healing or dorsal closure processes, as the surrounding epidermis is drawn in from around the placode to cover the patch where cells are invaginating into the embryo (Roper, 2012).

All three processes have in common that the patch of cells 'disappearing' from the plane of the epithelium is surrounded by a contractile actomyosin cable. In contrast to wound healing and dorsal closure, the cable in the case of salivary gland tubulogenesis is assembled within the cells on the inside, whereas it is assembled in the surrounding epithelial cells in the former two instances. Thus, the signal for cable assembly is provided by the 'inside' cells in the salivary gland placode (Roper, 2012).

The laser ablation data presented in this study clearly demonstrate that the cable around the placode is under increased tension, even when compared to other myosin enriched edges. The tension is in magnitude comparable to the tension determined for shorter supracellular myosin cables observed during germband extension in the fly embryo. This increased tension indicates active involvement in the invagination process. Previous modeling studies on sea urchin invagination have shown that a contractile apical ring surrounding a placode could be a driving force for invagination. Interestingly, upon laser ablation the cable around the salivary gland placode was very quickly repaired, suggesting a continuous signal to assemble myosin at the outermost surface of the placode. This fast repair precluded laser ablation as a means of probing function of the cable in the invagination in contrast to medial and junctional myosin (Roper, 2012).

Crumbs, the transmembrane component of the apical protein complex, shows a very striking anisotropic localization at the border of the placode, that is complementary to the accumulation of myosin II forming the cable. The data strongly support a model whereby Crumbs intracellular tails at cell edges facing toward the inside of the placode recruit aPKC, which can act as a negative regulatory factor impinging on Rok, thus preventing cable assembly at edges containing high levels of Crumbs tails. This leaves active Rok at the cell edges forming the placode boundary, where it acts to recruit myosin into the cable.

Interestingly, only the presence or artificial introduction of cortical anisotropy of Crumbs and downstream aPKC has this effect. The central cells of the placode that are not forming the boundary all show strongly upregulated levels of Crumbs, aPKC, Rok, and myosin II, but in these cells a high density of Crumbs tails does not preclude accumulation of junctional membrane-proximal myosin. Thus, the change in density of Crumbs tails, not the overall concentration, is instructive in this system (Roper, 2012).

Crumbs has previously been shown to have an effect on salivary gland morphogenesis through a proposed regulation of the apical membrane domain and has been implicated in tracheal pit invagination through regulation of phospho-Moesin). Also, members of the Crumbs polarity complex have been shown to be able to interact with the Par3 (Bazooka)/Par-6/aPKC complex (e.g., Par-6 can bind to Crumbsintra; aPKC can phosphorylate Crumbsintra. Also, an anticorrelation between localization of Crb and aPKC compared to Lgl and myosin has been described in the denticle belts of the fly epidermis (Kaplan, 2010). This work now describes a potential link from Crumbs through aPKC to Rok and myosin II, which would link the interaction of two different polarity factors directly with the coordination of morphogenesis through myosin II at a molecular level (Roper, 2012).

The large extracellular domain of Crumbs has long posed an enigma with regard to its role. Crumbs' function in epithelial polarity can mostly be mediated by its intracellular domain (Klebes, 2000). Only for photoreceptor morphogenesis, the extracellular domain appears required within the fly, though its molecular role is unclear. The protein domains present in the extracellular domain, namely EGF repeats and lamG domains, are both found in many classical and nonclassical cadherins. Data presented in this study suggest that Crumbs could be organized in the plasma membrane through homophilic interactions of the extracellular domains between molecules on neighboring cells: Crumbs shows highly anisotropic localization within the wild-type placode but also within wild-type cells bordering a crumbs mutant clone (Chen, 2010) or in clusters of Crumbs expressing cells in a null mutant embryo and within cells at the edge of an ectopic step change in Crumbs expression levels. Also in vitro, Crumbs accumulates at contact zones between expressing cells. The extracellular domain appears the ideal candidate to mediate this anisotropy, which is supported by the following findings: (1) the CrbTMextra-GFP shows anisotropic localization at borders with cells not expressing the construct; (2) endogenous Crumbs in wild-type cells is induced to localize in an anisotropic fashion when neighboring cells are depleted of endogenous Crumbs and only express the intracellular domain; and (3) the Crbintra-FLAG shows uniform expression in cells. These observations exclude that another transmembrane protein that interacts with the intracellular domain of Crumbs in equal stoichiometry could mediate the anisotropy, though it cannot formally exclude that another extracellular factor might act as an intermediary between two Crumbs extracellular domains. These data are strongly supported by recent evidence from zebrafish, where vertebrate Crumbs isoforms appear to mediate homophilic interactions to promote orderly arrays of photoreceptors. Also, recent data analyzing the establishment of polarity in the Drosophila follicular epithelium suggest a role for cis-interaction of Crumbs molecules within a single cell (Fletcher, 2012). Thus, a clear role for the Crumbs extracellular domain in organizing plasma membrane domains through homophilic interactions in cis and in trans is prominently emerging (Roper, 2012).

Data presented in this study describe a link between the transmembrane protein Crumbs and myosin II structures actively engaged in controlling morphogenesis. Crumbs' ability to interact in trans allows the step change in Crumbs levels between placode and surrounding cells to be translated into a subcellular asymmetry, the anisotropic localization of Crumbs. This mechanism provides the cells at the border of the salivary gland placode with the means of sensing this positional information and allows them to turn the positional information into a morphogenetic readout: myosin cable formation. In the future it will be interesting to determine if the arrangement of Crumbs and myosin II described in this study is conserved during topologically similar processes of tube invagination, such as, for instance, the side budding of branches during lung or mammary gland morphogenesis (Roper, 2012).

Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo

The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. This study found that the small GTPase Roughened/Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. The current linear model for polarity establishment was tested. Both Bazooka and aPKC regulate Canoe localization despite being 'downstream' of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data suggest that polarity establishment is regulated by a protein network rather than a linear pathway (Choi, 2013).

Polarity is a fundamental property of all cells, from polarized cell divisions in bacteria or fungi to the elaborate polarity of neurons. Among the most intensely studied forms of polarity in animal cells is epithelial apical-basal polarity. Polarity of epithelial sheets is key to their function as barriers between body compartments, and is also critical in collective cell migration and cell shape change during morphogenesis, as cytoskeletal and apical-basal polarity often go hand in hand. Loss of apical-basal polarity is a hallmark of metastasis. Significant advances have been made in defining the machinery required for cell polarity in many settings, but fundamental questions remain unanswered (Choi, 2013).

Cadherin-catenin complexes, which assemble into adherens junctions (AJs) near the apical end of the lateral cell interface, are critical polarity landmarks that define the boundary between apical and basolateral domains. Studies in C.elegans and Drosophila identified other key regulators of apical-basal polarity. In the textbook view, the apical domain is defined by the Par3/Par6/aPKC and Crumbs/Stardust(Pals1)/ PATJ complexes, while Scribble, Dlg, Lgl, and Par1 define the basolateral membrane (Choi, 2013).

Complex cross-regulatory interactions between apical and basolateral proteins maintain these mutually exclusive membrane territories. These proteins also regulate other types of polarity during morphogenesis; e.g., fly Par3 (Bazooka; Baz), aPKC, and AJ proteins are planar-polarized during fly convergent extension, thus regulating polarized cell movements (Choi, 2013).

Polarized cytoskeletal networks also play key roles in establishing and maintaining apical-basal and planar polarity. These networks are thought to be physically linked to apical junctional complexes. The earlier model suggesting that cadherin-catenin complexes link directly to actin via α-catenin is now viewed as over-simplified. Instead, different proteins are thought to mediate this connection in different tissues and at different times (Choi, 2013).

Among the linkers is Canoe (Cno)/Afadin, an actin-binding protein that binds transmembrane nectins via its PDZ domain. While originally hypothesized to be essential for cell adhesion, subsequent work supports a model in which afadin modulates adhesive and cytoskeletal machinery during cell migration in vitro and the complex events of mouse gastrulation. Afadin has two N-terminal Ras association domains for which the small GTPase Rap1 is the major binding partner, and Afadin and Rap1 are functionally linked in both flies and mice. Rap1, Cno, and the Rap1 GEF Dizzy/PDZGEF are all essential for maintaining effective linkage between AJs and the apical actomyosin cytoskeleton during apical constriction of Drosophila mesodermal cells during fly gastrulation. Rap1 regulates Cno localization to the membrane. Cno plays a related role during convergent extension, though its role is planar polarized during this process. Cno also regulates collective cell migration, signaling, and oriented asymmetric divisions. The Rap1/Cno regulatory module is also important in disease, as Afadin and Rap1 are implicated in congenital disorders of the cardiovascular system and cancer metastasis. It remains unclear whether these diverse roles all involve junction-cytoskeletal linkage or whether some are independent functions (Choi, 2013 and references therein).

The small GTPase Rap1 plays diverse cellular roles. Mammalian Rap1 isoforms are perhaps best known for regulating integrin-based cell matrix adhesion, but Rap1 also regulates cell-cell AJs in both Drosophila and mice. In murine endothelial cells, for example, Rap1, its effector Krit1, and VE-cadherin form a complex that regulates endothelial cell junctions and stabilizes apical-basal polarity (Choi, 2013 and references therein).

In Drosophila imaginal disc cells, Rap1 regulates the symmetric distribution of DE-cadherin (DEcad) around the apical circumference of each cell. Rap1 carries out these functions via a diverse set of effector proteins, including Krit1, TIAM, RIAM, and Cno/Afadin. Thus, Rap1 and its effectors are candidate proteins for regulating interactions between AJs, polarity proteins and the cytoskeleton during polarity establishment and maintenance (Choi, 2013).

The early Drosophila embryo provides among the best models of establishing and maintaining apical-basal polarity. Flies start embryogenesis as a syncytium, with 13 rounds of nuclear division without cytokinesis. Membranes then simultaneously invaginate around each nucleus, forming ~6000 cells in a process known as cellularization. Prior to cellularization, the egg membrane is already polarized and serves as a polarity cue for underlying nuclei. This ultimately becomes the apical end of the new cells. Epithelial apical-basal polarity is initiated during cellularization. In the absence of cadherin-catenin complexes, cells form normally but then lose adhesion and polarity as gastrulation begins. These data and earlier work from cell culture suggested AJs are the initial apical cue. However, it was found that Bazooka (Baz)/Par3 acts upstream of AJs in this process. Strikingly, Baz and DEcad apically co-localize in spot AJs from cellularization onset. In the absence of Baz, DEcad loses its apical enrichment and redistributes all along the lateral membrane, while in the absence of AJ proteins, Baz remains apically localized, and a subset of cells retain residual apical-basal polarity, although cell shapes are highly abnormal. Cadherin-catenin and Baz complexes form independently before cellularization, and Baz then helps position DEcad in the apicolateral position where spot AJs will form. This placed Baz atop of the polarization network, raising the question of how it is positioned apically. Two cytoskeletal networks play important roles in initial Baz positioning (Choi, 2013).

Disrupting dynein led to Baz spreading along the lateral membrane, suggesting polarized transport along microtubules (MTs) plays a role. Depolymerizing actin also destabilized apical Baz, as did significantly overexpressing Baz, suggesting an actin-based scaffold with a saturable number of binding sites anchors Baz apically. While both actin and MTs are required for initial Baz polarization, they are not the only cues. Mislocalized Baz is re-recruited or re-stabilized apically at gastrulation onset if either initial cue is disrupted, suggesting a third cue perhaps involving aPKC/Par6 or Par1. Thus, the current model for initial establishment of apical-basal polarity involves a relatively simple pathway in which Baz is positioned apically, and then positions other apical polarity players. However, once initial polarity is established, events become more complex, with a network of mutually reinforcing and inhibitory interactions between apical and basolateral polarity complexes leading to polarity elaboration and maintenance. These were significant advances, but the proteins directing apical accumulation of Baz remained unknown. Work on apical constriction in the fly mesoderm, convergent extension during gastrulation, establishment of anteriorposterior polarity in one cell C. elegans embryos, and on apically constricting Drosophila amnioserosal cells, suggested that a complex network of interactions link AJs, the apical polarity proteins Baz and aPKC, and the actomyosin cytoskeleton. Recent work on Canoe and Rap1's roles in mesoderm apical constriction and convergent elongation (Sawyer, 2011) suggested they also fit into this network. These data led to an exploration of whether Rap1 and Cno play roles in initial apical positioning of AJs and Baz and thus in the establishment and early maintenance of polarity (Choi, 2013).

In regulating polarity establishment, Rap1 and Cno could act by several possible mechanisms. Their role in AJ positioning may be solely due to their effects on Baz localization, or alternatively Rap1 and Cno may independently affect the localization of both Baz and AJs. In the latter case, Cno may directly link AJs to the apical actin scaffold, as it was suggested to act in apical constriction. Rap1 and Cno also clearly regulate Baz positioning. Since Baz apical positioning requires an apical actin scaffold and dynein based MT transport, whether Rap1 and Cno act indirectly by regulating cytoskeletal organization was examined. However, the data suggest this is not the case: both the MT and actomyosin cytoskeletons appear normal in mutants. Thus the most likely model is that Rap1 and Cno are required for anchoring Baz apically. Consistent with this, when Cno was ectopically localized to artificial cell-cell contacts in cultured fly cells, it was able to recruit Baz to that site. This could occur directly, for example, by Cno binding Baz, or indirectly, via unknown intermediaries. Strikingly, however, when Baz was over-expressed in cellularizing embryos, presumably saturating its apical binding sites, it accumulated basolaterally and recruited DEcad but not Cno to these ectopic sites. Thus Cno and Baz do not co-localize obligatorily. It likely that each has multiple binding partners and that when pools are limiting, as Cno may be in this latter experiment, ectopic Baz cannot recruit Cno away from a preferred binding site. Of course, it remains possible that Cno and Rap1 also regulate Baz positioning through effects on MT transport or, given Cno's apical localization, unloading at an apical docking site. It will be important to test these possibilities. As is discussed in more detail below, it will also be important to define the Cno- and Rap1-independent mechanisms that partially restore apical Baz localization after gastrulation onset (Choi, 2013).

Since Rap1 is uniformly distributed along the apical-basal axis during cellularization, the most likely hypothesis is that it is locally activated apically by a GEF. A number of Rap1GEFs exist, many of which are conserved between mammals and flies. Recent work from the Reuter lab demonstrated that, like Cno and Rap1, the Rap1 GEF Dizzy (Dzy/PDZ-GEF) plays an important role in coordinated mesodermal apical constriction, suggesting it is the GEF acting upstream of Cno and Rap1 in that process. They also suggest that Rap1 and Dzy help regulate establishment of AJs. While similar in outline, their analysis of AJs differs from this one in detail, as they see strong effects on DEcad localization without similar effects on Arm. This is surprising, since these two proteins of the cadherin-catenin complex generally localize very similarly at the cortex. However, these differences aside, their data are consistent with Dzy acting with Cno and Rap1 in AJ establishment-it will be important to examine the effects of Dzy on Baz localization. It will also be important to determine how pre-existing egg membrane polarity is translated into localized Rap1 activity (Choi, 2013).

In addition to the parallel roles of Rap1 and Cno in regulating initial apical-basal polarization, this study identified a second role for Rap1 in establishing and maintaining columnar cell shape. The data suggest that this is partially or completely Cno-independent, and thus one of the many other Rap1 effectors may play a role in this process. It will be exciting to examine embryos mutant for other Rap1 effectors, such as Krit1/Bili, TIAM/Still life, RIAM/Pico, or RhoL to see if they are required for establishing columnar cell shape. baz and aPKC mutants also had defects in establishing columnar cell architecture. It is possible that each protein provides an independent mechanistic input into this process. This is consistent with the observed differences in the details of how columnar cell shape is disrupted, with Baz and aPKC primarily regulating apical cell area, while Rap1 affects cell shape at multiple apical-basal positions. A more speculative but perhaps less likely possibility is that Rap1 uses Baz and aPKC as effectors in establishing columnar cell shape. Fly Rap1 can form a complex with aPKC and Par6, and Rap1 acts upstream of cdc42/Par3/aPKC in regulating polarity of cultured neurons (Choi, 2013 and references therein).

Having identified Rap1's direct effector(s) in regulating cell shape, it is necessary to move downstream. Based on analogies with other epithelial tissues in fly development, it is hypothesized establishing columnar cell shape involves regulating apical tension. Other small GTPases play key roles in this; e.g., Rho and cdc42 have striking and opposing roles in apical tension regulation during fly eye development. In that context, Rho acts via separate effectors to maintain AJs and apical tension-it regulates tension via Rok, Diaphanous, and ultimately myosin contractility. It will be interesting to determine whether the defects in apical cell shape in the absence of Rap1, Baz, or aPKC also reflect unbalanced contractility in different nascent cells, and which contractility regulators are involved. However, for now, this is speculative (Choi, 2013).

Previous work has suggested a linear hierarchy regulating polarity establishment, with Baz at the top, positioning AJs and aPKC. The current work extends this hierarchy, positioning Rap1 and Cno upstream of Baz in this process. However, the data further suggest that viewing polarity establishment as a linear process is significantly over-simplified. It is now known that all of the relevant players -- including the AJ proteins, Baz, Cno and aPKC -- are at the cortex in syncytial embryos, prior to cellularization and the initiation of apical-basal polarity. This places them in position to cross-regulate one another. Consistent with this, the data suggest that viewing relationships with an 'upstream-downstream' point of view misses important reciprocal interactions that occur as polarity is established. Two examples point this out most clearly. First, earlier work suggested that localization of aPKC occurs 'downstream' of Baz, as apical positioning of aPKC at gastrulation onset requires Baz function. The new data reveal that Rap1 and Cno are, in turn, 'upstream' of Baz, and thus, if things work in a strictly linear fashion, Rap1 and Cno should be 'upstream' of aPKC. However, in contrast to this simple view, this study found that precise positioning of Cno during cellularization requires aPKC - in its absence, Cno is not cleared from the apical region, and the apical-basal cables of Cno at tricellular junctions are not properly assembled. In a similar fashion, Baz, which in a linear model is 'downstream' of Cno, also regulates precise positioning of Cno during cellularization. aPKC and Baz also play important roles in Cno localization during the early polarity maintenance phase beginning at gastrulation onset. Together, these data suggest that initial positioning of proteins along the apical-basal axis involves a network of protein interactions, similar to that previously suggested to regulate polarity elaboration during the extended germband phase and beyond, as cells develop the full suite of epithelial junctions. It will now be important to define mechanisms by which aPKC and Baz act to precisely position Cno: two broad possibilities are that they act on Cno directly, or that they modulate the fine scale architecture of the actin cytoskeleton, with indirect effects on Cno. It will also be exciting to determine if other polarity determinants, like the basolateral proteins Discs Large, Scribble or Lgl, or the basolateral kinase Par1 also play roles in polarity establishment, as they do in polarity maintenance. Consistent with this possibility, recent work from the Harris lab suggests Par1 is important for the gastrulation onset rescue of Baz localization in embryos in which early cues are disrupted. Finally, it will be interesting to identify the cues that come into play at gastrulation onset, which partially restore apical Baz localization, as part of the increasingly complex network of partially redundant regulatory cues that give polarity its robustness (Choi, 2013).

Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains

Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. This study identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. This study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life (Goh, 2013).

In this study, Clu, a protein expressed in L3 brain NBs, was found to be involved in regulating aPKC levels. In the absence of Clu, both Mira and Numb were delocalized in small percentages of mitotic NBs. Clu preferably binds to aPKC and Baz but not Lgl. In addition, this study also showed that Clu promotes a tumorigenesis phenotype in the lgl mutant. In the absence of Lgl, the function of Clu to maintain aPKC levels was sensitized. Drastic rescue was seen when clu was removed from the lgl mutant. The Western blot analyses indicated that in the lgl clu double mutant, both aPKC and p-aPKC levels were reduced, which was responsible for the rescue of lgl tumorigenesis phenotype. These data are most consistent with a model in which Clu is a member of aPKC/Baz/Par6 complex and presumably functions to maintain its stability (Goh, 2013).

If Clu is indeed involved in the maintenance of the aPKC/Baz/Par6 active complex, why does deletion of clu alone fail to generate any drastic phenotype? Deletion of clu only caused a low percentage of Mira and Numb mislocalization. The L3 brain of clu did not form tumors and the mutant could even survive all the way to adulthood, albeit it could only survive for 3-7 days. The key to that question may lie in its genetic interactor, lgl (Goh, 2013).

In the Western Blot analyses of whole larval brain lysate and knockdown cell line lysate of clu, a decrease was observed in Lgl as compared with the control. This may suggest that in clu mutant, there might also be a lower level of the inactive aPKC/Lgl/Par6 inactive complex. It is possible that the lack of Clu not only exposes the aPKC/Baz/Par6 complex to perturbations such as dephosphorylation or degradation, it also lowers Lgl levels, which in turn results in less aPKC/Lgl/Par6 complex. As a consequence, more aPKC can be released and forms a complex with Baz to replenish the decreasing pool of active complex. Thus, Lgl is able to fulfill its role as a molecular buffer in the clu mutant, shifting the equilibrium toward the more active aPKC/Baz/Par6 complex and compensating Clu function (Goh, 2013).

The primary aim of asymmetric cell division in NBs is to localize cell fate determinants such as Pros, Brat and Numb to the basal cortex so that after cytokinesis, they would only be inherited by the GMC. Numb and Mira have been found to be phosphorylated by aPKC and this phosphorylation resulted in apical exclusion of the proteins. The optimal levels of phosphorylation of Numb depend heavily on the equilibrium of two complexes, the active aPKC/Baz/Par6 complex, and the inactive aPKC/Lgl/Par6. Mira phosphorylation may depend on a similar aPKC complex. Studies so far had pointed to Lgl as the major buffering molecule to maintain this equilibrium, together with other phosphatases like PP4 and PP2A modulating the equilibrium or activity of the two complexes. There should be, however, additional molecules that help maintain this equilibrium in the Drosophila NBs (Goh, 2013).

Clu exhibited preferential binding capacity to the aPKC/Baz/Par6 complex rather than aPKC/Lgl/Par6, suggesting its participation in the maintenance or regulation of the equilibrium between the two complexes. Under extreme conditions, such as in the lgl mutant, lack of Lgl not only shifts the equilibrium entirely to the aPKC/Baz/Par6 complex, resulting in hyper-phosphorylation and subsequent delocalization of Numb and Mira, but also sensitizes Clu function on stabilizing aPKC/Baz/Par6. When clu is further deleted in the lgl mutant, the aPKC/Baz/Par6 complex could be destabilized, hence resulting in a significant decrease in the levels of both aPKC and p-aPKC, which in turn rescues Numb and Mira localization phenotypes in dividing NBs and restores L3 brain sizes. Western Blot analyses of whole larval brain lysate and knockdown BG3C2 cell lines supported this hypothesis (Goh, 2013).

Both clu and park have been shown previously to cause mitochondrial defects in adults. An initial suspicion was that ATP levels in clu NBs might contribute to the defects of neuroblast asymmetric cell division, as well as the rescue phenotype in the lgl mutant. A recent paper indicated that localization of mitochondria did not affect levels of ATP or cause any oxidative damage in the 3L brain, although they observed abnormal clustering in the clu mutant (Sen, 2013). Measurements of ATP levels in clu, lgl and lgl clu mutant brains also did not indicate any differences among them or the wild type brains. Thus, it is concluded that the clu phenotype observed in this study is very likely to be independent of mitochondrial activity (Goh, 2013).

Parkinson's disease is a neurodegenerative disease of the central nervous system which occurs in aged individuals. In patients with Parkinson's disease, there is massive loss of dopaminergic neurons which results in impairment in movement. The Drosophila ortholog of park encodes an E3 ligase, and they were found to have defective flight muscles and loss of dopaminergic neurons in park null mutants (Goh, 2013).

This has directly linked genes involved in neurodegenerative disease, clu and park, with lgl, a gene that is involved in NB asymmetric division during early development. Since these three genes are expressed in the same cells and are involved in the same process during neurogenesis, it is speculated that early defects in neurogenesis, although weak, may become a susceptible factor contributing to neurodegenerative diseases such as Parkinson disease at a much late stage of life. More in-depth studies on the links among genes involved in two developmentally separated events, asymmetric division during early neurogenesis and neurodegeneration due to aging, need to be done before the final conclusion can be reached (Goh, 2013).

Protein Interactions

To test whether aPKC and Bazooka are associated in a protein complex, coimmunoprecipitation experiments were performed. Baz was immunoprecipitated from extracts of S2 cells overexpressing Baz. S2 cells express endogenous aPKC. The immune complex was subjected to SDS-PAGE and Western analysis with anti-aPKC antibody C20. A significant amount of aPKC coimmunoprecipitates with Baz. To determine whether binding of DaPKC to Baz is direct, interaction studies were performed with the yeast two-hybrid system. A construct containing full-length aPKC fused to the transactivation domain of GAL4 was cotransformed into yeast with bait constructs containing different regions of Baz fused to the GAL4 DNA-binding domain. Interaction of the bait constructs with DaPKC was assayed by X-Gal filter assays. Bait constructs containing the second and third PDZ domain of Baz show interaction with full length DaPKC, whereas all constructs lacking the second or third PDZ domain give negative results. It is concluded that binding of DaPKC to Baz is direct and that the region from amino acid 401 to 737 of Baz is sufficient for binding to DaPKC (Wodarz, 2000).

To generate different cell types, some cells can segregate protein determinants into one of their two daughter cells during mitosis. In Drosophila neuroblasts, the Par protein complex localizes apically and directs localization of the cell fate determinants Prospero and Numb and the adaptor proteins Miranda and Pon to the basal cell cortex, to ensure their segregation into the basal daughter cell. The Par protein complex has a conserved function in establishing cell polarity but how it directs proteins to the opposite side is unknown. A principal function of this complex is to phosphorylate the cytoskeletal protein Lethal (2) giant larvae [Lgl; also known as L(2)gl]. Phosphorylation by Drosophila atypical protein kinase C (aPKC), a member of the Par protein complex, releases Lgl from its association with membranes and the actin cytoskeleton. Genetic and biochemical experiments show that Lgl phosphorylation prevents the localization of cell fate determinants to the apical cell cortex. Lgl promotes cortical localization of Miranda, and it is proposed that phosphorylation of Lgl by aPKC at the apical neuroblast cortex restricts Lgl activity and Miranda localization to the opposite, basal side of the cell (Betschinger, 2003).

The Drosophila atypical protein kinase C-ref(2)p complex constitutes a conserved module for signaling in the toll pathway

Recent results have demonstrated the critical role of the mammalian p62-atypical protein kinase C (aPKC) complex in the activation of NF-kappaB in response to different stimuli. Using the RNA interference technique on Schneider cells it has been shown that Drosophila aPKC (DaPKC) is required for the stimulation of the Toll-signaling pathway, which activates the NF-kappaB homologs Dif and Dorsal. However, DaPKC does not appear to be important for the other Drosophila NF-kappaB signaling cascade, which activates the NF-kappaB homolog Relish in response to lipopolysaccharides. Interestingly, DaPKC functions downstream of the nuclear translocation of Dorsal or Dif, controlling the transcriptional activity of the Drosomycin promoter. The Drosophila Ref(2)P protein is the homolog of mammalian p62, since it binds to DaPKC: its overexpression is sufficient to activate the Drosomycin but not the Attacin promoter, and its depletion severely impairs Toll signaling. Collectively, these results demonstrate the conservation of the p62-aPKC complex for the control of innate immunity signal transduction in Drosophila melanogaster (Avila, 2002).

Drosophila represents an ideal system in which to determine the primary role of the aPKCs in NF-kappaB signal transduction because it encodes only one aPKC isoform. According to the data presented in this study, aPKC is selectively required for the innate immune Toll-signaling pathway, acting downstream of the translocation of Dorsal and Dif and playing a critical role in the induction (a typical NF-kappaB-dependent process) of the antimicrobial peptide gene for Drosomycin. Therefore, it can be argued that the primary role of the aPKCs, particularly that of zetaPKC in higher eukaryotic cells, is to somehow control the transcriptional activity of NF-kappaB through a still not completely understood mechanism that most likely involves the direct phosphorylation of RelA and Dif. Interestingly, in Drosophila it is well documented that the phosphorylation of Dorsal is required not only for its transcriptional activity but also for its nuclear translocation. In Drosophila aPKC-depleted cells, a strong inhibition of Dorsal or Dif nuclear translocation is not observed, suggesting that the role of Drosophila aPKC is independent of the previously characterized role for Dorsal phosphorylation in regulating nuclear translocation. Based on experiments in mammalian systems, which demonstrate that p65 transcriptional activity must be stimulated by phosphorylation, it is possible that the residues that control the transcriptional activities of both Dorsal and Dif are different from those controlling the nuclear import of the protein. It is also possible that Drosophila aPKC-mediated phosphorylation has a subtle, yet important, role in the nuclear translocation of Dif and/or Dorsal. Future studies will address this important issue (Avila, 2002).

These studies also demonstrate that Ref(2)P is most likely the functional homolog of p62 in Drosophila. Like p62, Ref(2)P interacts physically with the aPKCs. Therefore, it appears that the p62-aPKC signaling module, like the Par/aPKC complex, is highly conserved. Importantly, a functional role of Ref(2)P in Toll signaling is demonstrated. Thus, the ectopic expression of Ref(2)P is capable by itself of activating the Drosomycin promoter. More interestingly, its depletion severely impairs the Toll pathway (Drosomycin induction) but not the LPS pathway (Attacin induction). Thus, the Ref(2)P/DaPKC complex is critical for Toll signaling (Avila, 2002).

The results presented here also demonstrate that, similar to the p62-TRAF6 connection in mammals, Ref(2)P and Drosophila TRAF2 physically and functionally interact. Together with the results demonstrating that Drosophila aPKC and Ref(2)P are essential for a downstream event in the Toll-signaling pathway, this suggests that a putative Ref(2)P/aPKC/TRAF2 complex might function in the signal-induced stimulation of Dif or Dorsal transcriptional activity. In this regard, it is noteworthy that recent results suggest that TRAF6, in addition to its role in IKK recruitment and activation, may also be involved in the control of RelA transcriptional activity. However, the role of Drosophila TRAF2 in Toll signaling requires further investigation, since the effect of inhibiting (or mutating) TRAF2 has not yet been reported. Further studies will also address the precise mechanism whereby aPKC controls the Toll pathway. The data presented here clearly establish the conserved role of the homolog of the p62/aPKC cassette in NF-kappaB signaling in Drosophila (Avila, 2002).

Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. The Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. The absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors (Nam, 2003).

The strong dependence of Crb localization on Sdt and Dlt suggests that Crb may be destabilized or may not be targeted to the membrane in the absence of Sdt or Dlt. It is intriguing that Sdt and Dlt are lost only partially in the absence of Crb. The findings of a direct interaction between Dlt and Par-6 suggest that Sdt-Dlt can still be targeted to the membrane in the absence of Crb through the binding of Dlt to the Par-6 complex. However, it is important to note that Dlt is essentially lost in sdt mutant clones and vice versa. This raises an intriguing possibility that Dlt or Sdt are dependent on each other in vivo to be targeted to the apical membrane via binding to either Crb or Par-6. This mutual dependency between Dlt and Sdt may explain why Dlt and Sdt are lost in the absence of the other, rather than being associated with the Par-6 complex (Nam, 2003).

The interaction between the Crb and Par-6 complexes is mediated by the PDZ3 region of Dlt and the N-terminal domain of Par-6. The N-terminal domain of Par-6 is also used for binding aPKC. Therefore, a potential function of Dlt is to bind Par-6 in competition with aPKC or to facilitate the interaction of Par-6 with aPKC or other Par-6 binding proteins. Mutant analysis indicates that loss of Dlt and Sdt in sdt- clones causes mislocalization of both Crb and Par-6 complex proteins. This suggests that Sdt-Dlt interaction provides a scaffold to recruit Crb complex to the Par-6 complex and enhance the stability of these two complexes rather than functioning as a competitor for aPKC (Nam, 2003).

Proteins in Crb and Par-6 complexes consist of multiple functional domains which may be involved in diverse protein-protein interactions. A recent study has shown that in mammalian cell culture systems the PDZ domain of Par-6 binds not only Par-3 but also the N terminus of Pals1. These results suggest that the crosstalk between the Crb and Par-6 complexes is mediated by multiple domain-specific interactions. Evidence from genetic analysis using mutants suggests that the crosstalk between the two complexes is mutually required for normal organization of apical membranes and AJs in vivo, and also provides a basis for partial redundancy of these complexes in the organization of photoreceptor cell polarity. Interestingly, when either Crb or Sdt is lost, mislocalization or elimination of other associated components including Par-6 complex proteins becomes more severe in the age-dependent manner. This suggests that the Crb complex may be required for the maintenance rather than the formation of the Par-6 complex. The age-dependent degenerative phenotype may be related to the requirement of extensive apical membrane growth to make rhabdomeres and AJs along the growing axis of photoreceptors during pupal stage. Loss of any one component of the Crb complex is likely to be increasingly more detrimental as the process of membrane reorganization proceeds. In crb- or sdt- mutants, significant fractions of Par-6 complex proteins remain in the membrane despite the age-dependent and progressive mislocalization of apical markers. By contrast, loss of Par-6 or aPKC results in mislocalization of Dlt from the apical membrane. This suggests that the Par-6 complex plays essential functions for membrane localization of Crb complex proteins. Furthermore, both Par-6 and aPKC seem to be important for survival and/or proliferation of retinal cells because mutant clones were very small compared with adjacent twin spots and often completely disrupted, probably due to cell death. This is consistent with the findings of frequent apoptosis in aPKC- or par-6- embryos (Nam, 2003).

An important distinction of Par-6 complex in the photoreceptors from other epithelia is the localization of Baz. Baz localizes with Crb complex in the subapical membrane or both the subapical region and AJ in the Drosophila embryonic epithelia. Vertebrate Par-3 also localizes to the apical tight junction in vertebrate epithelial cells. By contrast, Baz in the photoreceptors is specifically positioned in the AJs basal to the all other proteins in the Crb/Par-6 complexes. Baz and Arm are recruited together to ectopic membrane sites by misexpression of CrbJM, suggesting that Baz is an integral component of AJ. However, Baz is not recruited by CrbPBM, whereas Par-6 and aPKC can be ectopically recruited by CrbPBM rather than CrbJM. Therefore, Baz appears to be recruited to AJ independently of Par-6/aPKC (Nam, 2003).

Intriguingly, despite its specific localization to AJs, loss of Baz results in most severe disruption of AJ as well as the more apical Dlt domain. It has been proposed that the Par-6/aPKC cassette is recruited to the site of cell-cell contact and then moves along the most apical zone of the developing cell-cell contact. In this process, an important step for cell polarity formation is to tether the cytoplasmic Par-6/aPKC complex to the site of cell-cell contact at the membrane, which is mediated by the interaction of Par-3 and a membrane protein JAM. Therefore, the results that baz mutation causes loss of Dlt and AJs support the crucial role of Baz in the initial step of cell polarization. However, the distinct localization of Baz from Par-6 and aPKC in the photoreceptors suggests that the mode of Baz localization varies in different systems. In photoreceptors, Baz may be targeted to the membrane with Par-6 but be sorted out from Par-6 in subsequent steps of polarization to remain in the AJs, whereas Par-6-aPKC-Baz cassette remains together in the complex in other epithelia. In contrast to Baz, aPKC localizes to both rhabdomere stalk and AJ, suggesting that Baz and Par-6 are completely separated during polarization while aPKC is not sorted from both Par-6 and Baz. The critical function of Baz in the localization of Crb complex in the rhabdomere stalk is consistent with the requirement of Baz for Crb localization in embryonic epithelia. However, the requirement of Baz in the embryo appears to be dependent on the stage of development since Crb distribution in the absence of Baz becomes normal in late embryos. On the contrary, such stage-dependent recovery of Crb complex localization has not been observed in baz- photoreceptor cells (Nam, 2003).

Recent studies have shown that mutations in human CRB1 cause RP12 and LCA, severe recessive retinal diseases, emphasizing the importance of Crb family proteins in the eyes of mammals including humans. The Drosophila Crb and human CRB1 are localized in analogous subcellular membrane domains of photoreceptors, the rhabdomere stalk and the inner segment in Drosophila and human photoreceptors, respectively. Besides similar subcellular localization, Crb and human CRB1 are functionally conserved. Age-dependent photoreceptor defects in the crb mutant also provide analogy to age-dependent retinal degeneration in RP12/LCA patients. These studies here imply that hCRB1 may function as a protein complex with homologs of Sdt and Dlt and such a complex may interact with a homologous Par-6 complex. Whether such homologous human genes are the targets of inherited retinal diseases such as RP remains to be studied (Nam, 2003).

Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. Polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell (Hutterer, 2004).

These results describe the first steps of a molecular pathway that leads to the establishment of polarity in epithelial cells of the Drosophila ectoderm. The Par-6 protein localizes to the apical cell cortex by binding to Cdc42. Par-6 recruits Bazooka and aPKC and is essential for establishment of the apical domain. Maintenance of Par-6 localization requires Lgl, a substrate of aPKC. Phosphorylation by aPKC inactivates Lgl at the apical cell cortex and restricts Lgl to the basolateral cortex to establish the basolateral domain (Hutterer, 2004).

Apical localization of Par-6 is a key event in the establishment of epithelial polarity. How is Par-6 recruited to the apical cell cortex? In C. elegans, the proteins Par-3, Par-6, and aPKC are localized to the anterior cell cortex before and during the first cell division. Their asymmetric localization is initiated by interaction of the sperm aster with the overlying cell cortex that excludes Par-6 from the posterior cell cortex. During Drosophila cellularization, centrosomes are located apically and it is therefore unlikely that a similar cortical microtubule interaction is responsible for the apical localization of Par-6 (Hutterer, 2004).

Although a distinct apical domain with sharp boundaries is established in epithelial cells only after cellularization, elegant membrane tracer experiments have revealed a subdivision of the plasma membrane into distinct regions already during cellularization. Are these membrane compartments prefiguring the future apical and basolateral domains and is Par-6 localizing apically by recognizing a preformed membrane domain? The first membrane domain is the furrow canal at the tip of the ingrowing cellularization front that is marked by Patj. This domain disintegrates after cellularization and is therefore unlikely to participate in Par-6 localization. During later stages, new membrane is preferentially inserted apically, then apicolaterally. At these stages, newly inserted membrane displaces the pre-existing membrane toward both the apical and basolateral side, indicating that a distinct apical membrane compartment is not established by the end of cellularization. It is therefore unlikely that Par-6 recognizes a preformed apical membrane compartment although these experiments do not rule out a more general role of the vesicle transport machinery in Par-6 localization (Hutterer, 2004).

The results indicate that Par-6 needs to bind to activated Cdc42 in order to localize apically. Since cdc42 mutants cannot be analyzed at this stage, a conserved proline in the CRIB domain was mutated to generate a Par-6 version that no longer binds Cdc42. The structure of the Par-6 Cdc42 complex shows that this residue comes to lie in a hydrophobic groove of the Cdc42 molecule. This may explain why it can be replaced by alanine without affecting Cdc42 binding. When it is deleted, however, one of the adjacent highly charged amino acids will occupy the position of the proline. This could strongly inhibit interaction with the hydrophobic pocket and eliminate binding to Cdc42 both in vertebrates and in flies. Since both Lgl and aPKC still bind Par-6-DeltaP and the protein is expressed at almost wild-type levels from the endogenous promoter in an otherwise null mutant background, par-6-DeltaP embryos are specifically defective in binding of Cdc42 to the Par-6/aPKC complex (Hutterer, 2004).

How does activated Cdc42 localize Par-6? Cdc42 might be required for association of an unidentified Par-6 binding partner that is essential for apical localization of the protein. The conformation of Par-6 changes upon binding to Cdc42, and this could affect interactions with other proteins. However, aPKC and Lgl are the only proteins identified in the Par-6 complex, and their interaction does not depend upon Cdc42 binding. In vertebrates, Par-6 interacts with the Stardust homolog Pals1, and this interaction is regulated by Cdc42. Stardust acts together with its binding partner Crumbs, but apical protein localization is initiated correctly in crumbs mutants. Therefore, it is unlikely that Stardust binding to Par-6 is critical for the initial apical localization of Par-6. It is more likely that Cdc42 activation provides an instructive cue for Par-6 localization. Cdc42 could be preferentially activated on the apical side, for example by localization of an exchange factor, and this could recruit Par-6 to the apical cell cortex. This hypothesis is supported by the ectopic patches of Par-6, which are observed after overexpression of constitutively active Cdc42. Asymmetric activation of Cdc42 is known to polarize other cell types. In yeast, the exchange factor Cdc24 is localized to the incipient bud site. This locally activates Cdc42 and polarizes the actin cytoskeleton toward the site. In migrating neutrophils, Cdc42 is locally activated in response to a chemoattractant gradient by the exchange factor PIXalpha. A clear Drosophila ortholog of PIXalpha exists, but whether it is involved in epithelial polarity remains to be determined (Hutterer, 2004).

Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Lgl acts at the basolateral cortex where it inhibits cortical localization of Par-6. How Lgl excludes Par-6 from the cortex is unclear, but it is remarkable that in other tissues, Lgl actually promotes cortical protein localization. In MDCK cells, Lgl was suggested to regulate basolateral exocytosis and it could recruit a Par-6 antagonizing factor to the basolateral plasma membrane. Since Lgl and Bazooka binding to Par-6 seem to be mutually exclusive, Lgl could also inactivate the Par protein complex by displacing Bazooka. To perform its role in epithelial polarity, Lgl needs to be phosphorylated by aPKC. This modification has been shown to inactivate the protein and release it from its association with membranes and the cytoskeleton. These results suggest that in epithelial cells, apically localized aPKC phosphorylates Lgl to displace the protein from the apical cell cortex. A simple model is proposed in which mutual inhibition between Par-6/aPKC on the apical and Lgl on the basolateral cell cortex maintains epithelial polarity. This model is in agreement with previous studies that demonstrate negative genetic interactions between lgl and proteins that localize to the apical domain. Furthermore, it provides a molecular explanation for the recently described suppression of the lgl mutant epithelial polarity phenotype by reduction of aPKC levels. Negative interactions between the apical and basolateral domains of epithelial cells have been described before. In the Drosophila follicular epithelium, Bazooka is phosphorylated and inhibited by Par-1, a protein kinase located on the basolateral domain, thus restricting the Par protein complex to the apical domain (Hutterer, 2004).

The proteins Par-6, Bazooka, and aPKC localize to the apical cell cortex of both neuroblasts and epithelial cells, but the mechanism of apical localization seems to be different in the two cell types. In epithelial cells, Lgl is required for maintaining Par proteins at the apical cell cortex, while Par protein localization in neuroblasts is Lgl independent. Expression of nonphosphorylatable Lgl disrupts asymmetric cell division in neuroblasts but is without effect in epithelial cells. In addition, overexpression of dominant-active or -negative Cdc42 disrupts epithelial polarity but has no effect on neuroblast division. What is the basis for these differences (Hutterer, 2004)?

Epithelial cells rely on adherens junctions for maintaining distinct membrane compartments. Such junctions are absent from neuroblasts, and in fact, distinct membrane compartments do not seem to exist. Instead, Par protein localization in neuroblasts requires a protein called Inscuteable that is recruited apically by binding to Bazooka and aPKC and activates heterotrimeric G proteins through an adaptor molecule called Pins. Both Inscuteable and G proteins are essential for maintaining Par protein localization in neuroblasts but not epithelial cells. It is possible that a feedback loop operates downstream of the G proteins to maintain polarity in the absence of diffusion barriers and cellular junctions. Mechanistic differences in the way Par proteins localize are also observed between species. In C. elegans, neither Lgl nor G proteins are required for Par-3 or Par-6 localization. Instead, a Ring finger protein called Par-2 maintains Par-3 and Par-6 at the anterior pole. Cdc42 plays a role, but only in maintenance and not establishment of polarity. Clearly, key players are missing that might help in an understanding of these mechanistic differences (Hutterer, 2004).

Cdc42 binds vertebrate Par-6. Both proteins are implicated in polarizing vertebrate epithelial cells, and their conserved interaction suggests that they achieve this via a conserved mechanism. Although in vertebrates both proteins primarily act on tight junctions, the role of Cdc42 in localizing the Par proteins seems conserved since overexpression of an activated form inhibits the localization of Par-3 to tight junctions in MDCK cells. However, current experiments do not confirm a previously demonstrated role of Cdc42 in activating Par-6-associated aPKC in vitro. Unlike in vertebrates, aPKC is shown to be equally active - at least toward Lgl - when bound to a form of Par-6 that does not interact with Cdc42. Whether species-specific differences or the different experimental setups are responsible for this apparent discrepancy remains unclear. Besides their function in polarity, the Par proteins are involved in proliferation control of vertebrate epithelial cells. Par-6 cooperates with Cdc42 in transforming cells, suggesting a role in oncogenic transformation. In Drosophila, Cdc42, Lgl, and Bazooka were shown to cooperate with activated ras in the formation of metastatic tumors. It can be anticipated that the powerful tool of Drosophila genetics will help to identify other components of this pathway that might clarify its role in carcinogenesis (Hutterer, 2004).

The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. A direct molecular link is described between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. Patj binds the cytoplasmic tail of Fz1 and is proposed to recruit aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. During PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. A model is proposed whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active (Djiane, 2005).

The C tail of Fz receptors regulates their localization and signaling activity. A short Fz Cterm governs apical localization, which is critical for effective Fz-PCP signaling. In contrast, a long Cterm (like that of Fz2) governs baso-lateral localization, promoting β-catenin signaling and preventing PCP activity. Thus, a striking feature of all core PCP proteins, including Fz1, is their apical localization within imaginal disc cells. Fz1 colocalizes partially with several components that regulate A/B polarity such as the Crumbs/Sdt/dPatj and Baz/aPKC/Par-6 complexes within the marginal domain, even though it is also present more basally relative to these complexes (Djiane, 2005).

Detailed sequence analysis of the Fz1 Cterm has revealed the presence of two clustered conserved PKC phosphorylation sites (Ser554 and Ser560 in Fz1). Given that aPKC expression in the apical domain overlaps with Fz1, a test was performed to see if aPKC can phosphorylate the Fz1 Cterm on the two conserved PKC sites in an in vitro kinase assay. Purified human aPKC protein phosphorylates in vitro a GST::Fz1 Cterm fusion protein. Furthermore, mutations of the two PKC consensus sites (Ser to Ala) prevent aPKC-mediated Fz1 phosphorylation, confirming that these sites are targets of aPKC (Djiane, 2005).

To investigate the importance of these phosphorylation sites in vivo, flies were generated carrying UAS-inducible transgenes of Fz1 mutant derivatives with either both serines mutated to alanine (Fz1-AA), inactivating the two prospective PKC sites, or both Serines mutated to Glutamic acid (Fz1-EE), mimicking phosphorylation. These transgenes were analyzed under sevenless (sev)-Gal4 control, which is expressed specifically in R3/R4 precursor cells just posterior to the MF during PCP establishment. Overexpression of wild-type Fz1 provides too much activity and interferes with the balance of Fz1 regulation within the R3/R4 pair, resulting in ommatidia with random R3/R4 cell fate decision and chirality, as well as symmetrical R3/R3 type ommatidia. Similarly, overexpression of Fz1-AA (with both aPKC sites inactivated; sev>Fz1-AA) induces ommatidia with random chirality and symmetrical clusters. In contrast, the phosphomimetic Fz1-EE (sev>Fz1-EE) shows hardly any effect. These data suggest that aPKC-mediated Fz1 phosphorylation inhibits Fz-PCP signaling activity (Djiane, 2005).

Since apical Fz1 localization is critical for its proper PCP signaling activity (Wu, 2004), it was hypothesized that the Fz1-EE mutation could affect the localization of the receptor. To investigate this possibility, the expression of the different myc tagged Fz1 transgenes was examined in imaginal discs (under en-Gal4 or dpp-Gal4 control). No difference between the expression of either Fz1-AA or Fz1-EE with that of wild-type Fz1 was found. These mutant Fz1 isoforms were expressed at similar levels and colocalized apically with aPKC, indicating that phosphorylation of Fz1 by aPKC does not affect Fz1's localization (Djiane, 2005).

A second feature critical to Fz1 signaling activity is its ability to recruit Dsh to the membrane. Interestingly, the aPKC sites partly overlap with the region of the Fz Cterm known to bind Dsh, raising the possibility that phosphorylation by aPKC could interfere with Dsh recruitment. Thus tests were performed to see whether Dsh recruitment is affected by phosphorylation of the Fz1 aPKC sites. S2 cells, which have no endogenous Fz, were transfected with Dsh-GFP and the different Fz1 mutants. In this assay, wild-type and both mutant forms of Fz1 recruit Dsh-GFP efficiently to the membrane. Then, whether overexpression of Fz1-AA and Fz1-EE can recruit Dsh-GFP to apical membranes like wild-type Fz1 in vivo (expressed with en-Gal4 in the posterior compartment of wing discs, where there is a sharp boundary between expressing and nonexpressing cells) was examined. Both Fz1-AA and Fz1-EE behave like wild-type Fz1, sequestering Dsh to the apical cell membrane in imaginal disc cells (Djiane, 2005).

In summary, it was shown that Fz1 can be phosphorylated in vitro by aPKC. Together with the in vitro results, the in vivo analysis of a phosphomimetic Fz1 mutant suggests that aPKC phosphorylation regulates Fz1 activity negatively and that this effect is not mediated by affecting Fz1 localization or Fz1-mediated Dsh membrane recruitment (Djiane, 2005).

In light of the importance of the aPKC sites in Fz1, it was of interest to determine how aPKC is recruited to the receptor. This could be mediated by direct binding or through a bridging factor, the most likely candidates being the A/B determinants that bind aPKC. Thus a two-hybrid interaction screen was conducted using the Fz1 Cterm as bait and components of different A/B protein complexes as prey. The closely related Fz2 Cterm was included as well as the Stbm Cterm as control baits. All components of the aPKC/Par-6/Bazooka apical complex were tested. For Baz, three different fragments were used: an N-terminal fragment involved in Baz dimerization (BazA), a central fragment with three PDZ domains involved in Par-6 binding (BazB), and a C-terminal fragment that binds aPKC (BazC). Similarly, all components of the Crb/Sdt/dPatj apical complex were tested except Crb (since Crb is a transmembrane protein) and the components of the more baso-lateral Scrib/Dlg/Lgl complex were analyzed. No direct interaction was detected between the Fz1 Cterm and aPKC, but, interestingly, Patj was found to be a specific binding partner of the Fz1 Cterm. No other protein was found to interact with the Fz1 Cterm, and in turn Patj did not interact with the Fz2 or Stbm Cterms. The interaction between the Stbm Cterm and Dlg was confirmed as was the interaction of aPKC with Par-6 and BazC (Djiane, 2005)

To confirm the Patj-Fz1 interaction in vivo, coimmunoprecipitation (CoIP) experiments were performed from Drosophila S2 cell extracts transfected with GFP fusion proteins with the Fz1 or Fz2 Cterms (GFP::Fz1 and GFP::Fz2, respectively). Patj could be co-immunoprecipitated from cells transfected with GFP::Fz1 but importantly not with GFP::Fz2 or GFP alone , demonstrating that Fz1 and Patj interact in Drosophila cells. A weak interaction was found between Fz1 and endogenous Baz and aPKC, suggesting the existence of one or several multiprotein complexes among Fz1, Patj, Baz, and aPKC. In contrast, other components of A/B protein complexes, such as Par-6 or Dlg, did not CoIP with either GFP::Fz1 or GFP::Fz2 (Djiane, 2005).

To map the Patj interaction domain with the Fz1 Cterm, GST pull-down experiments were performed. Patj is a modular protein containing a N-terminal L27 domain (previously referred to as MRE), mediating its interaction with Sdt, and four PDZ domains. Consistent with the yeast two-hybrid and CoIP results, in vitro translated full-length Patj bind the GST-Fz1 Cterm protein. The fourth PDZ domain of Patj is sufficient for direct binding to the Fz1 Cterm (Djiane, 2005).

Using the CoIP approach, the residues in the Fz1 Cterm required for Patj interaction were also mapped. Whereas the full-length Fz1 Cterm interacts with Patj, removing the last three residues (Fz1ΔBS) abolishes this interaction. The removal of an internal Cterm motif, encompassing the tryptophan critical for Dsh binding, retains Fz1 ability to bind Patj albeit to a lesser extent (Keyes, 2005).

These results support a direct interaction between the apical determinant Patj and the Fz1 Cterm and suggest that Patj could provide a link between Fz1 and aPKC, since Patj was shown in vitro to bind to aPKC either directly or indirectly through Par-6. The Fz1/Patj interaction is mediated by the fourth PDZ domain of Patj, requires the last three residues of Fz1, and is largely independent of the Fz motif that mediates Dsh binding (Djiane, 2005).

Apical localization is critical for PCP protein activity and particularly for Fz1, but until now no direct link between A/B polarity and PCP establishment has been described. This study shows that the apical determinants aPKC and Patj negatively regulate Fz-PCP signaling while Bazooka antagonizes this regulation. Patj binds directly to the Fz1 cytoplasmic tail, possibly recruiting aPKC, whose phosphorylation of two serine residues within the Fz1 Cterm inhibits the activity of the receptor in cells where signaling should not occur. This reveals a direct link between A/B polarity determinants and PCP establishment (Djiane, 2005).

This work provides the first evidence for a direct molecular link between A/B polarity determinants and PCP by demonstrating that the apical determinants aPKC, Patj, and Baz regulate Fz1 activity. This regulation is independent of Fz1 recruitment to the apical membrane, however, since none of the tested A/B determinants is actively responsible for it. For instance, deleting the Patj binding site in Fz1 or replacing the Fz1 Cterm for a shortened Fz2 Cterm, which cannot bind Patj, has no effect on Fz1 apical localization (Wu, 2004), excluding Patj as a recruiting or targeting factor in Fz1 apical localization. Furthermore, Fmi apical localization is unaffected in Patj and Baz mutants. Thus, although an intact A/B polarity is a prerequisite for PCP signaling, there is no mutual dependency for localizing the Patj/aPKC and the Fz-PCP complexes to the apical side of imaginal disc cells, where they can functionally interact (Djiane, 2005).

Other studies also support the existence of a link between A/B polarity and PCP. In the mouse, Looptail (Lp), the homolog of the Drosophila PCP gene stbm/Vang, interacts genetically with mScribble, a baso-lateral determinant conserved in flies. In particular, transheterozygous Lp/mScribble mice show PCP defects in the inner ear. In Drosophila, it has also been shown that PCP factors interact with A/B determinants. Recent work in the sensory organ precursor (SOP) cells has shown that the orientation of the two opposing domains of Dlg (anterior) and Baz (posterior) is dependent on Stbm and Fz activity (Djiane, 2005).

The downregulation of aPKC and Patj in the R3/R4 cells when Fz1 signals to induce PCP is consistent with a model whereby inhibitory phosphorylation of Fz1 mediated by aPKC is occurring throughout all eye disc cells, except in those that are required for PCP establishment at the time Fz1 signaling occurs. Fz1 activity is therefore always kept low outside of the PCP signaling window, and a release of this inhibition is required for PCP signaling to take place. It is noteworthy that overexpression of Fz1 always gives a robust GOF effect without requiring additional “input,” arguing that either the receptor is constitutively active or that a ligand is always present in nonlimiting amounts. In either scenario, it would be important to control Fz1 activity to prevent signaling at the wrong time and to allow limiting signaling components, such as Dsh, to be available for canonical Wnt/Fz-β-cat signaling when PCP signaling is not needed (Wu, 2004). This is particularly true in the eye disc, where cell fate determination and PCP occur almost simultaneously within a short time window. It is thus proposed that the downregulation of aPKC/Patj in the R3/R4 precursors, at the time of PCP establishment, determines when and in which cells Fz1 is active. A detailed analysis of the expression of Fz1 and Fmi in the non- R3/R4 cells reveals that they extend more basally than aPKC and Patj. Since the precise localization of the active Fz1 is unknown, it is possible that either another mechanism inactivates Fz1 more basally or that inactivation is not needed there (Djiane, 2005).

Furthermore, these results argue that high Baz levels in R3/R4 cells promote Fz1 signaling, possibly by antagonizing the inhibitory regulation of Fz1 by aPKC. Indeed, several lines of evidence suggest an inhibitory role of Baz on the activity of an aPKC complex. (1) In Drosophila embryonic neuroblasts, aPKC phosphorylates Lgl on the apical side of the cell to inhibit its function, restricting the active Lgl to the basal domain of the cell. This is mediated through direct binding of a Par-6/aPKC complex to Lgl, which can only occur after Baz is released from the Par-6/aPKC complex, suggesting a negative role of Baz on aPKC function. (2) Direct measurements of aPKC kinase activity on an exogenous substrate reveal that addition of purified Par-3, the vertebrate Baz homolog, inhibits aPKC kinase activity, whereas Par-6 enhances it. However, whether the aPKC inhibition by Par-3 is direct or indirect remains unclear. This antagonizing role of Bazooka on the aPKC-mediated inhibition of Fz1 activity in R3/R4 cells is further evidence of the tight regulation required for PCP establishment in the eye (Djiane, 2005).

In this model, the A/B determinants are acting upstream of PCP. Consistent with this, there is no effect on either aPKC or Patj expression in cell clones mutant for PCP genes. Similarly, the initial Baz enrichment in R3/R4 precursors is Fz/PCP independent. The later enrichment of Baz in R4 is, however, dependent on PCP signaling. This could correspond to a similar situation as observed in the SOP, in which the posterior relocalization of Baz is dependent on Fz1 activity (Djiane, 2005).

How does aPKC regulate Fz-PCP activity? The aPKC-mediated phosphorylation of the Fz1 Cterm inhibits its activity without affecting its apical localization or ability to recruit Dsh. The negative regulation must therefore occur by a different mechanism. One possibility is that Fz1 phosphorylation by aPKC inhibits a PCP-specific signal transduction to Dsh. Consistent with this hypothesis, similar point mutations in the conserved PKC sites of the canonical Wnt/β-cat-dedicated Fz2 (Fz2-AA and Fz2-EE), do not affect Fz2 ability to trigger a Wnt/β-cat response when overexpressed in the wing. Another possibility is that aPKC regulates Fz1 activity by promoting its destabilization or by increasing its turnover through the recycling pathway at the apical membrane. Further investigation will be required to answer these questions (Djiane, 2005).

The selective downregulation of Patj and upregulation of Baz in R3/R4 precursors define when and where Fz1, and therefore Fz-PCP signaling, is active. This scenario represents a permissive rather than an instructive requirement of aPKC, Patj, and Baz during PCP. Fz-PCP signaling components are widely expressed but only required at specific time points and in specific subsets of cells. As no activating PCP specific ligand is known, it is possible that alternate mechanisms control their activity. This study provides evidence for a negative regulation of PCP signaling by A/B polarity determinants, unveiling new mechanisms for regulating PCP. In addition to their importance during A/B polarity, a function has been revealed for the apical determinants Patj, Baz, and aPKC in regulating PCP and evidence is provided for a molecular link between apical-basal and planar cell polarity (Djiane, 2005).

Cdc42 acts downstream of Bazooka to regulate neuroblast polarity through Par-6 aPKC

Cdc42 recruits Par-6-aPKC to establish cell polarity from worms to mammals. Although Cdc42 is reported to have no function in Drosophila neuroblasts, a model for cell polarity and asymmetric cell division, this study shows that Cdc42 colocalizes with Par-6-aPKC at the apical cortex in a Bazooka-dependent manner, and is required for Par-6-aPKC localization. Loss of Cdc42 disrupts neuroblast polarity: cdc42 mutant neuroblasts have cytoplasmic Par-6-aPKC, and this phenotype is mimicked by neuroblast-specific expression of a dominant-negative Cdc42 protein or a Par-6 protein that lacks Cdc42-binding ability. Conversely, expression of constitutively active Cdc42 leads to ectopic Par-6-aPKC localization and corresponding cell polarity defects. Bazooka remains apically enriched in cdc42 mutants. Robust Cdc42 localization requires Par-6, indicating the presence of feedback in this pathway. In addition to regulating Par-6-aPKC localization, Cdc42 increases aPKC activity by relieving Par-6 inhibition. It is concluded that Cdc42 regulates aPKC localization and activity downstream of Bazooka, thereby directing neuroblast cell polarity and asymmetric cell division (Atwood, 2007).

Little is currently known about how the Par complex is localized or regulated in Drosophila neuroblasts, despite the importance of this complex for neuroblast polarity, asymmetric cell division and progenitor self-renewal. This study shows that Cdc42 plays an essential role in regulating neuroblast cell polarity and asymmetric cell division. Baz localizes Cdc42 to the apical cortex where it recruits Par-6-aPKC, leading to polarization of cortical kinase activity that is essential for directing neuroblast cell polarity, asymmetric cell division, and sibling cell fate (Atwood, 2007).

Asymmetric aPKC kinase activity is essential for the restriction of components such as Mira and Numb to the basal cortex. The aPKC substrates Lgl and Numb are thought to establish basal polarity either by antagonizing activity of myosin II or by direct displacement from the cortex. This study found that Cdc42 recruits Par-6-aPKC to the apical cortex and that Cdc42 relieves Par-6 inhibition of aPKC kinase activity. In the absence of Cdc42, aPKC is delocalized and has reduced activity, resulting in uniform cortical Mira. Expression of Cdc42-DN leads to cortical overlap of inactive Par-6-aPKC and Mira indicating the importance of Cdc42-dependent activation of aPKC kinase activity. Expression of Cdc42-CA leads to cortical aPKC that displaces Mira from the cortex, presumably because Lgl is phosphorylated at the entire cell cortex. This is similar to what is seen when a membrane-targeted aPKC is expressed (Atwood, 2007).

Baz, Par-6 and aPKC have been considered to be part of a single complex (the Par complex). This study found that, when Cdc42 function is perturbed, Par-6 and aPKC localization is disrupted but Baz is unaffected. Why is Baz unable to recruit Par-6-aPKC in the absence of Cdc42? One explanation is that Cdc42 modulates the Par-6-Baz interaction, although Cdc42 has no direct effect on Par-6-Baz affinity. Alternatively, Baz might only be transiently associated with the Par-6-aPKC complex (e.g. as an enzyme-substrate complex); this is consistent with the observation that Baz does not colocalize with Par-6-aPKC in Drosophila embryonic epithelia and its localization is not dependent on either protein. How does Baz recruit Cdc42 to the apical cortex? Like other Rho GTPases, Cdc42 is lipid modified (prenylated), which is sufficient for cortical localization. Baz is known to bind GDP-exchange factors (GEFs), which may induce accumulation of activated Cdc42 at the apical cortex (Atwood, 2007).

The requirement of Par-6 for robust Cdc42 apical enrichment suggests that positive feedback exists in this pathway, a signaling pathway property that is also found in polarized neutrophils. More work is required to test the role of feedback in neuroblast polarity but one attractive model is that Baz establishes an initial polarity landmark at the apical cortex in response to external cues, which leads to localized Par-6-aPKC activity through Cdc42. Phosphorylation of Baz by aPKC might further increase asymmetric Cdc42 activation, perhaps by increased GEF association, thereby reinforcing cell polarity. Such a mechanism could generate the robust polarity observed in neuroblasts and might explain why expression of dominant Cdc42 mutants late in embryogenesis does not lead to significant defects in polarity (Atwood, 2007).

This study argues that Cdc42 functions downstream of Baz. Cdc42 is required for Baz-Par-6-aPKC localization in C. elegans embryos and mammalian neural progenitors. In C. elegans embryos, RNA interference of cdc42 disrupts Par-6 localization, whereas PAR-3 localization is slightly perturbed. In this case, Cdc42 is required for the maintenance but not establishment of PAR-3-Par-6 asymmetry; however, other proteins have been shown to localize Par complex members independently of Cdc42. Conditional deletion of cdc42 in the mouse brain causes significant Par-3 localization defects, although this may be caused by the loss of adherens junctions. More work will be required in these systems to determine if the pathway that has been proposed is conserved (Atwood, 2007).

This study has identified at least two functions of Cdc42 in neuroblasts: first, to recruit Par-6-aPKC to the apical cortex by direct interaction with its CRIB domain and, second, to promote aPKC activity by relieving Par-6 repression. aPKC activity is required to partition Mira and associated differentiation factors into the basal GMC; this ensures maintenance of the apical neuroblast fate as well as the generation of differentiated neurons. Polarized Cdc42 activity may also have a third independent function in promoting physically asymmetric cell division, because uniform cortical localization of active Cdc42 leads to same-size sibling cells. Loss of active Cdc42 at the cortex by overexpression of Cdc42-DN still results in asymmetric cell division, suggesting that other factors also regulate cell-size asymmetry, such as Lgl and Pins. In conclusion, these data show that Cdc42 is essential for the establishment of neuroblast cell polarity and asymmetric cell division, and defines its role in recruiting and regulating Par-6-aPKC function. These findings now allow Drosophila neuroblasts to be used as a model system for investigating the regulation and function of Cdc42 in cell polarity, asymmetric cell division and neural stem cell self-renewal (Atwood, 2007).

Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization

Drosophila neural precursor cells divide asymmetrically by segregating the Numb protein into one of the two daughter cells. Numb is uniformly cortical in interphase but assumes a polarized localization in mitosis. This study shows that a phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis. Aurora-A phosphorylates Par-6, a regulatory subunit of atypical protein kinase C (aPKC). This activates aPKC, which initially phosphorylates Lethal (2) giant larvae (Lgl), a cytoskeletal protein that binds and inhibits aPKC during interphase. Phosphorylated Lgl is released from aPKC and thereby allows the PDZ domain protein Bazooka to enter the complex. This changes substrate specificity and allows aPKC to phosphorylate Numb and release the protein from one side of the cell cortex. These data reveal a molecular mechanism for the asymmetric localization of Numb and show how cell polarity can be coupled to cell-cycle progression (Wirtz-Peitz, 2008).

Since the discovery of Numb asymmetry, several proteins required for Numb localization have been identified, but how they cooperate remained unclear. This paper describes a cascade of interactions among these proteins that culminates in the asymmetric localization of Numb in mitosis. In interphase, Lgl localizes to the cell cortex, where it forms a complex with Par-6 and aPKC. At the onset of mitosis, AurA phosphorylates Par-6 in this complex, thereby releasing aPKC from inhibition by Par-6. Activated aPKC phosphorylates Lgl, causing its release from the cell cortex. Since Baz competes with Lgl for entry into the Par complex, the disassembly of the Lgl/Par-6/aPKC complex allows for the assembly of the Baz/Par-6/aPKC complex. Baz is a specificity factor that allows aPKC to phosphorylate Numb on one side of the cell cortex. Since p-Numb is released from the cortex (Nishimura, 2007; Smith, 2007), these events restrict Numb into a cortical crescent on the opposite side (Wirtz-Peitz, 2008).

The data show that Lgl acts as an inhibitory subunit of the Par complex. Given that Par-6 inhibits aPKC activity until the onset of mitosis, why would an additional layer of regulation be required? Like all phosphoproteins Numb is in a dynamic equilibrium between the phosphorylated and unphosphorylated states. Too high a rate of phosphorylation shifts this equilibrium toward the phosphorylated state, mislocalizing Numb into the cytoplasm. Too low a rate shifts it toward the unphosphorylated state, mislocalizing Numb around the cell cortex. Importantly, these data show that only the Baz complex can phosphorylate Numb. Assuming an abundance of Lgl over cortical Par-6, an increase in aPKC activity would translate into a comparatively small increase in the levels of Baz complex. This is because assembly of the Baz complex requires free subunits of Par-6 and aPKC, which become available only once the pool of cortical Lgl has been completely phosphorylated. Therefore, it is proposed that Lgl acts as a molecular buffer for the activity of the Par complex toward Numb. This maintains Numb phosphorylation within a range that is sufficiently high to exclude Numb from one side of the cell cortex but sufficiently low to permit the cortical localization of Numb to the other side (Wirtz-Peitz, 2008).

What is the evidence for this model? Lgl3A, a nonphosphorylatable mutant of Lgl in which the three aPKC phosphorylation sites are mutated to Ala, infinite buffering capacity, induces the mislocalization of Numb around the cell cortex. Conversely, in lgl mutants, having no buffering capacity, Numb is mislocalized into the cytoplasm. Moreover, the model predicts the loss of buffering capacity in the lgl mutant to be offset by an increase in the amount of substrate, since this would render the excess activity of the Par complex limiting. Indeed, overexpression of Numb in lgl mutants restores the cortical localization of Numb as well as its cortical asymmetry (Wirtz-Peitz, 2008).

The results indicate that Lgl gain- and loss-of-function phenotypes are entirely accounted for by the role of Lgl in inhibiting the assembly of the Baz complex. Previously, however, it was thought that the asymmetric phosphorylation of Lgl by aPKC restricts an activity of Lgl to the opposite side of the cell cortex. Based on this model, it was subsequently proposed that Lgl mediates the asymmetric localization of cell fate determinants by inhibiting the cortical localization of myosin-II. In addition, the role of the yeast orthologs of Lgl in exocytosis led to speculation that Lgl establishes an asymmetric binding site for cell fate determinants by promoting targeted vesicle fusion. However, the data show that Lgl asymmetry is extremely transient, and that the protein is completely cytoplasmic from NEBD onward. Lgl cannot therefore interact with any cortical proteins in prometaphase or metaphase, when myosin-II was reported to localize asymmetrically, or establish a stable landmark for vesicle fusion. Interestingly, a recent study demonstrated that yeast Lgl inhibits the assembly of SNARE complexes by sequestering a plasma membrane SNARE (Hattendorf, 2007). This mechanism is reminiscent of fly Lgl sequestering Par-6 and aPKC from interaction with Baz, suggesting that the defining property of Lgl-family members is not a specific role in exocytosis, but a more generic role in regulating the assembly of protein complexes (Wirtz-Peitz, 2008).

The data identify Numb as a key target of aPKC in tumor formation and suggest that Lgl acts as a tumor suppressor in the larval brain by inhibiting the aPKC-dependent phosphorylation of Numb. Although it is tempting to conclude that tumor formation in lgl mutants results from the missegregation of Numb, missegregation of Numb in numbS52F or upon expression of Lgl3A does not cause neuroblast tumors. How might this be explained? During mitosis, unphosphorylated cortical Numb is inherited by the differentiating daughter. At the same time, Baz and aPKC are excluded from this daughter, which limits Numb phosphorylation after exit from mitosis. In the subsequent interphase, some differentiating daughters reexpress members of the Baz complex (Bowman, 2008), but Numb continues to be protected from phosphorylation since cortical Lgl prevents the reassembly of the Baz complex. Thus, Lgl acts both in mitosis and interphase to maximize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).

In lgl mutants, Numb phosphorylation is increased in mitosis, and less unphosphorylated Numb is segregated into the basal daughter cell. Moreover, the assembly of the Baz complex is unrestrained in the subsequent interphase, which is exacerbated by the missegregation of aPKC into both daughter cells. Together, these defects minimize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).

Why is the amount of unphosphorylated Numb critical for differentiation? Recently, it was shown that aPKC-dependent phosphorylation of Numb inhibits not only its cortical localization, but also its activity, owing to the reduced affinity of p-Numb for its endocytic targets (Nishimura, 2007). Therefore, ectopic phosphorylation of Numb leads to its inactivation, transforming the basal daughter cell into a neuroblast in a manner similar to mutation of numb. Consistent with this model, studies in SOP cells have documented ectopic Notch signaling in lgl mutants. Although the numbS52F mutant and Lgl3A overexpression also lead to missegregation of Numb, the levels of active unphosphorylated Numb are increased rather than decreased in these cases and are sufficient to support differentiation (Wirtz-Peitz, 2008).

The data also provide additional insight into the mechanism of tumor formation in aurA mutants. In aurA mutants, the differentiating daughter cell inherits less Numb because Numb is mislocalized around the cell cortex. At the same time, aPKC is missegregated into the differentiating daughter cell, where it promotes Numb phosphorylation in the subsequent interphase. Together, these events result in subthreshold amounts of unphosphorylated Numb in some basal daughter cells, transforming these into neuroblasts. This model explains why aurA mutants are characterized by reduced aPKC activity in mitosis, but are nonetheless suppressed by aPKC mutations, since a lack of aPKC in the differentiating daughter cell restores threshold amounts of unphosphorylated Numb (Wirtz-Peitz, 2008).

The data reveal that Lgl inhibits Numb phosphorylation to maintain Numb activity, whereas AurA promotes Numb phosphorylation in mitosis to ensure its asymmetric segregation. It is concluded that Lgl and AurA act on opposite ends of a regulatory network that maintains appropriate levels of Numb phosphorylation at the appropriate time in the cell cycle (Wirtz-Peitz, 2008).

aPKC phosphorylates Miranda to polarize fate determinants during neuroblast asymmetric cell division

Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. Whereas Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC -| Lgl -| myosin II -| Miranda). This study provides biochemical, cellular, and genetic data showing that aPKC directly phosphorylates Miranda to exclude it from the cortex and that Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement, suggesting that the repressive-cascade model is incorrect. In investigating key results that led to this model, it was found that Y-27632, a Rho kinase inhibitor used to implicate myosin II, efficiently inhibits aPKC. Lgl3A, a nonphosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity. It is concluded that Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Rather than mediating Miranda cortical displacement, Lgl instead promotes aPKC asymmetry by regulating its activity. The role of myosin II in neuroblast polarization, if any, is unknown (Atwood, 2009).

This study examined the mechanism by which polarity is generated in Drosophila neuroblasts, a process required for the segregation of cell fate determinants during asymmetric cell division. This process utilizes aPKC, which is found in many polarized systems such as epithelia. Previously, polarization of the protein Miranda, which is normally restricted to the basal neuroblast cortex opposite aPKC, has been thought to occur by a complex cascade of repressive interactions involving the tumor suppressor Lgl and the motor protein myosin II. The finding that aPKC phosphorylation displaces Miranda from the cortex of neuroblasts and S2 cells led to the idea that the repressive-cascade model might not accurately describe Miranda displacement. This prompted a reexamination of key results supporting the repressive-cascade model (Atwood, 2009).

Based on previous results, it is proposed that studies suggesting that myosin II is involved in aPKC-mediated cortical displacement of Miranda are an artifact of inhibition of aPKC by the Rho kinase inhibitor Y-27632. Although the possibility cannot be excluded that the Miranda polarity defects observed in Y-27632-treated embryos are indeed the result of myosin II inhibition, the fact that this phenotype is identical to that exhibited by apkc mutants, the efficient inhibition of aPKC, and the high concentrations of this compound used in previous reports (~50 mM, compared to the IC50 < 10 μM for aPKC and 0.1 μM for Rho kinase) indicate that the simplest interpretation of the Y-27632 phenotype is direct inhibition of aPKC activity. The role of myosin II in Miranda cortical displacement, if any, is unclear (Atwood, 2009).

The central result that led to the placement of Lgl between aPKC and Miranda was reexamined. Expression of a form of Lgl in which the aPKC phosphorylation sites have been inactivated results in uniformly cortical Miranda in neuroblasts. This result can be interpreted in one of two ways: Lgl mediates Miranda cortical targeting and phosphorylation of Lgl represses this activity, or Lgl inhibits aPKC and this inhibition is repressed by aPKC phosphorylation (i.e., feedback). The key distinction between these two models is whether or not aPKC is repressed when Lgl3A is expressed. Several recent studies indicate that Lgl is a potent inhibitor of aPKC activity. Consistent with this, it was found that Lgl3A expression dramatically reduces the localization of aPKC to the neuroblast apical cortex. Furthermore, it was found that a form of aPKC that is not efficiently repressed by Lgl can overcome the effects of Lgl3A and drive Miranda into the cytoplasm, consistent only with Lgl phosphorylation not being a requirement for Miranda cortical displacement. In addition, it was shown that Lgl alone is sufficient for inhibition of aPKC activity. Thus, it is concluded that Lgl can directly inhibit aPKC and is not required for Miranda cortical targeting (Atwood, 2009).

A simpler mechanism than the repressive-cascade model for Miranda polarization by aPKC is favored: aPKC phosphorylates Miranda, causing it to be displaced from the cortex. The identification of Miranda as a direct aPKC substrate, the requirement of these phosphorylation events for cortical displacement in both S2 cells and neuroblasts, and the necessity of these phosphorylation events for normal development and viability support this model. The sufficiency of phosphorylation (phosphomimetic Miranda is cytoplasmic in the absence of aPKC) indicates that other phosphorylation events (such as phosphorylation of Lgl in the repressive-cascade model) are not required for Miranda cortical displacement. This new model dramatically simplifies the understanding of how asymmetric aPKC activity, as generated by Baz and Cdc42, is translated into the segregation of cell fate determinants. Thus, polarization of three components downstream of aPKC, Numb, and Miranda (this work), appears to occur by direct aPKC phosphorylation. Further work will be required to determine whether this mechanism is utilized by all factors that are polarized by aPKC (Atwood, 2009).

A modifier screen in the Drosophila eye reveals that aPKC interacts with Glued during central synapse formation

The Glued gene of Drosophila encodes the homologue of the vertebrate p150Glued subunit of dynactin. The Glued1 mutation compromises the dynein-dynactin retrograde motor complex and causes disruptions to the adult eye and the CNS, including sensory neurons and the formation of the giant fiber system neural circuit. A 2-stage genetic screen was performed to identify mutations that modified phenotypes caused by over-expression of a dominant-negative Glued protein. Over 34,000 flies were screened and 41 mutations were isolated that enhanced or suppress an eye phenotype. Of these, 12 were assayed for interactions in the giant fiber system by which they altered a giant fiber morphological phenotype and/or altered synaptic function between the giant fiber and the tergotrochanteral muscle motorneuron. Six showed interactions including a new allele of atypical protein kinase C (aPKC). This cell polarity regulator interacts with Glued during central synapse formation. The five other interacting mutations were mapped to discrete chromosomal regions. This study has used a novel approach to screen for genes involved in central synapse formation by performing a primary screen, using a sensitized background, on the adult eye and then a secondary screen, on the isolated mutations, for synaptic phenotypes. This study shows that forward genetic screens are powerful tools for identifying genes with roles in CNS development. This has highlighted a role for aPKC in the formation of an identified central synapse (Ma, 2009).

The success of the two-stage screening approach may have been facilitated by the fact that Glued has a plethora of distinct roles during eye development, including organizing optic neural architecture and an involvement in the formation of sensory neuronal circuits. Therefore an eye phenotype was available on which to base the screen. However, this does not preclude such a method being used for identifying genes involved in other aspects of neural differentiation. It was found that 50% (6/12) of the isolated mutation-containing chromosomes that altered the eye phenotype also altered GFS phenotypes when tested (Ma, 2009).

The over-expression of the truncated Glued protein caused strong phenotypes in both the eye and GF neurons, greater than those caused by heterozygosity for the dominant Gl1 allele. This is likely to be due to the GAL4-UAS system producing many more molecules of the truncated product than Gl1/+ cells in which, theoretically, a maximum of half of the Glued molecules will be truncated. Consistent with this observation, both the suppressors and enhancers isolated during this screen showed stronger effects on GlDN eye phenotypes than on those produced by Gl1. Determining interactions with the Gl1allele also allowed confirmation of GAL4-independent interactions with the Glued locus. For all of the mutations (with the exception of EG162), the alterations of the weaker Gl1/+ eye phenotype were not obvious, however, SEM and sectioning was performed to show interactions with two of the mutations (EG37 & SG13) (Ma, 2009).

Two different disruptions of Glued function, one strong and the other weaker, were used to assay successfully the effects of both enhancer and suppressor mutations in the giant fiber system (GFS) using both morphological and electrophysiological criteria. The severe disruptions of GF morphology and synaptic function enabled the effects of suppressor mutations to be clearly observed. This was less reliable when assaying the effects of mutations isolated as enhancers as either no increase of the already severe phenotype was seen or the interaction was lethal. For the enhancers, therefore, double heterozygotes were generated with Gl1/+. As was the case in the eye, interactions were less pronounced and only two enhancers, EG37/+ and EG162/+ showed enhancement of the Gl1/+ electrophysiological phenotype. Indeed, the subtlety of some interactions with Gl1/+ may have resulted in the analyses being unable to detect some positive interacting loci in the GFS that altered the eye phenotype caused by GlDN (Ma, 2009).

Some EMS alleles were generated, two of which were mapped to known genetic loci and four of which were mapped to discrete chromosomal locations. However, these four complement all the available lethal alleles in these regions indicating that the mutations lie in loci for which there are few or no lethal alleles available. Identification of the location of these new alleles will require either new rounds of mutagenesis, such as via P-element excision in the mapped regions, finer mapping using SNPs or custom made deficiencies using stocks from the DrosDel project. Completion of the BDGP Gene Disruption Project may also enable mapping of the lesions along with more recent approaches using other transposable elements that may disrupt genes refractory to P-element disruption. Interestingly, no mutations were isolated in genes that encode known components of the retrograde motor complex including any further alleles of Glued. During some of the early genetic analysis of the Glued locus, dominant second-site suppressors of the Gl1 eye phenotype were isolated and reported. Of these, two were mapped to the X chromosome (Su [Gl]27 &Su [Gl]57, and the others, Su(Gl)77 &Su(Gl)102 are alleles of Dynein heavy chain 64C (Ma, 2009).

Two new alleles of known genes, Su(H) and aPKC were isolated. Of the two, this study showned that alleles of aPKC genetically interact with Glued in the GFS and suppress the abnormalities in GF-TTMn synapse formation seen when the retrograde motor complex is compromised by GlDN. These abnormalities are: a lack of the presynaptic 'bends'; a branching event that takes place after the two neurons have met; swollen axon tips and a weak or absent functional synaps. aPKC is part of a protein complex, with PAR-3 (Bazooka in Drosophila) and PAR6 that regulates cell polarity in a number of different tissues/cells of Drosophila and vertebrates including neurons. So what is the role of aPKC in the GF neuron? In vertebrate neurons aPKC is needed for neurite outgrowth. In contrast, aPKC in flies is an essential part of the machinery that polarizes dividing neuroblasts but is not needed postmitotically for outgrowth. The data also indicate that aPKC is not needed for neurite extension since the introduction of aPKC mutations into the sensitized background has no effect on GF outgrowth. aPKC is involved in memory formation in Drosophila and at the developing larval NMJ it regulates microtubules (MTs) both pre- and postsynaptically during synapse formation. Indeed MTs are one of the major targets of the PAR-3/PAR-6/aPKC complex in several contexts. aPKC regulates MT orientation in fibroblasts and MT organization in the early embryo. At the NMJ it controls MT stability with a reduction in aPKC activity causing a decreased association of MTs with the microtubule associated protein Futsch and MT fragmentation. Dynein-dynactin is known to be involved in MT organization during growth cone remodeling as well as polarizing MTs in axons. The data indicate that dynein-dynactin and aPKC are acting antagonistically during formation of the GF presynaptic structure and suggest that both are needed to control microtubule organization and dynamics in synapse formation but have opposing roles. One simple explanation is that one of the roles of dynein-dynactin in the GF is to alter MT dynamics at the tip of the axon, when it has reached its post-synaptic target, so that they are more mobile enabling the presynaptic bend to be formed. aPKC regulates the stability of MTs thereby confining axon branching to a single bend. Blocking dynein-dynactin function prevents the MT re-organization needed for formation of the bends and this is ameliorated when aPKC function is reduced (Ma, 2009).

Polarity proteins and Rho GTPases cooperate to spatially organise epithelial actin-based protrusions

Different actin-filament-based structures co-exist in many cells. This study characterises dynamic actin-based protrusions that form at distinct positions within columnar epithelial cells in the Drosophila pupal notum, focusing on basal filopodia and sheet-like intermediate-level protrusions that extend between surrounding epithelial cells. Using a genetic analysis, it was found that the form and distribution of these actin-filament-based structures depends on the activities of apical polarity determinants, not on basal integrin signalling. Bazooka/Par3 acts upstream of the RacGEF Sif/TIAM1 to limit filopodia to the basal domain, whereas Cdc42, aPKC and Par6 are required for normal protrusion morphology and dynamics. Downstream of these polarity regulators, Sif/TIAM1, Rac, SCAR and Arp2/3 complexes catalyse actin nucleation to generate lamellipodia and filopodia, whose form depends on the level of Rac activation. Taken together, these data reveal a role for Baz/Par3 in the establishment of an intercellular gradient of Rac inhibition, from apical to basal, and an intimate association between different apically concentrated Par proteins and Rho-family GTPases in the regulation of the distribution and structure of the polarised epithelial actin cytoskeleton (Georgiou, 2010).

Although many studies have used the segregation of apical, junctional and basolateral markers as a model of epithelial polarity, and a number of studies have reported the existence of cell protrusions in the notum and other epithelia, these structures and the genes regulating their formation have not been characterised in detail. This study used Neuralized-Gal4 to express GFP-fusion proteins in isolated epithelial cells to reveal the dynamic shape of cells within the dorsal thorax of the fly during pupal development. Using this method, distinct populations of protrusions were characterised based on their form, dynamics and location within the basolateral domain of columnar epithelial cells. The analysis reveals dynamic protrusions at three distinct locations within the epithelial cell: apical microvillus-like structures, intermediate-level sheet-like protrusions and basal-level lamellipodia and filopodia. Importantly, although these are all dependent on continued actin filament dynamics, these populations of protrusions rely on different gene activities for their formation (Georgiou, 2010).

Cdc42, Rac, SCAR/WAVE and the Arp2/3 complex are required for the formation of basal lamellipodia and filopodia, but not for the formation of the apical microvillus-like structures. This analysis also confirms that HSPC300 should be considered to be a functional component of the SCAR complex. Moreover, the SCAR and Arp2/3 complexes are required to induce the formation of both lamellipodia and filopodia in this system. Although many studies have suggested that Rac activates the SCAR complex to induce branched Arp2/3-dependent actin nucleation that underlies lamellipodial formation, whereas Cdc42 is required to induce filopodial formation, this analysis suggests that the macroscopic form of the protrusion in a tissue context is not dictated by the nucleator used. In this, the current results are in line with several recent studies in cell culture. Instead, the macroscopic structure generated depends on the local level of Rac activity, with high levels of Rac driving filopodial formation and low levels leading to lamellipodial formation. Since the forces required to distort the membrane to generate finger-like protrusions are likely to be greater than those required to generate the equivalent section of a sheet-like protrusion, protrusion morphology might be a product of a force balance between membrane tension, extracellular confinement and local actin-filament formation. Since wild-type cells have a graded distribution of protrusions, with lamellipodia predominating apically and filopodia basally, wild-type cell morphology might reflect a gradient in the level of Rac activation, from high basal levels to low apical levels (Georgiou, 2010).

Within this system, Cdc42-Par6-aPKC and Baz/Par3 appear to have antagonistic roles in the formation of basolateral protrusions. Cdc42-Par6-aPKC is required for actin filament formation and protrusion dynamics, whereas Baz/Par3 ensures the separation of basal and intermediate protrusions by limiting the extent of basal filopodia along the apical-basal axis. In this, the current analysis adds to the growing body of evidence that Baz/Par3 and Par6-aPKC have distinct molecular targets. Moreover, the data confirm that Par6-aPKC act together with the Rho-family GTPase Cdc42. Significantly, the loss of Baz/Par3 phenocopies gain-of-function mutations in Rac and the overexpression of the Rac-GEF Sif/TIAM1, a Par3-interacting protein. Baz/Par3 might therefore serve as a cell-intrinsic cue to polarise the dynamic actin cytoskeleton along the epithelial apical-basal axis, giving epithelial cells their characteristic polarised morphology (Georgiou, 2010).

Baz/Par3 has previously been implicated in the restriction of actin polymerisation to specific subcompartments within a cell, allowing for the formation of distinct populations of protrusions. This has been studied most extensively in hippocampal neurons, in which Par3 was shown to interact with TIAM1 to regulate the activation of Rac within distinct domains of the cell during axon specification and dendritic spine morphogenesis. Indeed, it has been suggested that the formation of a Cdc42-Par6-Par3-TIAM1-Rac1 complex is required to establish neuronal polarity. The current study suggests that Baz/Par3 acts in a similar fashion in the morphogenesis and positioning of dynamic protrusions in epithelia. However, this analysis reveals an antagonistic relationship between Sif/TIAM1 and Baz/Par3 in protrusion formation. Baz/Par3 might sequester Sif/TIAM1 to prevent its association with Rac. Furthermore, because the loss of Cdc42, Par6 or aPKC results in the loss of basolateral protrusions and a marked reduction in the GFP:Moe reporter (a phenotype that can be rescued by the coexpression of RacV12 or Sif) Cdc42, Par6 and aPKC are probably required for the basal activation of Rac in epithelial cells in the Drosophila notum. Thus, signals from apically concentrated polarity determinants appear to be communicated and translated into local protrusion formation within the basolateral domain. Whether this occurs through the diffusion of an apically localised regulator or via long-range transmission of polarity information e.g. via microtubules, will be an important area of future research. An intriguing correlation is the largely apical localisation of Baz and its proximity to intermediate-level sheet-like protrusions. This would suggest a possible gradient of Baz/Par3-mediated Rac inhibition, allowing sheet-like protrusions at an intermediate level and restricting filopodial protrusions to the very base of the cell. Since Baz/Par3 has been shown to localise PTEN to apical junctions, it is possible that Baz recruits PTEN, which acts on PtdIns(3,4,5)P3 to generate a PtdIns(3,4,5)P3 gradient from high levels basally to low levels apically. PIP3 could then act to aid in the recruitment and activation of Rac at the membrane (Georgiou, 2010).

Taken together, these data demonstrate that different components of the apical determinants of cell polarity act in conjunction with the Rho-family GTPases Cdc42 and Rac to regulate the positioning of lamellipodial and filopodial protrusions over the entire span of the apical-basal cell axis. Significantly, in this tissue context, Rac, SCAR and Arp2/3 complexes promote the formation of both lamellipodia and filopodia, whose structure appears to depend on the level of Rac activation (Georgiou, 2010).

Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability

Mutations in the human von Hippel-Lindau (VHL) genes are the cause of VHL disease, which displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF). However, many other HIF-independent functions of VHL have been identified and recent evidence indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes. Many of these functions have not been verified in genetically tractable systems. Using an established follicular epithelial model in Drosophila, this study shows that the Drosophila VHL gene is involved in epithelial morphogenesis via stabilizing microtubule bundles and aPKC. Microtubule defects in VHL mutants lead to mislocalization of aPKC and subsequent loss of epithelial integrity. Destabilizing microtubules in ex vivo culture of wild-type egg chambers can also result in aPKC mislocalization and epithelial defects. Importantly, paclitaxel-induced stabilization of microtubules can rescue the aPKC localization phenotype in Drosophila VHL mutant follicle cells. The results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).

Establishing and maintaining epithelial integrity is essential for embryonic development, organogenesis and tissue remodeling. The key characteristic of epithelial cells is asymmetrical specification of membrane domains marked by domain-specific proteins. The epithelial morphogenic mechanism, although with some variations in different epithelial tissues, is highly conserved from worm to mammal. The crucial initial step in establishing epithelial polarity is the specification of the apical domain, which is defined by the function of a complex containing atypical PKC (aPKC), Bazooka (Baz; mammalian and worm PAR-3) and PAR-6. The PAR complex is initially recruited by activated Cdc42 to the apical domain. The three proteins were originally thought to function as a complex; however, recent evidence indicates that Baz might be required first to recruit the aPKC-PAR-6 complex to the subapical domain juxtaposed to the future adherens junction (AJ). The PAR complex is required for the localization of another apical complex containing Crumbs (Crb), Stardust (Std; mammalian Pals1) and Discs lost (Dlt; mammalian Patj). The PAR- and Crb-containing complexes occupy the apical-most region of the lateral membrane, just apical to the AJs (Duchi, 2010).

The apical complexes in turn restrict the localization of a third complex comprising Scribble (Scrib; mammalian Scribble/Vartul), Discs large (Dlg) and Lethal(2) giant larvae (Lgl) to the basolateral domain, while Lgl also antagonizes the apical components and prevents their spreading to the basolateral side. The antagonistic action of apical and basolateral complexes helps define the apicolateral loci eventually occupied by AJs. It is not yet completely clear how the initial localization of the apical complex is achieved (Duchi, 2010).

The VHL tumor-suppressor gene mutations are the genetic cause of the familial VHL disease. Germline mutations in VHL predispose the patients to several benign and malignant tumors, including renal cell carcinoma (RCC, kidney cancer), hemangioblastoma (overgrowth of blood vessels in the retina and central nervous system) and pheochromocytoma (tumors in the adrenal glands). VHL protein has been shown to function as an E3 ubiquitin ligase. Among its best-documented targets is the alpha subunit of the hypoxia-inducible factor (HIF-α). Therefore, the canonical tumor-suppressor function of VHL is modulation of the normal oxygen-sensing mechanism that regulates angiogenic response and metabolic switch to glycolysis. However, how this function correlates with the origin of epithelial tumors such as RCC is unclear, although it is thought that HIF-independent mechanisms might be involved (Duchi, 2010).

VHL is evolutionarily conserved. In Drosophila, the VHL gene has been implicated in tracheal tubule development and HIF-α regulation in the embryos based on biochemical and RNA interference-mediated phenotypic studies. In this report, the first genomic Drosophila VHL mutant was generated, and the function of VHL in epithelial morphogenesis was examined using a model epithelium -- the follicle cells in the egg chamber. VHL is shown to regulate the proper localization and stability of aPKC in the follicle cells, and this function is, at least in part, mediated by the action of VHL on microtubule (MT) stability. Without VHL function, MTs and aPKC are destabilized, resulting in epithelial defects. These results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).

The VHL mutation was generated using the homologous recombination strategy. Homozygous VHL1 mutants are sluggish after hatching and die at the end of first instar larval stage. Wild-type, heterozygous and homozygous first instar larvae were hand-picked and subjected to genomic DNA PCR. The homozygous mutant animals show complete loss of the wild-type gene copy. The VHL1 allele, when paired with a deficiency chromosome encompassing the VHL locus (at 47E), shows the same late first instar lethality, suggesting that VHL1 is a null mutant. The lethal phenotype can be rescued by expressing a wild-type VHL cDNA under the control of the hsp70 promoter. Therefore, VHL gene truncation is the only major genetic defect in the VHL1 allele (Duchi, 2010).

The Drosophila follicle cells exhibit the typical epithelial polarity exemplified by markers such as the apical PAR complex, AJ components and the basolateral Lgl complex. Establishment of the epithelial polarity begins soon after follicle cells diverge from the somatic stem cells and encircle the germ cells (stage 1). The epithelium reaches maturity at mid-oogenic stages (after stage 6 at ~30 hours of egg chamber development; total developmental time ~70 hours), when follicle cells cease to proliferate. To examine the phenotype in the follicular epithelium, an adult tissue, mosaic mitotic mutant clones were generated using the Flp/FRT system. Mutant clones are identified by a lack of GFP expression. In an initial survey of potential egg chamber phenotypes, prominent epithelial defects were observed at stage 10 (about 50-55 hours into egg chamber development. The most notable morphological abnormalities were the piling-up of follicle cells and stretched follicular epithelium. The same phenotypes were observed with the VHL deficiency Df(2R)en-A. To quantify the phenotypes, 100 clones of various sizes at stage 9-10 were analyzed. Clone sizes were categorized based on the number of cells in an optical cross-section. As the penetrance in single-cell clones is variable, probably because of phenotypic rescue by the neighboring epithelial cells, only clones larger than two cells were considered. Thirty-eight percent of clones showed stacking of follicle cells and the more severe, multilayering phenotypes (more than three layers of cells); 42% showed flattening/stretching of epithelium; and the remaining 20% showed swelling. It was also determined that VHL loss-of-function mutation did not affect cell proliferation or viability. As mitotic recombination in a heterozygous progenitor cell (VHL1/ubi-GFP) generates one VHL homozygote (no GFP) and one wild type (two copies of GFP), the numbers of high GFP-expressing cells and GFP-negative cells should be equal if the mutant cells do not exhibit an altered rate of proliferation or viability. The ratio was calculated of the cell number within the VHL1 mutant clones versus the cell number within the sister wild-type clones, and a mean value of 0.96 was obtained. Importantly, the ratio between the cell number in mutant and wild-type sister clones did not change as a function of the clone size. Furthermore, staining for mitosis and apoptosis markers phospho-histone H3 and cleaved caspase 3, respectively, also showed that VHL mutation does not affect cell proliferation or caspase-mediated apoptosis (Duchi, 2010).

This report shows that Drosophila VHL is important for establishing and maintaining epithelial integrity via its regulation of MT and aPKC stability. MT disruption and epithelial phenotypes were observed early in oogenesis. This indicates that MT bundles in developing epithelial cells are crucial for epithelial development and are under pressure from dynamic instability. Without stabilizing activity provided by VHL, MTs are disorganized and ultimately disintegrate, resulting in loss of epithelial integrity. Disrupted MTs interfere with proper localization of aPKC, which in turn leads to mislocalization of downstream epithelial markers and epithelial defects. An ex vivo experiment also demonstrates that epithelial defects can occur within a short time (relative to the entire oogenesis time frame) after destabilizing MTs in non-proliferating epithelial cells. This indicates that the maintenance of epithelial integrity is a dynamic and continuous process even in a stable epithelium, for which MTs are crucially important. Previous studies using RNA interference-mediated knockdown demonstrated a morphogenic role of VHL in trachea development. The tracheal phenotypes appear to be the result of elevated cell motility and ectopic chemotactic signaling. Therefore, the tracheal function of VHL might be mediated via different VHL targets in a tissue-specific context. Alternatively, regulation of MT stabilization might also be the underlying mechanism. A separate, tissue-specific function for VHL is favored since the tracheal defects in VHL knockdown can be relieved by decreased expression of breathless, which encodes the chemotactic signaling receptor in the trachea. The two VHL functions, however, are not necessarily mutually exclusive. These different organ systems might in the future serve as a model for testing whether the various functions assigned to VHL are tissue-specific and context-dependent (Duchi, 2010).

Human VHL has been shown to translocate aPKC to MTs, thereby influencing MT reorganization. This study shows that the aPKC mutant can affect MT organization but not stability, whereas VHL can influence both. Conversely, disruption of MTs alone can result in aPKC mislocalization resembling that observed in VHL mutant cells. Importantly, paclitaxel-induced MT stabilization can rescue aPKC localization in VHL mutant follicle cells. It is therefore concluded that a major function of VHL in the follicular epithelium is regulation of MT stability. Loss of MTs leads to aPKC mislocalization and degradation. Conversely, the results also indicate that part of the VHL epithelial functions might be mediated by its direct effect on aPKC stability, as exogenously expressed aPKC-GFP fusion protein can partially rescue (not statistically significant) the VHL mutant phenotype. Indeed, it was also demonstrated that VHL can co-immunoprecipitate with tubulin or aPKC, and that, at least in S2 cells, aPKC levels can be affected by VHL levels without affecting tubulin. Taken together, it appears that the epithelial function of VHL is mediated through stabilization of MT, with an auxiliary role in directly stabilizing aPKC (Duchi, 2010).

It has been suggested that VHL interacts with MTs via the kinesin 2 family of motors. Future studies using the Drosophila epithelial system should also address this issue in vivo. Also interestingly, this study showed that the YH mutant can associate with MT but has little MT-stabilizing activity. This suggests that the YH mutant might be defective in recruiting other proteins, possibly including aPKC, that are important for regulating MT functions. In light of the role of Drosophila VHL in regulating MT stability, a function presumably important for all cells, it is curious that the tissue-specific btl-driven VHL expression can rescue the homozygous lethality of VHL1. This study has shown that tracheal defects are the major embryonic phenotype observed in VHL mutant. In the course of attempting to rescue the tracheal phenotype with btl-driven VHL, the appearance of rescued homozygous adults was noted. This indicates that the MT stabilizing function of VHL is not required in all tissues. It is possible that although VHL can enhance MT stability, by itself it is not an essential factor for MT polymerization. As such, some tissues might be less dependent on VHL levels. In the follicular environment, MT rearrangement, including depolymerization and repolymerization, is crucial when the entire epithelial sheet moves over the germ cell complex while the cells grow increasingly columnar. MT stabilization facilitated by VHL might be of particular importance during this process (Duchi, 2010).

The best-documented function of VHL is its E3 ubiquitin ligase activity that targets the alpha subunit of the HIF transcription factor. This activity provides an elegant mechanistic explanation for the hypervascularity of many of the VHL tumors and for a potential contributor to the metabolic switch to glycolysis, as HIF can upregulate pro-angiogenic factors such as vascular-endothelial growth factor and components in the glucose metabolic pathway. However, recent evidence has suggested that VHL is a multifunctional protein. It can function as a regulator of matrix deposition, integrin assembly, endocytosis, kinase activity, senescence, protein stabilities and tight junction formation, among many others. Whether tight junction disassembly in VHL mutant cells is HIF-dependent is still unresolved; however, other HIF-independent functions appear to facilitate protein stability or activity instead of destabilizing them as a ubiquitin ligase. Such chaperon/adaptor function has also been implicated in promoting stability of MTs. The MT-stabilizing function, although potentially highly significant, has so far only been linked to cilium biogenesis and mitotic spindle orientation in cultured RCC and renal tubule cells. The physiological and developmental significance of this function has not been elucidated in vivo. Indeed, it is unclear how loss of many of these HIF-independent functions contributes to VHL tumor formation because of a lack of tractable genetic models (Duchi, 2010 and references therein).

One crucial element in tumorigenesis is the breakdown of epithelial integrity that ultimately leads to epithelial-to-mesenchymal transition. This report provides the first demonstration of a potential tumor-suppressor function for VHL in regulating epithelial morphogenesis via its role in promoting MT stability. Future studies should exploit further this genetic system for elucidating how a myriad of disease-related VHL point mutations might differentially influence such function (Duchi, 2010).

Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described in this study, Crb was determined to be an interaction partner and cargo of the retromer complex (See Retromer-mediated sorting). Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals. Loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. These data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. A novel function is also shown for retromer in maintaining epithelial cell polarity (Pocha, 2011).

This study aimed to identify factors that interact with the cytoplasmic domain of the type I transmembrane protein Crumbs (Crb) and are involved in its trafficking. A strategy was devised to present the Crb cytoplasmic tail on liposomes, a method uniquely suited to recruit and identify coats, because it mimics the native configuration of a receptor tail at the membrane/cytosol interface (Pocha, 2011).

Proteoliposomes have been used successfully to identify coat complexes and their accessory proteins; however, these studies were restricted to short, chemically synthesized peptides, which severely limited the length of the cytoplasmic tail. To overcome this, this study redesigned the recruitment assay enabling the use of tails expressed and purified from E. coli. A bacterial expression plasmid was designed containing an N-terminal tandem affinity tag followed by a tobacco etch virus (TEV) protease cleavage site and a single cysteine for the chemical coupling to liposomes, to which the cytoplasmic tail of mouse Crb2 (amino acids R1246 to I1282) was fused (Pocha, 2011).

Because the levels of many transmembrane proteins are regulated by sorting decisions in the early (sorting) endosome, phosphatidylinositol 3-phosphate, the predominant inositol phospholipid of early endosomes, was incorporated into proteoliposomes to selectively enrich endosomal trafficking proteins. These proteoliposomes were used for recruiting cytosolic coat components and other interactors from brain extract], followed by protein identification by tandem mass spectrometry (MS/MS). Crb2 was chosen, because it is the predominant Crb gene expressed in the vertebrate brain. Importantly, the tails of all Crb proteins are highly conserved, suggesting that their trafficking mechanisms may also be conserved. Mass spectroscopic analysis confirmed that large amounts of Crb2 (∼600 MS2 spectra) were coupled onto the liposomes. The most abundant protein isolated (as determined by MS2 spectra) with an established role in the recognition and trafficking of transmembrane cargoes was the retromer subunit Vps35. In addition, Vps26B was identified. Western blotting confirmed the presence of Vps35 in our Crb2 recruitment reactions and showed it to be highly enriched relative to two independent controls (Pocha, 2011).

The mammalian retromer is composed of a cargo recognition subcomplex containing Vps35, Vps26, and Vps29 and a membrane interacting subcomplex consisting of SNX1/SNX2 and SNX5/SNX6 heterodimers. Because both Vps35 and Vps26 are crucial for cargo recognition and binding, the recruitment data suggest that Crb2 is a retromer cargo (Pocha, 2011).

To probe the hypothesis that Crb is a retromer cargo, internalization assays were performed by overexpressing Flag-hCrb2 in HeLa cells and analyzing the uptake of anti-Flag antibodies, visualizing compartments through which Crb2 traffics. Previous studies using the classical retromer cargo, the cation-independent mannose-6-phosphate receptor (ciMPR), have shown that retromer subunits and cargo decorate tubules that emanate from endosomes and travel toward the trans-Golgi network (TGN). This study observed colocalization of Crb2 with Vps35 on intracellular vesicles and tubules as well as an overlap with ciMPR- and galactosyltransferase (GalT) label. These data suggest that in HeLa cells, Crb2 travels in retromer-decorated tubules and can traffic via the TGN. However, it should be noted that it does not accumulate there like other retromer cargoes (e.g., ciMPR). Instead, Crb2 appears to undergo rapid transport back to the plasma membrane. RNA interference (RNAi) suppression of Vps35 in HeLa cells displays enhanced localization of Crb2 in lysosomal structures positive for Lamp-I, a phenotype described previously for other retromer cargoes. These data are all in line with Crb being a potential retromer cargo (Pocha, 2011).

To study the functional interaction between Crb and retromer in Drosophila, a previously generated null allele of Vps35, Vps35MH20 was used. As a result of strong maternal contribution, animals homozygous for this allele reach the third larval instar, allowing analysis of Crb in homozygous mutants. Because retromer is required for the retrieval of receptors from endosomes and thus the prevention of their lysosomal degradation, total Crb levels were analyzed and found to be reduced in Vps35MH20 heterozygote third-instar larvae compared to stage-matched wild-type (WT) larvae and dramatically reduced in Vps35MH20 homozygotes. Analysis of the mRNA levels of Crb showed that loss of Vps35 has very little effect on crb transcripts, suggesting that the dramatic reduction in Crb protein that was seem is due to posttranscriptional regulation of Crb by Vps35 (Pocha, 2011).

This led to an investigation of Crb at a cellular level. For this, two different epithelia, wing discs of third-instar larvae and the follicle epithelium, were chosen. Clones of Vps35MH20 mutant cells in wing disc epithelia, labeled with GFP using the mosaic analysis with a repressible cell marker (MARCM) system, were induced by heat shock-Flp at early larval stages. Crb localizes to the subapical region of wing disc epithelial cells. In agreement with results from western blot analysis, Crb staining is decreased in Vps35MH20 clones. Quantification of the fluorescence intensity in the clone and in surrounding tissues revealed that there is an ∼50% reduction in Crb signal within Vps35MH20 clones. The wing discs of Vps35MH20 homozygous animals are small and show variable morphological defects, presumably as a result of defective Wingless secretion. Analysis of Crb localization (by immunofluorescence) and protein levels (by western blotting) in Vps35MH20 hetero- and homozygous wing discs corroborated the data that were obtained using Vps35MH20 clones and larval lysate, respectively (Pocha, 2011).

The stability of the cargo-selective retromer subcomplex is dependent on the presence of all of its components [8 and 16]. To show that the loss of Crb is due to loss of retromer function rather than just the loss of Vps35, the effect was compared of Vps26 and Vps35 knockdown in the posterior compartment of the wing disc using engrailed-Gal4 to drive UAS-Vps26RNAi and UAS-Vps35RNAi. Hedgehog expression, which is unperturbed by loss of retromer, served to label the posterior compartment. Expression of either RNAi construct resulted in a clear reduction of Crb staining in the posterior compartment (∼50% reduction in fluorescence). Expression of engrailed-Gal4 alone had no effect on Crb. From these data, it is concluded that the retromer cargo recognition subcomplex is required for the maintenance of Crb levels (Pocha, 2011).

To further analyze the relation between Crb and retromer, the follicular epithelium, which surrounds the germline cysts of the Drosophila ovary, was examined. Previous work has identified key roles for Crb in polarization of the follicular epithelium. Crb localizes to the entire apical membrane of the follicle epithelial cells, with very little detectable in the cytoplasm. Vps35MH20 clones show strong reduction in Crb staining and protein loss from the apical membrane. Interestingly, although Crb staining at the apical membrane is strongly reduced, it is not detected at increased levels within the cytoplasm, suggesting that Crb is not merely mislocalized but reduced at the protein level, as shown in larvae. The cytoplasmic domain of Crb organizes an apical, membrane-associated protein complex by recruiting the scaffolding proteins Stardust (Sdt), DPATJ, and DLin-7. Therefore, the apical localization of Sdt in the follicular epithelium was assessed, and at was found to be heavily reduced in Vps35MH20 clones. Probing whole larval lysates from third-instar WT and Vps35MH20 hetero- and homozygotes for Sdt confirmed that at the protein level, like Crb, Sdt shows a dose dependence on Vps35. Thus, retromer function in maintaining Crb levels and function is conserved between wing and follicle epithelia (Pocha, 2011).

Interestingly, in some Vps35MH20 clones, the strict monolayer structure of the epithelium is disrupted and the tissue appears multilayered, an indication of polarity defects and characteristic of loss of Crb at early stages of follicle development, whereas loss at later stages results only in the mislocalization of other polarity proteins, without affecting tissue integrity. Multilayering was observed in 19% of Vps35MH20 clones in follicles between stages 7 and 10 and did not appear to be dependent on clone size or position. Given that various links between Crb and Notch have been reported, tests were performed to see whether the multilayering phenotype observed in the follicle epithelium upon loss of Vps35MH20 could be the result of defective Notch signaling. The expression of Notch and Hindsight, a transcription factor downstream of Notch signaling that represses proliferation in the follicle epithelium, were examined in Vps35MH20 mutant clones. Both showed wild-type expression, suggesting that Notch signaling is not affected by loss of retromer, similar to previous findings in the wing disc (Pocha, 2011).

To test whether the loss of Crb in retromer mutants is due to missorting of Crb to the lysosome, follicles harboring Vps35MH20 clones were incubated in leupeptin, a potent inhibitor of lysosomal proteases. After a 3 hr incubation, a dramatic accumulation of Crb was observed in punctae within the cytoplasm of Vps35MH20 cells, a phenomenon that was not seen in WT tissue or in follicles containing Vps35MH20 clones that were incubated in control medium lacking leupeptin. Additionally, colocalization of these intracellular Crb punctae with LysoTracker was observed. Together with the reduction of Crb protein levels and constant crb mRNA levels in Vps35MH20 larvae and tissue, these data strongly suggest that retromer ablation leads to lysosomal degradation of Crb, as observed for other retromer cargoes (Pocha, 2011).

To test whether retromer functions after endocytosis of Crb, internalization of Crb from the plasma membrane was blocked by expression of a dominant-negative construct of shibire (dynamin) or by incubating follicles in dynasore, a dynamin inhibitor. In Vps35MH20 clones, this resulted in the accumulation of Crb at the plasma membrane, confirming that retromer is indeed transporting Crb after internalization from the plasma membrane (Pocha, 2011).

Crb is required, together with atypical protein kinase C (aPKC), to restrict Bazooka/Par3 to the zonula adherens, an adhesion belt at the apex of epithelial cells, in the follicle epithelium, and in photoreceptor cells, thus excluding it from the apical membrane and specifying the border between apical and lateral domains. In previous studies, it was shown that the localization of aPKC and Par6 was dependent on Crb. To test whether loss of retromer phenocopies the loss of Crb, aPKC and Par6 localization were examined in follicles containing Vps35MH20 clones. Indeed, the level of both proteins is reduced at the apical surface in Vps35MH20 clones. Interestingly, unlike Crb and the Crb complex member Sdt, Par6 and aPKC protein levels are not reduced in Vps35MH20 mutant larvae. Therefore, it is likely that the loss of Par6 and aPKC from the apical membrane of Vps35MH20 clones in the follicle epithelium is due to loss of cell polarity in the absence of Crb rather than loss of the proteins themselves (Pocha, 2011).

To test this, Crb was overexpressed in Vps35MH20 clones. Because overexpression of Crb causes defects in epithelial cell polarity, Crb overexpression was induced using GABFc204 Gal4, a follicle epithelium-specific driver that starts expression late in follicle development (stage 8). Thereby, it was possible to rescue the apical localization of Par6 and Sdt. This rescue did not appear to be dependent on clone size or location. From these data, it is concluded that the loss of polarity observed in retromer mutant clones is the direct result of loss of Crb (Pocha, 2011).

The identification of Crb as a retromer cargo confirms the hypothesis that one crucial step in the regulation of Crb occurs at the early (sorting) endosome and, importantly, fills a gap in the current understanding of Crb trafficking. Previous reports showed that transport of Crb to the plasma membrane is reliant on Rab11, the exocyst and Cdc42 in Drosophila embryonic epithelia. Internalization of Crb from the plasma membrane into endosomes is mediated by the syntaxin Avalanche and Rab5. This study has shown that retromer is responsible for sorting Crb away from the degradative pathway and into a recycling one, thus allowing a high level of control over the amount of cellular Crb, previously shown to be vital for maintaining epithelial polarity and integrity, as demonstrated by numerous loss- and gain-of-function studies. Interestingly, retromer was previously shown to play a role in the apical delivery of the polymeric immunoglobulin receptor (pIgR) in Madin-Darby canine kidney cells. However, as for Crb, it remains unclear whether this transport occurs via the TGN, via recycling endosomes, or through alternative pathways. The exact trafficking itinerary of Crb following recycling by retromer remains unclear and may depend upon the purpose of Crb recycling (Pocha, 2011).

Which function of Crb is the prime target of retromer-driven retrieval? Is this a Crb level-sensing mechanism, in which retromer regulates the amount of protein at the plasma membrane, which is crucial for cell homeostasis? To date, all known functions of Crb require an intact Crb complex. By controlling the recycling of Crb and thereby its level at the plasma membrane, retromer could define the amount of Crb available for complex formation. Alternatively, it is tempting to speculate that Crb, much like Wntless (Wls), acts as a transport receptor and that apical delivery of its (yet to be identified) ligand or many ligands is the main purpose of its recycling to the TGN. These are fascinating hypotheses that will be the focus of future research (Pocha, 2011).

Assembly of Bazooka polarity landmarks through a multifaceted membrane-association mechanism

Epithelial cell polarity is essential for animal development. The scaffold protein Bazooka (Baz/PAR-3) forms apical polarity landmarks to organize epithelial cells. However, it is unclear how Baz is recruited to the plasma membrane and how this is coupled with downstream effects. Baz contains an oligomerization domain, three PDZ domains, and binding regions for the protein kinase aPKC and phosphoinositide lipids. With a structure-function approach, this study dissected the roles of these domains in the localization and function of Baz in the Drosophila embryonic ectoderm. A multifaceted membrane association mechanism localizes Baz to the apical circumference. Although none of the Baz protein domains are essential for cortical localization, it was determined that each contributes to cortical anchorage in a specific manner. It is proposed that the redundancies involved might provide plasticity and robustness to Baz polarity landmarks. Specific downstream effects were identified, including the promotion of epithelial structure, a positive-feedback loop that recruits aPKC, PAR-6 and Crumbs, and a negative-feedback loop that regulates Baz (McKinley, 2012).

The PDZ domains of Baz are dispensable for its localization. The current results show that this is due to redundant mechanisms in other parts of the protein. In fact, each PDZ domain plays a unique role in Baz positioning and activity in the Drosophila embryonic ectoderm. The following main roles were identified for the PDZ domains: PDZ1 and PDZ3 recruit Baz to the apical domain, PDZ2 mediates downstream effects on epithelial structure and PDZ1 promotes the turnover of Baz. Each domain also has minor effects that might result from distinct activities or secondary effects of their main activities: PDZ1 and PDZ3 have non-essential but detectable effects on epithelial structure and PDZ2 promotes weak membrane binding (McKinley, 2012).

PDZ1 and PDZ3 activities involve at least two sub-regions of the domains. PDZ domains typically use their peptide-binding pocket to bind the C-termini of their protein partners, but regions outside of these pockets can also mediate interactions. PDZ1 promotes apical surface and circumferential localization independently of its peptide-binding pocket, and its peptide-binding pocket plays a distinct role in promoting Baz turnover. These opposing activities might form a negative-feedback loop that regulates localization of Baz. By contrast, PDZ3 appears to solely promote Baz localization. It can promote apical surface and circumferential localization independently of its peptide-binding pocket, whereas its peptide-binding pocket specifically promotes circumferential anchorage. The binding partners that engage these sites are unknown. However, in vitro studies have shown that the C-termini of Arm and Ed can bind Baz PDZ1-3 in tandem, and that the C-terminus of PTEN can bind Baz PDZ2-3. Binding partners for regions outside the peptide-binding pockets have not been identified for Baz PDZ domains, but the binding of rat PAR-3 PDZ2 to PIPs involves outside regions, as does the binding of C. elegans PAR-3 PDZ1 to PAR-6 (McKinley, 2012).

A major function of Baz PDZ domains is to maintain the protein around the apical circumference. PDZ1 and PDZ3 use peptide-binding-pocket-independent mechanisms to generally localize Baz to the apical domain, but Baz is focused around the apical circumference through mechanisms involving the peptide-binding pockets of these domains. Without these pockets, Baz can saturate its remaining apical anchors and mislocalizes in puncta over the apical surface. PDZ1 appears to prevent this mislocalization by reducing protein levels below saturation, but PDZ2 and PDZ3 might directly bind circumferential proteins or promote an active redistribution of Baz. This activity is weaker for PDZ2 versus PDZ3 (with the former requiring oligomerization and the latter not) and it is possible that the localization activity of PDZ2 is a by-product of its binding to downstream effectors localized to the apical circumference. Thus, it is proposed that PDZ3 has the most direct role in anchoring Baz around the apical circumference (McKinley, 2012).

Because Baz can localize to the apical membrane without its PDZ domains, other localization mechanisms are also involved. The results clarify the importance of two additional mechanisms. The first involves dynamic interactions with apical polarity proteins. Baz has been shown to recruit aPKC to the apical domain as epithelial polarity is first established and to maintain aPKC during later stages. However, aPKC normally localizes at the apical surface with PAR-6 and the Crb complex above Baz and adherens junctions. When BazδPDZ1-3 forms puncta over the apical surface domain it recruits aPKC, PAR-6 and Crb, but the proteins then segregate locally, mimicking their associations around the apical circumference. Indeed, BazδPDZ1-3 might separate from the apical polarity proteins by two known mechanisms: the release of aPKC after it phosphorylates its binding site on Baz, and the loss of PAR-6 binding as a result of competition with Crb. The segregation of aPKC, PAR-6 and Crb from BazδPDZ1-3 suggests that they would not form a stable anchorage site for Baz. However, removal of the aPKC binding region from BazδPDZ1-3 (but not full-length Baz) severely weakens its cortical localization. This suggests that a positive feedback loop exists between Baz and aPKC to maintain localization of each protein. It is proposed that the proteins undergo continuous cycles of attraction and local repulsion to maintain their close but non-overlapping positioning around the apical domain (McKinley, 2012).

An additional Baz localization mechanism involves PIPs. A conserved region of the C-terminal tail of Baz has been shown to bind PIPs (Krahn, 2010), and it was found that the apical surface puncta of BazδPDZ1-3 colocalize with plasma membrane domains enriched with PIPs. Although deletion of the PIP binding region had no effect on full-length Baz (Krahn, 2010), deleting it and the PDZ domains together strongly disrupts plasma membrane binding. Thus, the aPKC binding region and the PIP binding region might both mediate apical localization of Baz in the absence of the PDZ domains. These anchorage mechanisms are also dependent on the oligomerization of Baz, because BazδOD+δPDZ1-3 shows minimal cortical localization. Moreover, the mechanisms appear to support each other because the aPKC binding region cannot compensate for the loss of the PIP binding region from BazδPDZ1-3 and vice versa. Also, membrane binding is abrogated with deletion of the Baz C-terminus, including the aPKC and PIP binding regions (Krahn, 2010). Perhaps the continuous cycles of attraction and local repulsion between Baz, aPKC, PAR-6 and Crb are partly staged on a platform of PIPs (McKinley, 2012).

Interactions with these proteins and lipids might also explain the ability of Baz to partially maintain epithelial structure without its PDZ domains. Indeed, deletion of the aPKC binding region from full-length Baz abrogates its rescue activity (Krahn, 2010), as does deletion of the OD and the PIP binding region, when expressed with a weaker driver, but not a stronger driver (McKinley, 2012).

The results indicate that there are at least five sites in Baz, in addition to its OD, that are involved in membrane localization. No single site is essential, and different combinations of interactions are sufficient for anchorage. This suggests that the individual anchorage mechanisms are relatively weak, as has been shown for the PIP binding region (Krahn, 2010). Cortical localization through multiple weak interactions might provide plasticity and robustness for the role of Baz/PAR-3 as a multifunctional polarity landmark (McKinley, 2012).

The membrane-association mechanism of Baz would allow fine regulation of protein positioning. For example, Baz becomes planar polarized around the apical domain to regulate germband extension in the Drosophila embryo. Rho kinase has been shown to reduce Baz at anterior and posterior cell edges by phosphorylating the Baz C-terminus and inhibiting PIP binding. However, Baz is not fully lost from these edges. Thus, planar polarity might arise from a partial set of membrane-association mechanisms acting along anterior-posterior edges and a more complete set acting at dorsal-ventral edges. Apical localization of Baz is also altered in amnioserosa cells to regulate apical constriction during dorsal closure. Here, Baz forms apical surface puncta in addition to its circumferential localization. Although the mechanisms for this redistribution are unclear, the work suggests that it might involve weakening of PDZ domain activities. Intriguingly, Ed, an in vitro binding partner of the PDZ domains, is specifically absent in the amnioserosa. However, this might not fully explain the redistribution because Baz appears to be localized normally in most ectodermal cells of ed mutants. A more dramatic cellular reorganization occurs as neuroblasts delaminate from the epithelium. As this occurs, adherens junctions and Crb are lost from the cells, but Baz is retained apically and engages with new partners to direct asymmetric cell division after delamination. The mechanisms regulating Baz during this transition are unknown, but its multifaceted membrane-association mechanism might ensure robust apical localization as Baz exchanges molecular interaction networks (McKinley, 2012).

Redundancies in Baz/PAR-3 scaffold activity might also have permitted co-evolution with polarity networks to organize eggs, single-cell embryos, epithelial cells, neurons and stem cells. Indeed, roles for Baz/PAR-3 PDZ domains appear to have diverged. In C. elegans, PDZ2, but not PDZ1 or PDZ3, is essential for embryogenesis, as in Drosophila, but PDZ2 of C. elegans PAR-3 was also shown to be required for proper localization, in contrast to Baz PDZ2. Also, mammalian PDZ2 has also been shown to mediate membrane binding through PIPs, but key residues involved in the interaction are not conserved in Drosophila or C. elegans (McKinley, 2012).

The Baz localization mechanism appears to be unique among characterized polarity scaffold proteins. Other scaffolds also involve multiple mechanisms, but typically there is a primary mechanism that localizes the scaffold to the membrane and secondary mechanisms that focus localization to a particular site. For example, the leucine-rich repeats of Scribble are crucial for its cortical localization in Drosophila epithelia, whereas its second PDZ domain promotes septate junction localization. In Drosophila, the Hook domain of Discs Large is crucial for plasma membrane targeting, whereas particular PDZ domains promote septate junction localization in epithelia and synapse localization in neurons. Similar 'twostep' localization mechanisms have been described for C. elegans and mammalian Discs large, mammalian PSD-95, Drosophila Inscuteable and Pins and Drosophila Stardust. Contrasting these mechanisms, no single site in Baz is essential for membrane recruitment in ectodermal cells. However, any of these mechanisms could be context dependent. Scaffolds shown to localize through a two-step mechanism in one context might use a multifaceted membrane-association mechanism in another, and Baz could localize by one-step or two-step mechanisms in other cell types or developmental stages (McKinley, 2012).

This study has identified a multifaceted membrane-association mechanism that localizes Baz to the apical circumference in epithelial cells. This mechanism integrates with downstream pathways, involving both negative- and positive-feedback loops, which regulate Baz and epithelial polarity. It is important to define the partners for the interaction sites involved, and to dissect how these interactions are controlled (McKinley, 2012).

Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction

Cell shape changes drive tissue morphogenesis during animal development. An important example is the apical cell constriction that initiates tissue internalisation. Apical constriction can occur through a phase of cyclic assembly and disassembly of apicomedial actomyosin networks, followed by stabilisation of these networks. Delayed negative-feedback mechanisms typically underlie cyclic behaviour, but the mechanisms regulating cyclic actomyosin networks remain obscure, as do mechanisms that transform overall network behaviour. This study shows that a known inhibitor of apicomedial actomyosin networks in Drosophila amnioserosa cells, the Par-6-aPKC complex, is recruited to the apicomedial domain by actomyosin networks during dorsal closure of the embryo. This finding establishes an actomyosin-aPKC negative-feedback loop in the system. Additionally, aPKC was found to recruit Bazooka to the apicomedial domain, and phosphorylates Bazooka for a dynamic interaction. Remarkably, stabilising aPKC-Bazooka interactions can inhibit the antagonism of actomyosin by aPKC, suggesting that Bazooka acts as an aPKC inhibitor, and providing a possible mechanism for delaying the actomyosin-aPKC negative-feedback loop. These data also implicate an increasing degree of Par-6-aPKC-Bazooka interactions as dorsal closure progresses, potentially explaining a developmental transition in actomyosin behaviour from cyclic to persistent networks. This later impact of aPKC inhibition is supported by mathematical modelling of the system. Overall, this work illustrates how shifting chemical signals can tune actomyosin network behaviour during development (David, 2013).

These data outline a regulatory circuit for guiding amnioserosa apical constriction. The circuit controls both the localisation and activity of its components. In terms of protein localisation, it was found that amnioserosa actomyosin networks recruit the Par proteins to the apicomedial domain. Although Par protein puncta are not continually dependent on the actomyosin networks, their numbers build over developmental time, apparently owing to the cumulative effect of multiple rounds of actomyosin network assembly. The networks appear to impact aPKC directly, and in turn, aPKC recruits Baz to the apical domain. This recruitment depends on the C-terminal aPKC-binding region of Baz, which aPKC phosphorylates for a dynamic relationship with Baz in the apical domain of amnioserosa cells (David, 2013).

Par-6-aPKC activity inhibits amnioserosa actomyosin networks (David, 2010), and the recruitment of aPKC by the networks implicates a negative-feedback loop. As delayed negative feedback tied to a continual input signal can produce an oscillatory output, the actomyosin-aPKC negative-feedback loop might explain how aPKC regulates actomyosin network assembly-disassembly cycles (David, 2010). However, apical populations of Par-6-aPKC puncta are not fully recruited and fully removed with each actomyosin cycle, suggesting additional mechanisms. Importantly, Par-6-aPKC activity can be tempered by Baz. Thus, aPKC inhibition by Baz might delay the actomyosin-aPKC negative-feedback loop during early DC, promoting the actomyosin assembly-disassembly cycles. As DC proceeds, the additive effects of actomyosin assembly-disassembly cycles could increase apical Par protein levels; additionally, the gradual apical constriction of the cells decreases their apical surface areas and could thus increase apical surface Par protein concentrations. It is proposed that a gradual increase to apicomedial aPKC-Baz interactions inhibits aPKC and thus leads to the stabilisation of actomyosin networks. Simulations indicate that this transition in network behaviour can occur abruptly following incremental reductions to myosin inhibition during earlier DC. It is proposed that Baz acts as a competitive inhibitor to reduce aPKC phosphorylation of cytoskeletal regulators. This idea is consistent with reports of Par-3 inhibiting aPKC in kinase assays in vitro. However, Baz is also known to promote aPKC localisation in the epidermis and amnioserosa. Thus, Baz appears to both promote and inhibit aPKC activity, potentially forming a paradoxical circuit (or incoherent feed-forward loop) in which Baz and aPKC promote each other's recruitment, and in which Baz competitively inhibits aPKC activity. Significantly, Baz has multiple binding sites for the Par-6-aPKC complex [Par-6 binds Baz PDZ1; aPKC binds Baz PDZ2-3; aPKC binds the Baz C-terminal aPKC-binding region], suggesting cooperative binding and that Baz interactions with the Par-6-aPKC complex are stronger than those between the Par-6-aPKC complex and its cytoskeleton targets. Notably, this study found that Baz apical surface levels are ~66% lower than those of Par-6, suggesting that the inhibitory effect of Baz must be dynamic; Baz cannot simply sequester all Par-6-aPKC complexes by outnumbering them. The inhibitory effect must also depend on phosphatases because aPKC interactions with Baz are weakened following phosphorylation (Morais-de-Sá, 2010). Baz/Par-3 is known to be regulated by Protein phosphatase 1 and Protein phosphatase 2A with Protein phosphatase 1 de-phosphorylating the aPKC phosphorylation site of Par-3. Thus, Baz may act as a strong and dynamic inhibitor of Par-6-aPKC to buffer and eventually overcome the actomyosin-aPKC negative-feedback loop (David, 2013).

A crucial unknown is the identity of the cytoskeletal target(s) of aPKC. Cytoskeletal targets of aPKC have been identified but have not been examined during amnioserosa apical constriction. In mammalian cells, Par-6-aPKC can phosphorylate Smurf1, an E3 ubiquitin ligase, in turn leading to RhoA degradation in cellular protrusions (Wang, 2003). During dendritic spine morphogenesis, Par-6-aPKC acts though p190RhoGAP to inhibit RhoA (Zhang, 2008). As well, aPKC phosphorylation of Rho kinase leads to its cortical dissociation in mammalian cell culture (Ishiuchi, 2011), and apparently during salivary gland tubulogenesis in Drosophila (Röper, 2012). Of note, the persistent Par-6-aPKC puncta could actively downregulate actomyosin activity, or prolong the lull between actomyosin activations, or do both. Another question is how actomyosin networks recruit aPKC. The recruitment of Par proteins by actomyosin networks has been documented during Drosophila cellularisation and C. elegans one-cell polarisation, and Baz and aPKC have been shown to co-immunoprecipitate with myosin regulatory light chain from Drosophila egg chambers, but specific linkages have yet to be identified. Defining further components of the actomyosin-aPKC negative-feedback loop will be crucial for understanding its regulation and its effects on actomyosin network dynamics. In particular, despite identifying a potential delay mechanism for the loop, it is unclear how the loop and the delay mechanism could translate into oscillatory network behaviour. Perhaps the cytoskeletal target(s) of aPKC are co-recruited with the assembling networks, which in combination with the buffering effect of Baz, could delay their phosphorylation by aPKC. It is also possible that the clustering of Par protein puncta with each network assembly event could somehow modify the Baz buffering effect (David, 2013).

Another unanswered question is the influence of circumferential anchors for Baz or Par-6-aPKC, as weakening of these anchors could contribute to apicomedial Par protein accumulation over DC. Echinoid (Ed), a transmembrane AJ-associated protein that can directly bind Baz, is normally lost from the amnioserosa during DC. It is hypothesised that this loss might promote the loss of Baz from AJs and its apicomedial accumulation. However, ectopic expression of Ed in the amnioserosa leading to circumferential Ed levels higher than those seen in the epidermis had no apparent effect on apicomedial Baz localisation. Thus, differences in Ed expression alone cannot account for the differential localisation of Par proteins between the amnioserosa and epidermis. It is possible that the effects of actomyosin can overpower ectopic Ed, or that other changes to the apical circumference of amnioserosa cells are involved. More generally, other Par protein interaction partners should be considered. For example, Baz and Stardust also interact and, together with Crumbs and Patj, they form the apical Crumbs complex (Tepass, 2012). Recent results suggest Patj can activate myosin by suppressing myosin light chain phosphatase. Intriguingly, amnioserosa BazS980A apical surface puncta also recruit Patj, suggesting that this pathway might contribute to myosin activity as well (David, 2013).

In summary, the data argue that the differential regulation of amnioserosa actomyosin networks by Baz and Par-6-aPKC can be explained by a single pathway in which Baz inhibits Par-6-aPKC antagonism of the cytoskeletal networks. It was also found that the actomyosin networks recruit aPKC, forming a negative-feedback loop. It is proposed that the inhibition of aPKC by Baz delays the negative feedback at earlier DC for cycling actomyosin networks, and with increased inhibition of aPKC by later DC, the actomyosin networks persist. These findings provide an example of how chemical signalling, and changes to this signalling, can modify the behaviour of actomyosin networks during embryo development (David, 2013).

The F-box protein Slmb restricts the activity of aPKC to polarize epithelial cells

The Par-3/Par-6/aPKC complex is the primary determinant of apical polarity in epithelia across animal species, but how the activity of this complex is restricted to allow polarization of the basolateral domain is less well understood. In Drosophila, several multiprotein modules antagonize the Par complex through a variety of means. This study identified a new mechanism involving regulated protein degradation. Strong mutations in supernumerary limbs (slmb), which encodes the substrate adaptor of an SCF-class E3 ubiquitin ligase, cause dramatic loss of polarity in imaginal discs accompanied by tumorous proliferation defects. Slmb function is required to restrain apical aPKC activity in a manner that is independent of endolysosomal trafficking and parallel to the Scribble module of junctional scaffolding proteins. The involvement of the Slmb E3 ligase in epithelial polarity, specifically limiting Par complex activity to distinguish the basolateral domain, points to parallels with polarization of the C. elegans zygote (Skwarek, 2014).

This study extends the mechanisms involved in epithelial polarity to include a new function: targeted protein degradation. Targeted degradation can create spatial asymmetries in protein distributions, and there is precedent for roles of E3 ubiquitin ligases, including SCFSlmb, in polarizing different aspects of cells. The involvement of Slmb in Drosophila apicobasal polarity has gone unnoticed due to the previous use of hypomorphic alleles. The strong alleles described in this study display potent expansion of the apical pole of imaginal epithelia, demonstrating that Slmb is a new polarity regulator that functions to restrict the apical domain (Skwarek, 2014).

Loss of Slmb phenocopies the polarity defects associated with mutations in two classes of 'apical antagonists': the Scrib module of core polarity regulators, and endocytic regulators that control trafficking through the early endosome. Despite the similar polarity defects, slmb mutations do not alter endolysosomal cargo traffic, nor do they display protein recruitment defects characteristic of Scrib module mutants; furthermore, no genetic interactions are seen with either pathway. Nevertheless, the downstream consequences of polarity misregulation - including tumor-like transformation and the upregulation of specific target genes - are again shared between slmb and the other apical antagonists, and, moreover, slmb and Scrib module mutant cells share a distinctive trafficking defect associated with elevated aPKC activity. It is therefore suggestrf that Slmb acts in parallel to the Scrib module to antagonize the Par complex and other apical regulators (Skwarek, 2014).

The role for Slmb defined in this study points to the existence of an apical polarity-regulating protein substrate, the levels of which must be controlled. A number of validated Slmb substrates have been ruled out as the relevant target. Bioinformatic scans of Drosophila proteins for Slmb degron sequences suggest other candidates, including Expanded (Ex), but overexpression of Ex is not sufficient to induce polarity defects resembling those of slmb. Although a contribution from the elevation of multiple substrates cannot be ruled out, slmb-like polarity phenotypes can be induced by the elevated activity of individual proteins, including Crb or aPKC. Despite evidence that aPKC undergoes ubiquitin-mediated degradation in embryos, neither aPKC nor Crb levels appear to be controlled by Slmb-mediated degradation in imaginal discs. Nevertheless, the data together suggest that the substrate of Slmb in polarity regulation will function as a positive regulator of aPKC-driven outcomes (Skwarek, 2014).

The demonstration that Slmb limits aPKC activity to distinguish the epithelial basolateral domain reveals intriguing parallels to polarization of the worm zygote. In this context, Par-2 is the primary antagonist that restricts aPKC/Par activity, while Lgl homologs function in a parallel, redundant role. Par-2 contains a RING finger domain that is characteristic of single-subunit E3 ligases, but Par-2 homologs have not been identified outside of nematodes, Par-2 does not affect aPKC/Par levels, and a degraded substrate in polarity regulation has yet to be identified. The discovery of a Drosophila E3 ligase with a similar function to Par-2 raises the possibility of a conserved molecular logic to polarity in these two paradigmatic systems; determination of the relevant substrate will shed further light on this question (Skwarek, 2014).

Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity

The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. This study shows that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl (Morais-de-Sa, 2014).

Very little is known about how the posterior follicle cells signal to polarise the AP axis of the oocyte, except that signalling is disrupted when the germline is mutant for components of the exon junction complex, such as Mago nashi. The current results reveal that Slmb also plays an essential role in this pathway, where it acts to establish the complementary cortical domains of Baz/Par-6/aPKC and Par-1. Although Slmb might act in a variety of ways to establish this asymmetry, the observation that it regulates the levels of the Par-6/aPKC complex suggests a simple model in which Slmb directly or indirectly targets a component of the complex for degradation at the posterior of the oocyte. Since aPKC phosphorylates Par-1 to exclude the latter from the cortex, the degradation of aPKC would allow the posterior recruitment of Par-1, which would then maintain polarity by phosphorylating and antagonising Baz. Indeed, this might explain the observation that Par-6 is excluded from the posterior cortex before Baz. The polarisation of the oocyte therefore appears to occur in two phases. During the initiation phase, Slmb removes the Par-6/aPKC complex from the posterior cortex to allow the recruitment of Par-1. Par-1 then maintains and reinforces this asymmetry by phosphorylating Baz to exclude it from the posterior cortex, thereby removing the cortical anchor for the Par-6/aPKC complex (Morais-de-Sa, 2014).

Slmb is usually recruited to its targets by binding to phosphorylated residues that lie 9-14 amino acids downstream from the ubiquitylated lysine. Although both aPKC and Par-6 contain several sequences that could serve as atypical Slmb binding sites, neither contains a classic Slmb-dependent degron sequence. It is therefore unclear whether the SCFSlmb complex directly ubiquitylates either protein to target it for degradation or whether it targets another, unknown component of the complex that is required for the stability of Par-6 and aPKC. Nevertheless, this model leads to the prediction that the polarising signal from the follicle cells will induce the activation of a kinase that phosphorylates a Slmb substrate at the posterior of the oocyte, thereby triggering the local degradation of the Par-6/aPKC complex (Morais-de-Sa, 2014).

The demonstration that Slmb is required for the exclusion of the Par-6/aPKC complex from the posterior of the Drosophila oocyte raises interesting parallels with AP axis formation in C. elegans. Although Drosophila does not have an equivalent of the main symmetry-breaking step in the worm, in which a contraction of the actomyosin cortex removes the anterior PAR proteins from the posterior, the function of Slmb is analogous to that of PAR-2 in the alternative polarity induction pathway. Both proteins act to remove the Par-6/aPKC complex from the posterior cortex to allow the posterior recruitment of Par-1, which then reinforces polarity by excluding Baz/PAR-3 by phosphorylation. Furthermore, the polarity phenotypes of both slmb and par-2 mutants can be rescued by the overexpression of Lgl. Slmb and PAR-2 act by different mechanisms, since the former is a subunit of the SCF ubiquitin ligase complex and promotes the degradation of the Par-6/aPKC complex, whereas the latter functions by recruiting PAR-1. Nevertheless, it is intriguing that PAR-2 contains a RING finger domain that is typically found in ubiquitin ligases, suggesting that it might have lost this activity during evolution (Morais-de-Sa, 2014).

Jiang, K., Liu, Y., Fan, J., Epperly, G., Gao, T., Jiang, J. and Jia, J. (2014). Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila. Proc Natl Acad Sci U S A 111(45):E4842-50. PubMed ID: 25349414

Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila

Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. This study demonstrates that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. These findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity (Jiang, 2014).

Aurora A triggers Lgl cortical release during symmetric division to control planar spindle orientation

Mitotic spindle orientation is essential to control cell-fate specification and epithelial architecture. The tumor suppressor Lgl localizes to the basolateral cortex of epithelial cells, where it acts together with Dlg and Scrib to organize apicobasal polarity. Dlg and Scrib also control planar spindle orientation but how the organization of polarity complexes is adjusted to control symmetric division is largely unknown. Lgl redistribution during epithelial mitosis is reminiscent of asymmetric cell division, where it is proposed that Aurora A promotes aPKC activation to control the localization of Lgl and cell-fate determinants. This study shows that the Dlg complex is remodeled during Drosophila follicular epithelium cell division, when Lgl is released to the cytoplasm. Aurora A controlled Lgl localization directly, triggering its cortical release at early prophase in both epithelial and S2 cells. This relied on double phosphorylation within the putative aPKC phosphorylation site, which was required and sufficient for Lgl cortical release during mitosis and could be achieved by a combination of aPKC and Aurora A activities. Cortical retention of Lgl disrupted planar spindle orientation, but only when Lgl mutants that could bind Dlg were expressed. Taken together, Lgl mitotic cortical release is not specifically linked to the asymmetric segregation of fate determinants, and the study proposes that Aurora A activation breaks the Dlg/Lgl interaction to allow planar spindle orientation during symmetric division via the Pins (LGN)/Dlg pathway (Carvalho, 2015).

Evolutionarily conserved polarity complexes establish distinct membrane domains and the polarized assembly of junctions along the apicobasal axis has been extensively characterized. One general feature is that it relies on mutual antagonism between apical atypical protein kinase C (aPKC) and Crumbs complexes and a basolateral complex formed by Scribble (Scrib), Lethal giant larvae (Lgl), and Discs large (Dlg). This study used the Drosophila follicular epithelium as an epithelial polarity model to address how polarity is coordinated during symmetric division. Dlg and Scrib have been shown to provide a lateral cue for planar spindle orientation. Accordingly, Scrib and Dlg remain at the cortex during follicle cell division. In contrast, Lgl is released from the lateral cortex to the cytoplasm during mitosis. This subcellular reallocation begins during early prophase, since Lgl starts to be excluded from the cortex prior to cell rounding, one of the earliest mitotic events, and is completely cytoplasmic before nuclear envelope breakdown (NEB). Thus, the Dlg complex is remodeled at mitosis onset in epithelia (Carvalho, 2015).

The subcellular localization of Lgl is controlled by aPKC-mediated phosphorylation of a conserved motif, which blocks Lgl interaction with the apical cortex. To address the mechanism of cortical release during mitosis, nonphosphorytable form Lgl3A-GFP was expressed in the follicular epithelium. Lgl3A-GFP remains at the cortex throughout mitosis indicating that Lgl dynamics during epithelial mitosis also rely on the aPKC phosphorylation motif. Although the apical aPKC complex depolarizes during follicle cell division, Lgl cortical release precedes aPKC depolarization. Using Par-6-GFP as a marker for the aPKC complex and the Lgl cytoplasmic accumulation as readout of its cortical release, it was found that maximum cytoplasmic accumulation of Lgl occurs when most Par-6 is still apically localized (~70% relative to interphase levels). Thus, Lgl cortical release is the first event of the depolarization that characterizes follicle cell division, indicating that Lgl reallocation does not require extension of aPKC along the lateral cortex (Carvalho, 2015).

Although the major pools of Lgl and aPKC are segregated during interphase, Lgl has a dynamic cytoplasmic pool that rapidly exchanges with the cortex. Thus, further activation of aPKC at mitosis onset would be expected to shift the equilibrium toward cytoplasmic localization. Lgl dynamic redistribution in epithelia is similar to the neuroblast, where activation of Aurora A (AurA) leads to Par-6 phosphorylation and subsequent aPKC activation. To test whether a similar mechanism induced Lgl cortical release during epithelial mitosis, Lgl subcellular localization was analyzed in aPKC mutants and in par-6 mutants unphosphorylatable by AurA. Lgl cytoplasmic accumulation is unaffected in par-6; par-6S34A mutant cells. Temperature-sensitive aPKCts/aPKCk06403 mutants display strong cytoplasmic accumulation of Lgl during prophase, with a minor delay relatively to the wild-type). Moreover, homozygous mutant clones for null (aPKCk06403) and kinase-defective (aPKCpsu141) alleles also display Lgl cortical release during mitosis. These results implicate that although aPKC activity may contribute for Lgl mitotic dynamics, the putative aPKC phosphorylation motif is under the control of a different kinase, which triggers Lgl cortical release in the absence of aPKC (Carvalho, 2015).

AurA is a good candidate to induce Lgl cortical release as it controls polarity during asymmetric division. Furthermore, Drosophila AurA is activated at the beginning of prophase, which coincides with the timing of Lgl cytoplasmic reallocation. To examine whether AurA controls Lgl dynamics in the follicular epithelium, homozygous mutant clones were generated for the kinase-defective allele aurA37. In contrast to wild-type cells, only low amounts of cytoplasmic Lgl were detected during prophase in aurA37 mutants, which display a pronounced delay in the cytoplasmic reallocation of Lgl during mitosis. This delayed cortical release of Lgl has been previously reported during asymmetric cell division in aurA37 mutants, possibly resulting from residual kinase activity. Thus, AurA is essential to trigger Lgl cortical exclusion at epithelial mitosis onset (Carvalho, 2015).

The idea that Lgl mitotic reallocation is directly controlled by a mitotic kinase implies that Lgl should display similar dynamics regardless of the polarized status of the cell. Consistently, Lgl-GFP is also released from the cortex before NEB in nonpolarized Drosophila S2 cells. Furthermore, Lgl3A-GFP is retained in the cortex during mitosis, revealing that Lgl cortical release is also phosphorylation dependent in S2 cells. Treatment with a specific AurA inhibitor (MLN8237), or with aurA RNAi, strongly impairs Lgl cortical release during prophase, as Lgl is present in the cortex at NEB. However, inhibition of AurA still allows later cortical exclusion, which could result from the activity of another kinase. Despite their distinct roles, AurA and Aurora B (AurB) phosphorylate common substrates in vitro. Therefore, whether AurB could act redundantly with AurA was analyzed. Inactivation of AurB with a specific inhibitor, Binucleine 2, enables normal Lgl cytoplasmic accumulation before NEB and still allows later cortical exclusion in cells treated simultaneously with the AurA inhibitor As AurB does not seem to participate on Lgl mitotic dynamics, RNAi directed against aPKC was used to examine whether it could act redundantly with AurA. aPKC depletion did not block Lgl cortical exclusion, but it was slightly delayed. However, simultaneous AurA inhibition and aPKC RNAi produced almost complete cortical retention of Lgl during mitosis. Thus, AurA induces Lgl release during early prophase, but aPKC retains its ability to phosphorylate Lgl during mitosis (Carvalho, 2015).

To address which serine(s) within the phosphorylation motif of Lgl control its dynamics during mitosis, individual and double mutants were enerated. As complete cortical release occurs before NEB, the ratio of cytoplasmic to cortical mean intensity of Lgl-GFP at NEB was quantified to compare each different mutant. All the single mutants displayed similar dynamics to LglWT, exiting to the cytoplasm prior to NEB. In contrast, all double mutants were cortically retained during mitosis, indicating that double phosphorylation is both sufficient and required to efficiently block Lgl cortical localization (Carvalho, 2015).

The ability to doubly phosphorylate Lgl would explain how AurA drives Lgl cortical release. Accordingly, the sequence surrounding S656 perfectly matches AurA phosphorylation consensus, whereas the S664 surrounding sequence shows an exception in the -3 position. In contrast, the sequence surrounding S660 does not resemble AurA phosphorylation consensus, and AurA does not directly phosphorylate S660 in vitro as detected by phosphospecific antibodies against S660. That S656 is directly phosphorylated by recombinant AurA was confirmed in vitro using a phosphospecific antibody for S656. Moreover, AurA inhibition or aurA RNAi results in a similar cortical retention at NEB to LglS656A,S664A, suggesting that AurA also controls S664 phosphorylation during mitosis, whereas aPKC would be the only kinase active on S660. Consistent with this, aPKC RNAi increases the cortical retention of LglS656A,S664A, mimicking the localization of Lgl3A. Furthermore, whereas S660A mutation does not significantly affect the cytoplasmic accumulation of Lgl in aPKC RNAi, S656A and S664A mutations disrupt Lgl cortical release in aPKC-depleted cells, leading to the degree of cortical retention of LglS656A,S660A and LglS660A,S664A, respectively. Altogether, these results support that AurA controls S656 and S664 and that these phosphorylations are partially redundant with aPKC phosphorylation to produce doubly phosphorylated Lgl, which is released from the cortex (Carvalho, 2015).

RNAi-mediated knockdown of Lgl in vertebrate HEK293 cells results in defective chromosome segregation. Furthermore, overexpressed Lgl-GFP shows a slight enrichment on the mitotic spindle suggesting that relocalization of Lgl could be important to control chromosome segregation. However, lgl mutant follicle cells assemble normal bipolar spindles, and although it was possible to detect minor defects on chromosome segregation, the mitotic timing (time between NEB and anaphase) is indistinguishable between lgl and wild-type cells. Additionally, loss of Lgl activity allows proper chromosome segregation in both Drosophila S2 cells and syncytial embryos. Thus, Lgl does not seem to have a general role in the control of faithful chromosome segregation in Drosophila (Carvalho, 2015).

Nevertheless, Lgl cortical release could per se play a mitotic function, as key mitotic events are controlled at the cortex. In fact, the orientation of cell division requires the precise connection between cortical attachment sites and astral microtubules, which relies on the plasma membrane associated protein Pins (vertebrate LGN). Pins uses its TPR repeat domain to bind Mud (vertebrate NUMA), which recruits the dynein complex to pull on astral microtubules, and its linker domain to interact with Dlg, which participates on the capture of microtubule plus ends. Notably, Pins/LGN localizes apically during interphase in Drosophila and vertebrate epithelia, being reallocated to the lateral cortex to orient cell division. Pins relocalization relies on aPKC in some epithelial tissues, but not in chick neuroepithelium and in the Drosophila follicular epithelium, where Dlg provides a polarity cue to restrict Pins to the lateral cortex. Dlg controls Pins localization during both asymmetric and symmetric division, and a recent study has shown that vertebrate Dlg1 recruits LGN to cortex via a direct interaction. However, Dlg uses the same phosphoserine binding region within its guanylate kinase (GUK) domain to interact with Pins/LGN and Lgl. Thus, maintenance of a cortical Dlg/Lgl complex during mitosis is expected to impair the ability of Dlg to bind Pins and control spindle orientation (Carvalho, 2015).

Interaction between the Dlg's GUK domain and Lgl requires phosphorylation of at least one serine within the aPKC phosphorylation site. Although the phosphorylation-dependent binding of Lgl to Dlg remains to be shown in Drosophila, crystallographic studies revealed that all residues directly involved in the interaction with p-Lgl are evolutionarily conserved from C. elegans to humans. Thus, whereas Lgl3A does not form a fully functional Dlg/Lgl polarity complex, double mutants should bind Dlg's GUK domain and are significantly retained at the cortex during mitosis due to the inability to be double phosphorylated. This led to an examination of their ability to support epithelial polarization during interphase and to interfere with mitotic spindle orientation. Rescue experiments were performed in mosaic egg chambers containing lgl27S3 null follicle cell clones. lgl mutant clones display multilayered cells with delocalization of aPKC. This phenotype is rescued by Lgl-GFP, but not by Lgl3A-GFP. More importantly, in contrast to LglS660A,S664A, which extends to the apical domain in wild-type cells and fails to rescue epithelial polarity in lgl mutant cells, LglS656A,S660A and LglS656A,S664A can rescue epithelial polarity, localizing with Dlg at the lateral cortex and below aPKC. Hence, aPKC-mediated phosphorylation of S660 or S664 is sufficient on its own to control epithelial polarity and to confine Lgl to the lateral cortex (Carvalho, 2015).

Whether exclusion of Lgl from the cortex and the consequent release from Dlg would be functionally relevant for oriented cell division was examined. Expression of Lgl-GFP or Lgl3A-GFP does not affect planar spindle orientation during follicle cell division. In contrast, Lgl double mutants display metaphasic cells in which the spindle axis, determined by centrosome position, is nearly perpendicular to the epithelial layer. Live imaging revealed that these spindle orientation defects were maintained throughout division as it was possible to follow daughter cells separating along oblique and perpendicular angles to the epithelia. Moreover, equivalent defects on planar spindle orientation were detected upon expression of LglS656A,S664A in the lgl or wild-type background, indicating that cortical retention of Lgl exerts a dominant effect. Interestingly, LglS656A,S660A and LglS656A,S664A induce higher randomization of angles, whereas LglS660A,S664A, which is less efficiently restricted to the lateral cortex, produces a milder phenotype. Altogether, these results indicate that retention of Lgl at the lateral cortex disrupts planar spindle orientation only if Lgl can interact with Dlg (Carvalho, 2015).

Despite the ability of LglS656A,S660A-GFP to rescue epithelial polarity in lgl mutants, strong overexpression of LglS656A,S660A-GFP, but not of other Lgl double mutants, can dominantly disrupt epithelial polarity during the proliferative stages of oogenesis. One interpretation is that LglS656A,S660A forms the most active lateral complex of the mutant transgenes, disrupting the balance between apical and lateral domains. Therefore whether the dominant effect of Lgl cortical retention on spindle orientation could solely result from Dlg mislocalization was assessed. Dlg is properly localized at the lateral cortex in LglS656A,S660A-expressing cells presenting misoriented spindles, but this position does not correlate with the orientation of the centrosomes. Thus, cortical retention of Lgl interferes with Dlg's ability to transmit its lateral cue to instruct spindle orientation, which may result from an impairment of the Dlg/Pins interaction (Carvalho, 2015).

In conclusion, these findings outline a mechanism that explains how the lateral domain is remodeled to accomplish oriented epithelial cell division, unveiling that AurA has a central role in controlling the subcellular distribution of Lgl. AurA regulates the activity of aPKC at mitotic entry during asymmetric division, and these results are consistent with the ability of aPKC to phosphorylate and collaborate in Lgl cortical release. However, in epithelia, aPKC accumulates in the apical side during interphase, where it induces apical exclusion of Lgl, in part by generating a phosphorylated form that binds Dlg. Consequently, aPKC has a reduced access to the cortical pool of Lgl at mitotic entry and would be unable to rapidly induce Lgl cortical exclusion. These data show that cell-cycle-dependent activation of AurA removes Lgl from the lateral cortex through AurA's ability to control Lgl phosphorylation on S656 and S664 independently of aPKC. Thus, AurA and aPKC exert the spatiotemporal control of Lgl distribution to achieve unique cell polarity roles in distinct cell types (Carvalho, 2015).

It is proposed that release of Lgl from the cortex allows Dlg interaction with Pins to promote planar cell division in Drosophila epithelia. Lgl cortical release requires double phosphorylation, indicating that whereas Lgl-Dlg association involves aPKC phosphorylation, multiple phosphorylations break this interaction, acting as an off switch on Lgl-Dlg binding. Triple phosphomimetic Lgl mutants display weak interactions with Dlg, suggesting that multiple phosphorylations could directly block Lgl-Dlg interaction. Alternatively, the negative charge of two phosphate groups may suffice to induce association between the N- and C-terminal domains of Lgl, impairing its ability to interact with the cytoskeleton and plasma membrane as previously proposed. This would reduce the local concentration of Lgl available to interact with Dlg, enabling the interaction of Dlg's GUK domain with the pool of Pins phosphorylated by AurA. Therefore, AurA converts the Lgl/Dlg polarity complex generated upon aPKC phosphorylation into the Pins/Dlg spindle orientation complex. This study, underlines the critical requirement of synchronizing the cell cycle with the reorganization of polarity complexes to achieve precise control of spindle orientation in epithelia (Carvalho, 2015).

The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC

The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, this study examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb was necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. This study found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis (Sherrard, 2015).

These results reveal novel functional interactions between Crumbs, Moesin and aPKC in the follicular epithelium during late stages of Drosophila oogenesis. Loss of Crb, or mutation of its JM domain, results in reduction of both aPKC and active Moe at the MZ. Surprisingly, it was also found that whereas aPKC promotes MZ accumulation of Crb, Moe does the opposite - loss of Moe results in increased MZ accumulation of Crb in the follicular epithelium. These opposing phenotypes suggest that Moe and aPKC function antagonistically to regulate accumulation of Crb in the MZ at these stages (Sherrard, 2015).

How do Moe and aPKC regulate levels of Crb in the MZ? First, although aPKC is generally thought to interact with the Crb complex through binding to Patj and Par-6 via its C-terminal PDZ binding motif, it has been recently suggested that aPKC also interacts with the Crb JM domain. Therefore, aPKC might promote junctional localization of Crb through interaction with two Crb domains, consistent with the observation that MZ localization of Crb is partially lost in crbJMM cells. In contrast to loss of aPKC, excessive accumulation of Crb+ (and aPKC), but not CrbJMM, was observed in the absence of Moe. A previous study has proposed that Moe also interacts with the JM domain, suggesting that Moe and aPKC could compete for Crb binding. Overall, it is hypothesized that aPKC promotes Crb stability through interactions with the Crb JM domain, whereas Moe reduces these stabilizing interactions at the MZ through competition with aPKC (Sherrard, 2015).

A second possible contributing factor in MZ accumulation of Crb is competition between the apical and junctional pools of Crb, with Moe functioning to stabilize apical Crb. This study observed that whereas loss of aPKC does not appear to affect Crb localization at the apical membrane, reduction in Moe function caused decreased Crb on the apical membrane and increased junctional Crb. Moe might stabilize Crb at the apical membrane by linking Crb to cortical actin or through more general effects on the actin cytoskeleton. Destabilized Crb on the apical membrane could then move within the plasma membrane and/or be recycled to the MZ, where it is stabilized by interactions through its PDZ-binding domain (Sherrard, 2015).

The results suggest that, in addition to the previously proposed interactions between Moe and the Crb JM domain, the more general effects of Moe on cortical actin also affect Crb localization and dynamics. In support of this idea, loss of Moe affected the localization of CrbJMM, which should not be able to interact directly with Moe, and this study observed pleiotropic effects on F-actin staining in late follicular cells depleted for Moe protein. Studies in yeast have demonstrated a crucial role for cortical actin in regulating early endocytic events. Actin dynamics are necessary for apical endocytosis in mammalian cells Furthermore, the mammalian ERM protein Ezrin provides a necessary linkage to actin in the recycling of a β-adrenergic receptor. Further disentangling the details of the interactions between Crb, Moe and F-actin will require more specific reagents to identify and manipulate the Crb/Moe binding sites to disrupt specific functions (Sherrard, 2015).

Strikingly, interactions among Crb, pMoe and aPKC were evident after stage 10, coincident with an increase in Crb trafficking and MZ accumulation. Cells undergoing shape changes, such as rapid squamous expansion, must remodel their junctions and membrane domains. Similar to the expansion of amnioserosal cells, but unlike the follicle stretch cells of stage 9, the main-body FCs maintained continuous AJs throughout their expansion. After stage 10, MZ localization of aPKC, F-actin and pMoe, and continuity of Baz, an AJ component, required Crb. This latter result is consistent with a previous study, in which Crb was required for the addition of AJ material to the expanding rhabdomere of the pupal eye. However, another rhabodmere study using transgenic overexpression implicated the Crb JM domain in AJ continuity, in contrast to the current finding that AJs were intact with the crbJMM allele (Sherrard, 2015).

An intriguing possibility is that the putative competition between aPKC and Moe contributes to the role of Crb in morphogenesis, the mechanisms of which are not fully known. In salivary placode invagination, the Crb JM domain recruits Moe and actin to a supercellular, contractile purse string, which is specified by the anisotropic distribution of Crb and aPKC away from the site of the purse string Additional evidence that the JM domain can regulate actin cable formation comes from the defective dorsal closure of crbJMmutants (Sherrard, 2015).

The results suggest a mechanism whereby competition between Moe and aPKC for Crb JM binding could help specify an actin cable. The apical Crb-Par complex might favor aPKC binding in regions of high Crb density, but Moe binding in regions of low Crb density (such as the apical surface, or the site of the purse string in the salivary placode). Thus, competition for binding Crb could amplify the anisotropy of aPKC and Crb, and Moe could recruit the actin for the supercellular cable. The upregulation of Crb endocytosis observed after stage 10 probably plays a role in regulating Crb dynamics during stages 9-11. In other contexts, the retromer regulates Crb membrane levels by influencing its recycling versus degradation. Intercellular homophilic binding of Crb might favor its MZ localization in most tissues (Sherrard, 2015).

However, in egg chambers, the germline cells, which are tightly apposed to the apical surfaces of FCs, also express Crb, which could stabilize Crb apically. Thus, the outgrowth and subsequent regression of microvilli in stages 9-10 could drive the movement of Crb apically and back to the MZ, but this hypothesis remains untested (Sherrard, 2015).

A striking aspect of the effect of Crb loss on Moesin localization was that pMoe and F-actin staining appeared to be noticeably excluded from the MZ in Crb-deficient or CrbJMM-expressing cells. In addition, the width of the zone of pMoe and F-actin loss appeared greater than the width of the MZ Crb staining. It is somewhat puzzling how pMoe and actin could be either excluded from, or fail to be recruited, to this rather broad region in the absence of the relatively narrow JM domain. One possibility is that the zone of pMoe absence represents newly added membrane material, which cannot recruit pMoe or F-actin in the absence of Crb or the Crb JM domain. Alternately, Crb might 'nucleate' addition of F-actin and/or pMoe at the MZ, and it could spread outwards from there (Sherrard, 2015).

The late-stage follicular epithelium displays an unexpectedly dynamic deployment of MZ components, revealing functional relationships not readily apparent in other developmental contexts. Whereas the interactions between Moe, aPKC and Crb uncover a novel connection between the apical PAR network and the apical actin cortex, much remains to be learned about how multifaceted interactions among these proteins and other junctional components direct tissue homeostasis and morphogenesis (Sherrard, 2015).

A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion

To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy, but AJs are dynamic during epithelial development and remodeling, and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation. This study shows that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer, and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization. Par-1 was found to promote the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference (Jiang, 2015).

The identification of Par-1 as an inducer of Baz-centrosome co-recruitment is surprising given its well-established role in inhibiting Baz complex formation in Drosophila, C. elegans, and mammalian systems. It is proposed that Par-1 contributes to both global inhibition and local promotion of Baz complex assembly, providing a simple and potent Baz polarization mechanism (Jiang, 2015).

The Baz-centrosome positive feedback loop is evident from the specific accumulation of Baz next to cortical centrosomes, the MT requirement for Baz accumulation, the Baz requirement for centrosome recruitment, and the dynein role for drawing Baz and centrosomes together. Significantly, Par-1 is also necessary and sufficient for the loop and seems to have two direct roles. One is promotion of astral microtubules around the centrosome, an effect consistent with known effects of Par-1 on MT regulators, but requiring further elucidation in the Drosophila embryo. The other is the phosphorylation of Baz at Ser-151 and Ser-1085. These modifications have well-characterized inhibitory effects on Baz cortical association, but strikingly, they are also enriched where the Baz-centrosome positive feedback loop occurs and appear necessary for Baz entry into the loop. It is speculated that phospho-regulated Baz-14-3-3 protein interactions mediate further protein interactions, or induce conformational changes, important for Baz-MT association. Indeed, 14-3-3 proteins can bridge MT motors, a Par-3 conformational change induces direct MT binding, Par-3 directly binds a dynein subunit, and other links to MTs are known (Jiang, 2015).

Although the Par-1-Par-3-centrosome pathway can be a potent Baz polarization mechanism, it is normally attenuated within a homeostatic system. During early cellularization, Par-1 localizes over the entire PM and presumably phosphorylates Baz and MT regulators. In response, it is proposed that Baz is continually displaced and diffuses over the PM but is additionally primed for MT interactions. Simultaneously, the two centrosomes found atop each nucleus would provide the positional information for localizing Baz around the apical circumference through dynein-mediated MT associations. As Baz accumulates, it recruits aPKC to the apical domain, from where aPKC then displaces Par-1. Normally, this Baz-aPKC-Par-1 negative feedback loop seems to keep the Par-1-Baz-centrosome pathway in check. In the absence of aPKC, the Par-1- Baz-centrosome pathway continues unabated, leading to excessive Baz and centrosome polarization, loss of AJ isotropy, and later epithelial dissociation (Jiang, 2015).

Intriguingly, focused accumulations of Par-3 and AJs colocalize with cortical centrosomes during C. elegans intestinal development and during zebrafish collective cell migration. Moreover, Par-1 induces centrosomal MT interactions with AJs during human liver lumen formation in vitro and is needed for Baz-centrosome associations during the asymmetric division of Drosophila germline stem cells. Thus, induction of the Par- 1-Par-3-centrosome pathway, with regulated shifts to aPKC or Par-1 activities, may be generally relevant to developmental transitions of animal tissues (Jiang, 2015).

Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex

The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. This study investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. A Magi null mutant was generated and found to be viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi results in the displacement of Baz/Par3 and aPKC and leads to an increase in the level of PIP3. Interestingly, it was found that Magi and Baz function in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex (Padash Barmchi, 2016).

A common component of junctional and polarity complexes is modular scaffolding proteins that are capable of binding to each other as well as recruiting other proteins to the complex. Magi proteins are evolutionarily conserved scaffolding proteins and contain multiple domains including a N-terminal catalytically inactive GUK domain, two WW domains and five to six PDZ (PSD95/Dlg/ZO-1) domains (Dobrosotskaya, 1997). There are three MAGI proteins in vertebrates (MAGI-1,2,3) all with multiple splice isoforms. MAGI-1 and MAGI-3 are relatively ubiquitously expressed and localize to a range of junctions including epithelial tight junctions. MAGI-2 (also known as AIP1/S-SCAM/ARIP1) is expressed in the nervous system as a synaptic protein and within glomerular podocytes in the kidney and plays important role in scaffolding synaptic proteins such as NMDA receptors and Neuroligin, the tip-link protocadherin Cadherin23, the Kir4.1 K(+) channel, as well as kidney proteins such as nephrin and JAM4 (Padash Barmchi, 2016).

Within epithelia and endothelia, MAGI-1 and -3 are localized at tight junctions and form a structural scaffold for the assembly of junctional complexes. MAGI-1 also localizes and plays a role in modulating adherens junction adhesion through scaffolding beta-catenin and PTEN. MAGI-1 overexpression stabilizes adherens junctions and epithelial cell morphology through increased E-cadherin and β-catenin recruitment. Silencing of MAGI-1 has the opposite effect with decreased adherens junction adhesion and reduced focal adhesion formation leading to anchorage-independent growth and migration in vitro. MAGI-1 overexpression suppresses the invasiveness of MDCK cells, as well as suppresses tumor growth and spontaneous lung metastasis through the increased recruitment of PTEN or β-catenin and E-cadherin (Padash Barmchi, 2016).

Overall, MAGI proteins play important roles in the stabilization of cell-cell interactions and as such Magi is a key target in polarized epithelia during cell death and viral infection. For instance, MAGI-1 is cleaved by activated caspases during apoptosis, a process thought to mediate the disassembly of cell-cell contacts (Gregorc, 2007). MAGI proteins are also targeted by a number of oncogenic viruses: it is aberrantly sequestered in the cytoplasm by Adenovirus E4orf1, and is targeted for degradation by the E6 oncoprotein of high-risk human papillomavirus. E6-mediated degradation of MAGI-1 in cultured epithelial cells leads to loss of tight-junction integrity (Padash Barmchi, 2016 and references therein).

There is a high degree of conservation of protein structure and function in the invertebrate homologues of Magi in particular with regards to epithelial junction formation and maintenance. In C. elegans, Magi-1 plays a role in the segregation of different cell adhesion complexes into distinct membrane domains along the lateral plasma membrane. In Drosophila, Magi binds Ras association domain protein 8 (RASSF8) and modulates adherens junctions remodeling in late eye development during interommatidial cell (IOC) rearrangements. In this context Magi function is necessary to recruit the polarity protein Par-3 (Drosophila Bazooka, Baz) to the remodeling adherens junction. However, the association of Drosophila Magi or any Magi homologue with any components of the Par polarity complex in stable epithelia has not been determined (Padash Barmchi, 2016).

The Par complex consisting of Par-3/Par-6/aPKC localizes to tight junctions where MAGI is present in vertebrate epithelial cells and is necessary for assembly of this junctional complex as well as for separation of the apical region of the plasma membrane from the basolateral domain. In Drosophila epithelial cells, the Par complex localizes to the apicolateral membrane and demarcates the boundary between the apical and basolateral membrane regions. Mutant embryos for any member of this complex show loss of apicobasal polarity and disruption in the integrity of epithelia. Although the members of the Par complex are important for the establishment of cell polarity, some of the core components of this complex such as Baz are dispensable for the maintenance of cell polarity during later stages of development. Baz localizes to adherens junction and mutant clones of baz in wing imaginal discs are fully viable with no polarity or adherens junction defects. Similarly, Magi function in AJ stability has been determined in many systems, but surprisingly loss of Drosophila Magi has no effect on established, stable AJs (Zaessinger, 2015). Little is known about the convergence of Magi and Par complex function at the adherens junctions and it is possible that Baz and Magi function in established epithelia are redundant. Therefore this study investigated the role of Magi in the established and stable epithelia of the Drosophila wing imaginal disc to test the potential interactions between Magi and members of the Par complex (Padash Barmchi, 2016).

Drosophila Magi was found associated with the PAR polarity complex and is localized at the adherens junction with Baz, Par-6, and aPKC. Overexpression of Magi resulted in the reduction of apical polarity proteins from the membrane and these interactions required the second half of the Magi protein containing the four PDZ domains. Overexpression of Baz resulted in a reduction of Magi from the membrane but an increase in aPKC and Par-6. While Magi mutants were viable with no polarity defects, Magi levels were found to be antagonistic with Baz, and a balance between the two was found to be necessary to regulate the level and localization of Par complex (Padash Barmchi, 2016).

PDZ domain-containing proteins form scaffolding protein complexes with a wide range of roles including cell polarity and signaling. As a MAGUK protein, Magi is part of a scaffold that interacts with members of the polarity complex at the adherens junctions in the epithelia of the imaginal disc. The scaffolding function of Magi has been well established in other systems. In vertebrates epithelial cells MAGI-1 has been shown to act as structural scaffold at tight junctions and adherens junctions. In C. elegans, Magi-1 localizes apical to adherens junction and functions as an organizer to ensure that different cell adhesion complexes are segregated into distinct membrane domains along the lateral plasma membrane. In neuronal cells MAGI-2/S-SCAM was also shown to cluster the cell adhesion molecule Sidekick, and the AMPA and NMDA glutamate receptors at the synapse (Padash Barmchi, 2016).

Given the strong conservation of the Magi protein it is surprising that null mutants of Drosophila Magi exhibit no lasting cellular defects (other than transient defects in the interommatidial cells of the pupal eye and null animals are fully viable. Similarly in C. elegans, magi-1 null worms are healthy with only a few embryos (1.3%) with defects during the ventral enclosure stage. As Magi is highly conserved, it is plausible that Magi may only act in response to cell stress, DNA damage or some other trigger. For example, loss of p53 does not disrupt cellular function under normal conditions and p53 null flies or mice are viable with no cellular defects. However, the role of p53 in response to DNA damage is well established and when these animals are exposed to irradiation apoptosis is not induced. Alternatively, Magi function might be redundant with other components of the apical polarity complex or another protein and that loss of both is necessary for the disruption of cellular function. Core scaffolding components of the apicobasal polarity complex are dispensable for maintaining polarity in the wing imaginal disc epithelia supporting the idea of redundancy in this system. For instance, somatic clones of loss of function mutations in crb, sdt and baz have no effect on the polarity in the wing disc epithelia of the 3rd instar larvae. Baz is a strong candidate for redundancy with Magi given the localization to the adherens junction and function as a PDZ scaffolding protein. As loss of baz in the wing imaginal disc does not disrupt the polarity of wing disc epithelia this leads to the hypothesis that Baz and Magi are redundant. However, somatic clones of a baz null mutant in a Magi mutant background did not lead to a loss of cell polarity or apoptosis. While the two scaffolding proteins do not appear to functionally interact, it was observed that Magi and Baz are in a protein complex and their close proximity within the wing columnar epithelia also suggests a common complex. Overexpression of Magi displaces Baz and aPKC from the apical membrane and, likewise overexpression of Baz displaces Magi from the membrane. The simultaneous over-expression of Magi and Baz suppresses the changes caused by their individual expression, suggesting a balance or competition between the two proteins. The maintenance of a balance between Magi and Baz might be due to a direct physical competition between these two proteins or opposite effects on a common mediator or interactor (Padash Barmchi, 2016).

Baz and vertebrate MAGI proteins bind the lipid phosphatase PTEN and thus the Magi-Baz interaction and balance could be influenced by changes in the level of phosphoinositides such as PtdIns(4,5)P2 (PIP2) or PtdIns(3,4,5)P3 (PIP3). In polarized epithelia, PIP2 is found within the apical domain and PIP3 restricted to the basal-lateral domain. Baz localization in polarized epithelia depends on PIP2 and on the PI4P5 kinase Skittles. Baz in turn can be a positive regulator of PIP2 levels at the plasma membrane by local recruitment of the lipid phosphatase PTEN. This study observed an increase in PIP3 levels with increased expression of Magi, which may reflect the loss of Baz and a loss of PTEN recruitment to the membrane. This study was not able to assess changes in PTEN levels at the membrane with available antibodies. However it was observe that the recruitment of Magi or Baz was not affected in Pten mutant cells. Similarly the changes in PIP3 levels are unlikely to be the cause of Baz loss in the presence of increased Magi as co-expression of PTEN and Magi still resulted in the loss of Baz from the membrane. Prior studies on Magi in Drosophila in the pupal eye did not detect any physical interaction between Drosophila Magi and Pten, and the phenotypes generated by overexpression of Magi in the Drosophila eye were not affected by Pten mutants. Therefore it is likely that loss of Baz in the presence of increased Magi in the wing imaginal disc and vice versa is through competition for a protein component (Padash Barmchi, 2016).

In the developing eye Magi forms a protein complex with RASSF8 (the N-terminal Ras association domain-containing protein) and ASPP (Ankyrin-repeat, SH3-domain, and proline-rich-region containing protein), and this complex plays a role during remodeling of the adherens junctions in the interommatidial cells (IOCs) (Zaessinger, 2015). When IOCs rearrange to create the pupal lattice, this process requires regulation of the E-Cadherin complex where RASSF8 and ASPP regulate adherens junction remodeling and integrity through regulation of Src kinase activity. Magi recruits the RASSF8-ASPP complex in the process of adherens junction remodeling and there are defects in IOC rearrangement in Magi mutants where AJs are frequently interrupted. In the eye the Magi-RASSF8-ASPP complex is necessary for the cortical recruitment of Baz and of the adherens junction proteins α- and β-catenin. A model has been proposed where Magi-RASSF8-ASPP complex functions to localize Baz to remodeling junctions to promote the recruitment or stabilization of E-Cad complexes (Zaessinger, 2015). However, it is not thought that the RASSF8-ASPP complex is the point of competition between Magi and Baz within the wing imaginal disc. In the wing imaginal disc Magi and the RASSF8-ASPP complex are localized to the adherens junction domain independently (Zaessinger, 2015) and while RASSF8 mutants have a wing rounding phenotype, Magi mutants do not. Furthermore no differences were observed in Baz, Ecad or Arm distribution in Magi somatic loss of function clones in the wing imaginal disc. Finally the Magi WW domains are required for the interaction with RASSF8 (Zaessinger, 2015), while the overexpression of the Magi transgene that contains the PDZ domains led to a reduction in Baz suggesting that second half of the Magi protein containing the PDZ domains contains the important sites for this competition (Padash Barmchi, 2016).

Therefore, a strong possibility to explain the reciprocal effects of overexpression is that Baz and Magi compete for a common binding site. Magi was found to interacte with both Baz and aPKC; the latter two are known to interact directly. However, it is unlikely that the shared site is through physical scaffolding of aPKC, as high levels of wild type aPKC had no effect on either Magi or Baz and was not able rescue the changes in Baz levels and localization caused by Magi overexpression. In addition the overexpression of Magi also led to a reduction in aPKC. It is unlikely that the loss of Baz is responsible for this displacement as aPKC is not mislocalized in Baz clones and Baz is not mislocalized in Par-6, aPKC or Cdc42 null clones. Further investigation is required to explore the mechanisms that underlie Magi interactions with components of the apical polarity complex and the adherens junction complex (Padash Barmchi, 2016).

atypical protein kinase C: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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