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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
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).
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).
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).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.