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).
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).
To see where aPKC is expressed during embryonic development, the mRNA distribution was analyzed by RNA in situ hybridization. aPKC mRNA is already detectable in freshly laid eggs before the onset of zygotic transcription and thus must be deposited maternally during oogenesis. At the cellular blastoderm stage, aPKC mRNA is present in all cells except for the pole cells. During gastrulation, strong expression of aPKC is detectable in tissues that undergo morphogenetic movements; e.g., the invaginating mesoderm, the proctodeum, and the cephalic furrow. In embryos at the extended germ band stage, prominent aPKC expression is detectable in neuroblasts. In several epithelial tissues, in particular in the fore- and hind-gut and in the Malpighian tubules, aPKC mRNA is highly enriched in the apical cytocortex, reminiscent of the polarized localization of baz and crumbs (crb) mRNAs (Wodarz, 2000).
The distribution of aPKC protein was analyzed using anti-PKCzeta antibody C20. During cellularization of the embryo, aPKC becomes localized to the apical cytocortex of all cells except for the pole cells, which do not contain detectable amounts of aPKC. Already at this stage aPKC is enriched at apico-lateral cell borders, giving rise to a honeycomb pattern in en face views of the blastoderm. After completion of cellularization, DaPKC is highly concentrated in the apico-lateral cortex and shows little overlap with the basolateral marker Nrt. Apical localization is maintained throughout embryonic development in most epithelia that are derived from the ectoderm (e. g. epidermis, fore-, and hind-gut), Malpighian tubules, and the tracheal system. The only ectodermal epithelium devoid of DaPKC expression is the amnioserosa (Wodarz, 2000).
Strong expression of DaPKC is also detected in neuroblasts, the stem cells of the embryonic CNS. During delamination of neuroblasts, aPKC is localized in the apical stalk that is wedged between adjacent cells of the neuroectodermal epithelium. In pro- and meta-phase, aPKC forms apical cortical crescents. In anaphase, aPKC staining is strongly diminished and expands over a broader region of the neuroblast cortex, but is clearly excluded from the budding ganglion mother cell. Thus, from delamination through pro- and meta-phase, localization of DaPKC in neuroblasts is very similar to that of Baz and Insc. Simultaneously with aPKC, cell outlines of epithelial cells and neuroblasts were visualized with the Nrt antibody. Similar to epithelial cells, Nrt expression is clearly polarized in neuroblasts. Strong staining for Nrt is detectable in the basal and lateral membrane of neuroblasts, whereas staining is strongly reduced in apical regions where DaPKC is expressed (Wodarz, 2000).
To study the consequences of aPKC loss-of-function on epithelial polarity and asymmetric division of neuroblasts, the phenotype of mutants in the aPKC locus was analyzed. BLAST searches with the genomic sequence of the aPKC gene revealed that the P-element insertion l(2)k06403 is located in the third intron of the aPKC gene. This insertion line is homozygous lethal, contains a single PlacW P-element, and does not complement Df(2R)Jp1, which removes the cytological interval 51C3;52F5-9. Staining intensity with anti-PKCzeta antibody C20 was reduced to background level in embryos homozygous mutant for l(2)k06403. The P-element was mobilized using the transposase source P[delta2-3] and several revertants lacking the w+ marker of the original P-element insertion were recovered. Of seven revertant lines tested, 3 were homozygous viable, demonstrating that insertion of the P-element into the aPKC locus was the cause of lethality and of the observed phenotype. It was thus conclude that l(2)k06403 is an allele of aPKC and it was renamed DaPKCk06403 (Wodarz, 2000).
Homozygous mutant DaPKCk06403 embryos do not produce any cuticle. The reason for this phenotype is early embryonic lethality before the onset of cuticle secretion. Transheterozygous DaPKCk06403/Df(2R)Jp1 embryos show exactly the same cuticle phenotype, suggesting that DaPKCk06403 is a strong hypomorphic or null allele. The analysis of the aPKC loss-of-function phenotype was complicated by the fact that aPKC mutants show maternal haploinsufficiency with incomplete penetrance. The evidence for this conclusion comes from the following observations: it was noticed that, in egg collections from the DaPKCk06403/CyO stock, significantly more than 50% of embryos (including CyO/CyO homozygotes) fail to hatch, which means that even embryos with a zygotic wild-type allele of aPKC frequently show developmental defects. This finding was confirmed in crosses of heterozygous DaPKCk06403/CyO females to wild-type males, where considerable embryonic lethality was observed. The reciprocal cross does not show an increase of embryonic lethality compared with wild-type controls. It is therefore concluded that the decreased viability of the progeny from DaPKCk06403/CyO mothers is caused by the reduction of the maternal DaPKC level during oogenesis (Wodarz, 2000).
To produce embryos with the wild-type maternal contribution of aPKC, but lacking any zygotic aPKC expression, C(2)v females were crossed to DaPKCk06403/CyO males and the cuticle phenotype of their progeny was analyzed. One quarter of the embryos derived from that cross completely lacked zygotic expression of aPKC and produced cuticles with characteristic defects. In most cases, head structures were missing and the ventral cuticle either showed large holes or was missing altogether. These phenotypes are strikingly similar to those of baz mutants. baz null embryos derived from germ-line clones produce very little or no cuticle and resemble DaPKCk06403 mutant embryos derived from heterozygous mothers. baz mutant embryos lacking only the zygotic expression produce cuticles with characteristic head defects and ventral holes that are very similar to DaPKCk06403 mutant embryos derived from C(2)v mothers (Wodarz, 2000).
The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote, subsequently have been shown to regulate cellular polarity in diverse cell types by means of an evolutionarily conserved protein complex including PAR-3, PAR-6, and atypical protein kinase C (aPKC). The Drosophila homologs of par-1, par-3 (bazooka [baz]), par-6 (DmPar-6), and pkc-3 (Drosophila aPKC; DaPKC) each are known to play conserved roles in the generation of cell polarity in the germ line as well as in epithelial and neural precursor cells within the embryo. In light of this functional conservation, the potential role of baz and DaPKC in the regulation of oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of initial anterior-posterior polarity within germ-line cysts and maintenance of oocyte cell fate. Germ-line clonal analyses indicate both proteins are essential for two key aspects of oocyte determination: the posterior translocation of oocyte specification factors and the posterior establishment of the microtubule organizing center within the presumptive oocyte. Baz and DaPKC colocalize to belt-like structures between germarial cyst cells. However, in contrast to their regulatory relationship in the Drosophila and C. elegans embryos, these proteins are not mutually dependent for their germ-line localization, nor is either protein specifically required for PAR-1 localization to the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).
To examine the potential oogenic function of baz and DaPKC, protein null germ-line mutant clones were generated for both baz and DaPKC. Germ-line clones, identified by the absence of nuclear GFP expression, were counterstained with the chromatin marker propidium iodide to examine the number and ploidy of the germ-line nuclei. In contrast to wild-type egg chambers, which invariably contain 15 nurse cells and a single oocyte, baz mutant germ-line clones, while containing the normal complement of germ-line nuclei, fail to differentiate an oocyte, resulting in a 16-nurse cell phenotype as revealed by the polyploid state of all 16 germ-line nuclei. Similarly, DaPKCk06403 germ-line clones also fail to differentiate an oocyte as indicated by the presence of 16 polyploid nurse cell nuclei in germ-line mutant egg chambers. These results reveal a germ-line autonomous requirement for DaPKC and confirm the role of Baz in oocyte differentiation and/or maintenance (Cox, 2001).
These analyses further reveal that germ-line depletion of either DaPKC or baz function from the follicle cells leads to their multilayering, which disrupts the normal partitioning of germ-line nuclei to successively mature egg chambers caused by mispositioning of mutant follicle cells. These mispartitioned baz+ nurse cell nuclei, into an otherwise baz null germ-line clone, may provide the threshold of germ-line Baz required to rescue the oocyte differentiation defect and allow production of a mature egg. Alternatively, the Baz protein may exhibit a long perdurance after mitotic clone induction, which depletes over a period of days, resulting in the cessation of egg production in these mosaic females. These results, however, in no way invalidate the previous conclusions that maternally provided Baz masks the severity of the embryonic polarity phenotype in both epithelial cells and neuroblasts. Rather, the results indicate that these embryos are not likely to represent a complete maternal depletion for Baz (Cox, 2001).
Oocyte differentiation requires the polarized accumulation of oocyte specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in the localization of these factors, mutant germ-line clones for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst cells in region 2a, and then both molecules are targeted first to the two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In contrast, ORB fails to translocate from the anterior to a posterior crescent in both baz and DaPKC null germ-line cysts in germarial region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in baz and DaPKC null germ-line clones in germarial region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).
Furthermore, in contrast to wild-type germ-line cysts in which DHC64C localizes to a single posterior cell, in DaPKC null germ-line clones DHC64C fails to localize to a single cell posteriorly, but rather accumulates in the two posterior-most presumptive pro-oocytes of the mutant germ-line cyst. Therefore, baz and DaPKC display essentially identical phenotypes in germ-line mutant clones with regards to oocyte differentiation and the establishment of initial A-P polarity within the oocyte. The failure to maintain oocyte identity in either baz or DaPKC mutant cysts can therefore be directly correlated with defects in the A-P translocation of oocyte specification factors within a single posterior cell of a germ-line cyst, suggesting oocyte differentiation depends on this early polarization event (Cox, 2001).
The posterior assembly of a functional MTOC has been directly implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the failure to translocate these factors to a posterior crescent in region 3 baz or DaPKC mutant cysts may result from a defect in microtubule reorganization within these mutant cysts. In contrast to wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of oocyte specification factors in these mutants are likely caused, at least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).
In addition to the microtubule network, both ring canals and the fusome play critical roles in cyst polarization and oocyte differentiation. The formation and spatial distribution of ring canals as well as fusome morphogenesis was examined in both baz and DaPKC mutant germ-line cysts. In baz mutant cysts, ring canal formation and spatial distribution are indistinguishable from wild-type germ-line cysts. Furthermore, the wild-type spatial arrangement of ring canals in baz null germ-line cysts suggests there is no apparent disruption in germ cell adhesion within the cyst. Similarly, no defects were observed in ring canal formation or spatial distribution in DaPKC mutant cysts (Cox, 2001).
These analyses indicate baz mutant cysts display relatively normal fusome morphology, although mutant fusome branches appear slightly thinner when compared with wild-type fusomes within the same germarium. Similarly, no apparent defect is observed in fusome morphology in DaPKC null germ-line cysts, indicating DaPKC is dispensable in the germ line for proper fusome morphogenesis (Cox, 2001).
To investigate the mechanism by which Baz and DaPKC exert their effects on oocyte differentiation, the localization of these proteins was analyzed in the germ line and soma during oogenesis. The specificity of the Baz and DaPKC antibodies was verified by using mosaic ovaries containing baz or DaPKC null mutant clones. Baz is first detected in the germarium as a belt-like specialization on germ cell membranes at sites of germ cell interconnection. These belt structures are reminiscent of ring canals with respect to their position between germ-line cyst cells; however, in contrast to ring canals these "Baz belts" are approximately 2-fold greater in diameter. Germaria double-labeled for Baz and rhodamine phalloidin reveal that the Baz belts localize adjacent to ring canals and further reveal an approximate 1:1 ratio between the two structures within germ-line cysts. The microtubule cytoskeleton as well as the fusome were observed projecting through individual cystocytes coincident with the site of Baz belt expression on the germ cell membrane. Later in oogenesis, Baz is transiently enriched in the oocyte cytoplasm at stages 5-6 before the onset of vitellogenesis and is subsequently undetectable in the germ line of vitellogenic egg chambers. As with Baz localization to the apical junctional zone in the embryonic epithelium, tight apical localization of Baz is also observed in follicular epithelia (Cox, 2001).
DaPKC localizes to the Baz belts in the germarium, whereas in follicle cells DaPKC is apically constricted consistent with Baz localization in these cells (Cox, 2001).
In embryonic epithelia, Baz, DmPAR-6, and DaPKC apically colocalize and partially overlap with the apico-laterally enriched Arm and DE-cadherin proteins in the region of the apical zonula adherens. Furthermore, these proteins are required to maintain epithelial cell polarity and are mutually dependent for their proper localization. DE-cadherin is localized to belt-like structures adjacent to ring canals in region 2 germarial cysts reminiscent of Baz belt localization, suggesting that these adherens junction components may similarly colocalize in the germ line. To further investigate the molecular and functional nature of Baz belt expression in the germarium, germaria were labeled with anti-Baz, anti-Arm, and anti-DE-cadherin antibodies and their potential colocalization within the germarium was examined. These analyses reveal Baz colocalizes with both DE-cadherin and Arm to the Baz belts within the germarium. Furthermore, consistent with the colocalization of Baz and DaPKC to Baz belts within the germarium, partial colocalization of DaPKC with DE-cadherin is observed in wild-type germarial cysts. To assay whether these proteins are mutually dependent for their germ-line localization, the localization of DE-cadherin and Arm was examined in baz null germ-line clones. In contrast to their mutual dependence in embryonic epithelia, these analyses indicate germ-line Baz function is dispensable for the localization of either DE-cadherin or Arm to the Baz belts in the germarium. DaPKC is dispensable for the localization of either DE-cadherin or Arm to these structures. These results indicate Baz and DaPKC function are not required for the formation of these structures because both Arm and DE-cadherin localization to these belts in baz or DaPKC mutant cysts is indistinguishable from that observed in wild-type cysts. Previous studies have revealed that germ-line clones of a strong allele of shotgun (shgIG29), the gene encoding DE-cadherin, disrupts the arrangement of germ cells in region 2b germarial cysts, suggesting DE-cadherin may mediate germ cell adhesion. In contrast, these analyses of ring canal spatial distribution in baz and DaPKC mosaic cysts strongly suggests Baz and DaPKC function are dispensable for normal germ cell adhesion. Furthermore, germ-line clonal analyses of either shg or arm reveal neither gene is required for oocyte differentiation, whereas both baz and DaPKC are essential in oocyte determination. These results suggest the components of the Baz belts likely mediate diverse cellular functions essential for germ-line cyst development, which may include cell adhesion, cell signaling, and cyst polarization (Cox, 2001).
In the C. elegans zygote, the PAR-3/PAR-6/PKC-3 complex is localized to the anterior where it is required for the posterior localization of PAR-1. To investigate the potential regulatory relationship between baz and par-1 in the Drosophila germ line, baz null germ-line clones were generated and PAR-1 expression and localization was analyzed. In wild-type germ-line cysts, PAR-1 is localized to the spectrosome and fusome in germarial regions 1 and 2a and subsequently is down-regulated on fusomes in regions 2b and 3. In baz mutant germ-line cysts, PAR-1 localization to the spectrosome is unaffected and is likewise present on fusomes although somewhat weaker localization is observed on fusome branches of baz mutant germ-line cysts when compared with wild-type germ-line cysts. Taken together, these results suggest germ-line baz is dispensable in the germarium for normal PAR-1 expression and localization (Cox, 2001).
To determine whether par-1 may regulate Baz expression, par-1 null germ-line clones were generated and Baz localization was examined. No defect was observed in Baz belt expression in par-1 mutant germ-line cysts. Furthermore, in contrast to wild-type stage 5-6 egg chambers in which Baz is transiently enriched in the oocyte cytoplasm, in par-1 mutant egg chambers Baz localization is abolished from the posterior presumably due to the defect in oocyte differentiation observed in par-1 mutant egg chambers. These results indicate germ-line par-1 is dispensable for normal Baz belt expression and localization (Cox, 2001).
To assay the regulatory relationship between baz and DaPKC the localization of DaPKC was analyzed in baz mutant germ-line cysts, as well as the localization of Baz in DaPKC mutant germ-line cysts. In contrast to their mutual dependence for localization in the embryo, both Baz and DaPKC localization within the germ line is mutually independent. These results indicate that despite the apparent functional conservation of Baz, DaPKC, and PAR-1 in generating oocyte polarity, the regulatory relationships among these genes are not conserved in the germ line (Cox, 2001).
The colocalization of Baz, DaPKC, Arm, and DE-cadherin to the Baz belt structures in the germarium strongly suggests the components of the Baz belts are capable of mediating a multiplicity of functions in germ-line cysts. In embryonic epithelia, these proteins function in the formation of the apical zonula adherens junction and are mutually dependent for their apical localization. The germ-line colocalization of these molecules to the Baz belts suggests these structures may represent a potential polarity cue on the germ cell plasma membrane. The restricted localization of Baz belt components to germ cell membranes at points of cell-cell contact represents an asymmetry on the plasma membrane of germ-line cyst cells with regard to the A-P axis of the cyst and stage 1 oocyte. The asymmetric localization of these molecules to one side of the germ cell plasma membrane may act as a polarity cue in defining anterior versus posterior within individual cystocytes of the 16-cell germ-line cyst and thus contribute to the establishment of an initial A-P axis and to subsequent germ-line cyst polarization. These results further suggest that Baz and DaPKC likely function in a signaling capacity, rather than a structural one, to mediate oocyte differentiation, whereas DE-cadherin and Arm are more likely to function in maintaining germ cell adhesion and cyst integrity (Cox, 2001).
Consistent with par gene function in other systems, these results indicate that Baz, DaPKC, and PAR-1 are required for the establishment and maintenance of cellular polarity in the Drosophila germ line; however, the regulatory relationships observed between these genes in the germ line versus that observed in embryonic blastomeres, epithelial cells, and neural precursor cells indicates that, while these molecules are functionally conserved, the mechanisms by which these genes act appear to be less well conserved. In contrast to their mutual dependence for localization in the embryo, Baz, DaPKC, and PAR-1 each are mutually independent for their localization in the germ line. Taken together, these results underscore the functional utility of the par genes and their effectors as a molecular module for generating cellular polarity in diverse cell types. Furthermore, the independence of Baz, DaPKC, and PAR-1 in their germ-line localization provides a unique opportunity to probe new mechanisms by which these highly conserved proteins function in regulating diverse processes such as cellular polarity, asymmetric cell division, or growth control (Cox, 2001).
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date revised: 10 May 2004
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