Patj negatively regulates Fz1 activity by recruiting aPKC to Fz1. is that Patj and aPKC should be either downregulated or absent in cells where Fz-PCP signaling is active. To investigate this, immunostainings were performed with Patj- and aPKC-specific antibodies in third instar larval eye discs during PCP establishment. Patj is expressed at the most apical lateral membrane of third instar eye imaginal disc cells anterior to the MF. Posterior to the furrow, as photoreceptor preclusters emerge and begin to differentiate, Patj is still detected apically in all intercluster cells but shows a complex pattern within the preclusters. In developing preclusters, Patj is enriched in R2/R5 precursors and dramatically downregulated in R3/R4 precursors between rows 1 and 7. This reduction in expression is complementary to an increase in apical Fz1 localization (monitored by Fz-GFP staining), which shows the typical double horseshoe pattern specific for PCP factors in early R3/R4 pairs. Posterior to row 7, Patj is found in R3/R4 as well but remains enriched in R2 and R5. Similarly, although not as dramatic as for Patj, aPKC expression is weaker apically in R3/R4 cells as compared to the neighboring R2 and R5 (Djiane, 2005).
The downregulation of aPKC and Patj from the R3/R4 cell border during PCP establishment raised the possibility that the PCP determinants could control expression or localization of aPKC/Patj. Clonal analyses of PCP genes revealed, however, that the Patj and aPKC characteristic expression patterns in the preclusters are unaffected in fmi, dgo, pk, stbm, and fz mutant clones. The aPKC and Patj expression patterns are therefore independent of PCP signaling, consistent with an upstream early role of these A/B polarity determinants (Djiane, 2005).
To investigate a possible role for aPKC in regulating PCP, aPKC mutant clones were generated using the eyFLP technique. No clones were recovered in adult eye tissue, making it impossible to score for PCP defects. In the eye imaginal disc, only few very small clones anterior and posterior to the furrow were recovered, probably reflecting the aPKC requirement for A/B polarity and cell division (Djiane, 2005).
Since aPKC mutant clones are cell lethal, the effect of aPKC on PCP were analyzed by overexpressing aPKC under sev-Gal4 control. Full-length aPKC caused no PCP defects, but constitutively active aPKC (aPKCΔN) driven by sep-Gal4 (a weaker version of sev-Gal4, which caused massive cell lethality) resulted in PCP defects and photoreceptor loss. These results suggest that aPKC is subject to regulation, since excessive amounts of wild-type protein do not produce a gain-of-function (GOF) phenotype, while a deregulated active form of aPKC induces PCP defects. This is consistent with aPKC activity described in other contexts (Djiane, 2005).
To overcome the technical difficulties of aPKC mutants resulting from its early function in A/B polarity, the genetic contribution of Patj for PCP establishment was analyzed. Since the results indicate that Patj links aPKC to Fz1, removing Patj should hinder aPKC from regulating Fz1, but aPKC should still be able to perform its function in A/B polarity. There is no Patj mutant currently available, a small deficiency uncovering Patj, Df(3L)My10 was used, in combination with rescue constructs for all other genes disrupted by this deficiency. This combination encodes for a truncated version of Patj lacking the Fz1-interacting PDZ domain 4 but containing the N-terminal part of Patj with the L27 Sdt binding domain and the first PDZ domain. Although externally these flies have a largely normal appearance, analysis of their eyes revealed PCP defects with many symmetrical R3/R3 type ommatidia, implicating Patj in the regulation of PCP establishment in the Drosophila eye (Djiane, 2005).
Wild-type Patj was overexpressed in the eye (sev>Patj) and, similarly to full-length aPKC, this caused no PCP defects. However, expression of a Fz1 transgene that lacks the Patj binding site (Fz1ΔBS) induced a significantly stronger GOF effect than wild-type Fz1. In particular, the number of symmetrical R3/R3 type ommatidia, reflecting an elevation in Fz1 activity, was markedly increased (rising from 9% in sev>Fz1 to 51% in sev>Fz1ΔBS). Finally, an enhancement of the sev>Fz1 GOF phenotype was observed by the removal of one copy of Df(3L)My10 uncovering the Patj locus (Djiane, 2005).
These results show that the aPKC/Patj complex is required for PCP establishment and are consistent with previous observations showing a negative role of aPKC phosphorylation on Fz1 activity (Djiane, 2005).
The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Patj (the Drosophila homolog of mammalian Patj) is multi-PDZ domain protein that localizes to the apical cell membrane and form a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (St). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the patj gene. Genetic analysis of hypomorph, null, and RNA inerference reveals distinct dual functions of Patj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Patj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Patj is essential for animal viability and photoreceptor morphgenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced level of Patj. Patj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. This study provides evidence that Drosophila Patj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells (Nam, 2006).
Dynamic changes take place in developing photoreceptors to reorganize the apical cell membrane during pupal stage. Crb and Sdt are required for growth and maintenance of rhabdomeres and AJs during this time of photoreceptor morphogenesis. Patj binds Sdt to form a conserved heterotrimeric Crb complex (Roh, 2002), but its function in vivo has been unclear. In this study, a key question whether Patj is an essential member of the Crb complex was addressed in vivo (Nam, 2006).
This study made three new observations that demonstrate important functions of Patj: (1) hypomorphic patj shows severe late-onset degeneration of photoreceptor cells in adult eye although the mutant eyes develop relatively normally; (2) analysis of patj null mutant patj RNAi demonstrate that Patj is essential for early development of the animal and for morphogenesis of AJ and apical membrane domains of photoreceptor cells during pupal development (Nam, 2006).
Consistent with these results on the late-onset degeneration of photoreceptor cells in hypomorphic adult eyes, (3) degeneration of adult photoreceptor cells was found. However, despite the findings on the phenotypes of the hypomorphic mutant, it is worth noting that there are important differences between these two studies. First, a second study on patj function (Richard, 2005) is limited to the analysis of a viable hypomorphic condition patj that shows no obvious defects in the pattern of AJs and rhabdomeres in the eyes until approximately 70% late pupal development. In contrast, the current study with the newly generated null mutant and RNAi reveals important developmental functions of Patj in the eye as well as animal viability. Second, the hypomorpic mutant is not only deleted in the PDZ2-4 domain portion of the dpatj gene but also deficient for JTBR and partially CG32327 that are adjacent to the 3' end of patj. In this study, it was shown that patj RNAi causes similar phenotypes of patj null mutant in the eye, suggesting that JTBR and CG32327 do not affect eye development and, thus, have no detectable influence on analysis of hypomorphic and null mutants (Nam, 2006).
The complete loss of patj causes several significant defects in early pupal eyes at approximately 45% pupal development. (1) Both Crb and Sdt are strongly reduced or mislocalized in the absence of Patj, although the loss of patj shows slightly weaker phenotypes than those of crb and sdt mutants. Furthermore, and in contrast to the hypomorphic mutant, (2) null clones or the eyes expressing patj RNAi reveal striking basolateral displacement or expansion of AJ markers such as Arm and Baz. These results suggest that the N- and C-terminal region of Patj protein may have distinct functions in the photoreceptor cells. Because the hypomorphic mutant shows relatively normal development of photoreceptors until later pupal stage or eclosion, the PDZ2-4 domains are not required during early photoreceptor development but are necessary for the maintenance of fully differentiated photoreceptors during very late pupal and adult stage. Of interest, a significant level of Crb and Sdt are apically localized in 45% pupal photoreceptors in hypomorphic mutant eyes (Richard, 2005) but are lost later in adult eyes (Richard, 2005), suggesting that PDZ2-4 domains are required to maintain the Crb complex in late pupal and adult stages. In contrast, the N-terminal region of Patj containing MRE and PDZ1 is crucial for morphogenesis of AJ and rhabdomere during the first half of pupal stage. The MRE motif of Patj interacts with the L27 domain of Sdt. Thus, it is likely that the N-terminal region expressed in hypomorphic mutants might be sufficient for interaction with Sdt, allowing normal development. Conversely, Sdt cannot bind Patj in the null mutant, resulting in the failure of normal development as in crb or sdt mutants (Nam, 2006).
Recently, it has been shown that reduction of Patj by RNAi in MDCK epithelial cell culture leads to delayed tight junction formation and defects in cell polarization. Similarly, mammalian Patj is required for localizing tight junction proteins and stabilizing the Crb3 complex in human intestinal cells. Thus, the findings on the important function of Drosophila Patj in developing photoreceptors are consistent with the results from these mammalian studies, and further provide evidence for the developmental function of Patj in the organization of apical cell membrane in the in vivo animal model. Mouse Patj is closely related to Drosophila Patj, but it has 10 PDZ domains in contrast to the presence of 4 PDZ domains in Drosphila Patj. It will be interesting to see whether the mammalian Patj also show distinct functions of the N-terminal MRE and the multiple PDZ domains in organizing cell polarity and maintaining the stability of the Crb complex, respectively (Nam, 2006).
Mutations in the human CRB1 gene cause retinal diseases such as retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis. The current results that Patj has dual functions in photoreceptor morphogenesis and maintenance in developing and adult animals, respectively, suggest that like CRB1, human Patj may be a target for early- and late-onset retinal diseases. It has been shown that the extracellular domain of Crb is required for preventing photoreceptor degeneration in ageing adult fly eyes. Results from this study and Richard (2005) indicate that PDZ2-4 domains of Drosophila Patj are required for blocking late-onset photoreceptor degeneration. It is currently unknown whether the requirement of Crb extracellular domain and the intracellular function of Drosophila Patj PDZ2-4 domains are related events in the maintenance of adult photoreceptors (Nam, 2006).
The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including Patj (formerly known as Discs lost). Taking advantage of a Patj mutant line in which only a truncated form of the protein is synthesized, Patj was demonstrated to be necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, Patj protects against light-induced photoreceptor degeneration (Richard, 2006).
This study presents evidence to show that Patj plays an important role in stabilizing the Crb complex in photoreceptor cells (PRCs). A truncated form of Patj, consisting of the N-terminal L27 and the first PDZ domain, is produced in Patj mutant eyes. It is further shown that the truncated Patj protein fails to stabilize Crb and Sdt at the stalk membrane during late pupal development and in adult eyes. It has been demonstrated that Crb is required for proper localization of Sdt and Patj at the stalk membrane of PRCs and that, in sdtXP96, the maintenance of Crb and Patj is compromised. These data together with the results presented in this study support the view that lack of any component of the Crb complex leads to mislocalization and/or dysfunction of the whole complex in the Drosophila eye (Richard, 2006).
To understand how the Crb complex may ensure a proper morphogenesis of photoreceptor cells, two major events during photoreceptor development have to be considered. In the first half of pupal development, stabilization of the ZAs is essential to maintain adhesion between PRCs during the tremendous cell shape changes that take place later when the cells undergo elongation. Furthermore, from 37% pupal development onward, the apical membrane differentiates into the rhabdomere and the stalk (Richard, 2006).
In crb and sdt mutants, the rhabdomeres are shorter and thicker, suggesting a failure to stabilize adhesion in early stages of pupal development, which in turn prevents proper elongation. This interpretation is consistent with the observation that, in eyes lacking crb function, the continuity of the ZA is interrupted at early stages of pupal development. Patj mutants do not exhibit any obvious defects in ZA development or PRC elongation, which can be explained by the fact that components of the Crb complex are still correctly localized until 70% pupal development. This timing contrasts with crb or sdt mutant PRCs, in which the integrity of the Crb complex is lost at an early stage of pupal development. Several explanations may account for this different behavior of Patj mutant PRCs. (1) The N-terminal portion of Patj may still retain some function during early pupal development, which stabilizes the Crb complex and, hence, the ZA. (2) Alternatively, Patj does not play a major function for the stability of the complex at early stages. (3) Finally, additional factors may interact with Patj and stabilize the Crb complex at early stages of development, and these interactions still occur with a truncated Patj. In fact, recent in vitro studies have suggested direct interactions between Patj and either Drosophila Par-6 or Drosophila PKC, two members of the other apically localized protein complex, which is essential for epithelial cell polarity in the embryo. However, it can be excluded that the suggested interaction between the third PDZ domain of Patj and the N-terminal domain of DmPar-6 plays any role in the stabilization of the complex during the first half of pupal development. The truncated Patj protein studied here lacks the third PDZ domain, yet it remains localized at the apical membrane at this stage (Richard, 2006).
The other major aspect of PRC maturation -- the differentiation of the apical membrane into rhabdomere and stalk -- is affected in crb, sdtXP96, and Patj mutant eyes, suggesting that all three components are necessary for this process. These mutations result in a shortening of the stalk membrane. The weaker phenotype of the Patj mutant relative to that of crb mutants is probably due to the hypomorphic nature of the former. Separation of the apical membrane of PRCs into two distinct domains, the rhabdomere and the stalk, becomes manifest at approximately 55% pupal development and coincides with the restriction of Crb and its associated proteins to this region. No other mutant affecting the length of stalk membrane has been described to date, although some mutants affect individual aspects of the crb or Patj morphogenetic phenotype, displaying thicker (bifocal; DSec61), malformed (Glued; WASp) or missing (overexpression of amphiphysin) rhabdomeres. Thus, the regulation of stalk membrane development seems to be a unique function for members of the Crb complex (Richard, 2006).
One phenotype of Patj mutants observed in this study, the progressive resorption of rhabdomeric microvilli, has not been described to date for any other mutant of the Crb complex. This raises the question whether Patj is involved in other processes in addition to those that are controlled by crb and sdt. The rhabdomere is composed of microvilli, each of which is supported by actin filaments. Rhabdomere morphogenesis and integrity depend on constant renewal of the membrane and on a highly organized actin cytoskeleton. Thus, it is not surprising that mutations in proteins involved in endo- or exocytosis, such as dynamin, Rab1, Rab6, Rab11, Sec6, Sec61, or Sunglasses, affect the integrity of the rhabdomere. It has been suggested that the addition of new membrane occurs at the base of the rhabdomere in Drosophila, while shedding occurs at the distal tip in tipulids. The further analysis of the function of these genes, the subcellular distribution of the respective proteins and their possible interactions with members of the Crb complex will be required to determine any involvement of the Crb complex in these processes. Rhabdomere integrity is also affected in eyes lacking proteins involved in actin structure and remodeling, such as NinaC, Chaoptin, Glued, Moesin or Rac1 but also in mutants for rhodopsin itself, which plays a structural role in addition to its function in signal transduction. In this scenario, Patj could help to stabilize the cytoskeleton and thereby maintain the integrity of the rhabdomere. Alternatively, the four PDZ domains in Patj may mediate the assembly of additional proteins. The identification and functional characterization of these proteins will shed light on the process by which Patj controls the stability of PRCs (Richard, 2006).
At present, the possibility that the defects observed in pigment cells in Patj mutant eyes contribute to the mutant phenotype observed in PRCs cannot be excluded. In vertebrate eyes, the pigment epithelium plays an active role in the renewal of rhodopsin, and defects in the pigment epithelium can lead to degeneration of PRCs. It is not yet known whether pigment cells in the Drosophila eye have a comparable function, although they certainly serve to insulate the PRCs of individual ommatidia from the light impinging on their neighbors. The accumulation and fusion of pigment granules in Patj mutant eyes may point to a defect in vesicular biogenesis and/or secretion. Whether this defect also affects interactions with the PRCs, in other words, whether pigment cells play an active role in the maintenance of the rhabdomeres or PRC function, and if so, whether Patj is involved in this process, is not known (Richard, 2006).
Finally, the results of this study demonstrate that Patj, like Crb, protects PRCs from the deleterious effects of excess light. The degeneration of PRCs observed in Patj mutant eyes may be a direct consequence of the failure to stabilize the components of the Crb complex at the stalk membrane. Previously published data have shown that the absence of crb in Drosophila eyes leads to retinal degeneration under similar lighting conditions. Similarly, mice deficient for CRB1 display signs of retinal degeneration upon exposure to light, which are reminiscent of defects seen in patients bearing mutations in the CRB1 gene. However, the penetrance of degeneration observed in crb eyes is much higher than that observed in Patj eyes, although the cellular features of degeneration observed in both mutants are similar. Taking into account that the crb clones were produced in a white background, whereas Patj eyes are red (due the transgenes that were introduced), it is not unlikely that the pigments could play a protective role in the latter case, as also shown previously for white mutants. Preliminary experiments suggest that the presence of pigments in crb mutant ommatidia indeed slows down the light-dependent degeneration (Richard, 2006).
Taken together, these results extend the knowledge of the genes involved in controlling retinal morphogenesis and preventing light-dependent PRC degeneration in the fly. Mutations in human CRB1 lead to RP12 and LCA, two severe forms of retinal dystrophy, raising the question whether mutations in the homologues of the other members of the complex might result in similar phenotypes. Understanding the molecular mechanisms leading to the mutant phenotype in the fly will certainly contribute to unraveling the pathogenesis of these retinal dystrophies in humans (Richard, 2006).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Patj
Djiane, A., Yogev, S. and Mlodzik, M. (2005). The apical determinants aPKC and Patj regulate Frizzled-dependent planar cell polarity in the Drosophila eye. Cell 121(4): 621-31. Medline abstract: 15907474
Feng, W., Long, J. F. and Zhang, M. (2005). A unified assembly mode revealed by the structures of tetrameric L27 domain complexes formed by mLin-2/mLin-7 and Patj/Pals1 scaffold proteins. Proc. Natl. Acad. Sci. 102(19): 6861-6. Medline abstract: 15863617
Lemmers, C., et al. (2002). hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J. Biol. Chem. 277(28): 25408-15. Medline abstract: 11964389
Makarova, O., Roh, M. H., Liu, C. J., Laurinec, S. and Margolis, B. (2003). Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Pals1). Gene 302: 21-29. Medline abstract: 12527193
Massey-Harroche, D., et al. (2007). Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex. Hum. Mol. Genet. 16(5): 529-36. Medline abstract: 17234746
Michel, D., et al. (2005). PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells. J. Cell Sci. 118(Pt 17): 4049-57. Medline abstract: 16129888
Nam, S.-C. and Choi, K.-W. (2003). Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila. Development 130: 4363-4372 . Medline abstract: 12900452
Nam, S. C. and Choi, K. W. (2006). Domain-specific early and late function of Patj in Drosophila photoreceptor cells. Dev. Dyn. 235(6): 1501-7. Medline abstract: 16518799
Pielage, J., Stork, T., Bunse, I. and Kl”mbt, C. (2003). The Drosophila cell survival Gene discs lost encodes a cytoplasmic Codanin-1-like protein, not a homolog of tight junction PDZ protein Patj. Dev. Cell 5: 841-851. Medline abstract: 14667407
Richard, M., Grawe, F. and Knust, E. (2006). Patj plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye. Dev. Dyn. 235(4): 895-907. Medline abstract: 16245332
Roh, M. H., et al. (2002). The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol. 157(1): 161-72. Medline abstract: 11927608
Roh, M. H., et al. (2003). The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J. Cell Sci. 116: 2895-2906. Medline abstract: 12771187
Shin, K., Straight, S. and Margolis, B. (2005). PATJ regulates tight junction formation and polarity in mammalian epithelial cells. J. Cell Biol. 168(5): 705-11. Medline abstract: 15738264
Shin, K., Wang, Q. and Margolis, B. (2007). PATJ regulates directional migration of mammalian epithelial cells. EMBO Rep. 8(2): 158-64. Medline abstract: 17235357
Sotillos, S., et al. (2004). DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila. J. Cell Biol. 166(4): 549-57. Medline abstract: 15302858
Storrs, C. H. and Silverstein, S. J. (2007). PATJ, a tight junction-associated PDZ protein, is a novel degradation target of high-risk human papillomavirus E6 and the alternatively spliced isoform 18 E6. J. Virol. 81(8): 4080-90. Medline abstract: 17287269
Straight, S. W., et al. (2006). Mammalian lin-7 stabilizes polarity protein complexes. J. Biol. Chem. 281(49): 37738-47. Medline abstract: 17012742
van Rossum, A. G., et al. (2006). Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Muller glia cells. Hum. Mol. Genet. 15(18): 2659-72. Medline abstract: 16885194
Wu, J., Klein, T. J. and Mlodzik, M. (2004). Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity. PLoS Biol. 2(7): E158. Medline abstract: 15252441
date revised: 28 May 2008
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.