Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).

To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).

This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).

Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P2, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).

The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).

The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).

A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).

A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).

In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).


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).

Effects of Mutation or Deletion

aPKC and Patj are required for Fz1-dependent PCP establishment

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).

Domain-specific early and late function of Patj in Drosophila photoreceptor cells

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 Drosophila 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).

Patj plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye

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).

Drosophila Patj plays a supporting role in apical-basal polarity but is essential for viability

Patj has been characterized as one of the so-called polarity proteins that play essential and conserved roles in regulating cell polarity in many different cell types. Studies of Drosophila and mammalian cells suggest that Patj is required for the apical polarity protein complex Crumbs-Stardust (Pals1 or Mpp5 in mammalian cells) to establish apical-basal polarity. However, owing to the lack of suitable genetic mutants, the exact in vivo function of Patj in regulating apical-basal polarity and development remains to be elucidated. This study generated molecularly defined null mutants of Drosophila Patj (dPatj). The data show conclusively that dPatj plays only supporting and non-essential roles in regulating apical-basal polarity, although such a supporting role may become crucial in cells such as photoreceptors that undergo complex cellular morphogenesis. In addition, the results confirm that dPatj possesses an as yet unidentified function that is essential for pupal development (Zhou, 2012).

The molecularly defined dPatj null alleles conclusively show that dPatj is not essential for establishing or maintaining apical-basal polarity. The mild reduction in Crb expression in the absence of dPatj suggests that dPatj plays a supporting role in maintaining the Crb-Sdt complex in polarized epithelial cells. Such a scenario exhibits similarities with the situation for p120catenin (Adherens junction protein p120 - FlyBase) mutants: despite RNAi results suggesting that p120catenin is essential for cell adhesion, genetic mutants revealed that it only plays a supporting and non-essential role in cell adhesion in Drosophila. It remains possible, however, that mammalian Patj might have evolved increasingly important roles in apical-basal polarity, which might explain the differences between the dPatj null mutant phenotypes and those of mammalian cells with RNAi knocked-down Patj levels (Zhou, 2012).


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

Feller, C., Prestel, M., Hartmann, H., Straub, T., Soding, J. and Becker, P. B. (2012). The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset. Nucleic Acids Res 40: 1509-1522. PubMed ID: 22039099

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

Hochheimer, A., Zhou, S., Zheng, S., Holmes, M. C. and Tjian, R. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420: 439-445. PubMed ID: 12459787

Lam, K. C., Muhlpfordt, F., Vaquerizas, J. M., Raja, S. J., Holz, H., Luscombe, N. M., Manke, T. and Akhtar, A. (2012). The NSL complex regulates housekeeping genes in Drosophila. PLoS Genet 8: e1002736. PubMed ID: 22723752

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

Mavrakis, M., Rikhy, R. and Lippincott-Schwartz, J. (2008). Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo. Dev. Cell 16: 93-104. PubMed Citation: 19154721

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

Merrill, P. T., Sweeton, D. and Wieschaus, E. (1988). Requirements for autosomal gene activity during precellular stages of Drosophila melanogaster. Development 104: 495-509. PubMed ID: 3151484

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 Klämbt, 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

Raja, S. J., et al. (2010). The nonspecific lethal complex is a transcriptional regulator in Drosophila. Mol. Cell. 38: 827-841. PubMed Citation: 20620954

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

Wenzl, C., Yan, S., Laupsien, P. and Grosshans, J. (2010). Localization of RhoGEF2 during Drosophila cellularization is developmentally controlled by Slam. Mech Dev 127: 371-384. PubMed ID: 20060902

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

Zhou, W. and Hong, Y. (2012). Drosophila Patj plays a supporting role in apical-basal polarity but is essential for viability. Development 139: 2891-2896. PubMed ID: 22791898

Patj: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 28 March 2013

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