MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the Non-Specific Lethal (NSL) complex (Raja, 2010) in gene regulation is still poorly characterized. This study reports a comprehensive ChIP-seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes (a sample of which include Bap170, CG6506, sec5, CG15011, Ent2, Incenp, tho2 and Patj) are constitutively expressed across different cell types. An increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions was found to be characteristic of NSL-targeted genes. Furthermore, these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP-seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, it was demonstrated that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication-related Element (DRE). Taken together, these findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription (Lam, 2012).
This study has revealed that the majority of the NSL-complex-bound targets are housekeeping genes in Drosophila. While chromatin-modifying complexes that regulate tissue-specific genes, such as SAGA, polycomb and trithorax complexes, have been studied extensively, global regulators of housekeeping genes are poorly understood. The NSL complex is the first identified major regulator of housekeeping genes (Feller, 2012; Lam, 2012).
The promoters of NSL target genes exhibit prominent enrichment of certain histone modifications (H4K16ac, H3K9ac, H3K4me2, H3K4me3) as well as specific core promoter elements (such as DRE, E-box and motif 1). Furthermore, these genes display distinct nucleosome occupancy and dispersed promoter configuration characterized by multiple transcription start sites. The correlation between these promoter characteristics (well-defined chromatin marks, TATA-less DNA sequences and broad initiation patterns) was previously identified for housekeeping genes in mammals and flies, but how these promoter features are translated into gene transcription had remained elusive. This study now conclusively demonstrates that the NSL complex modulates transcription at the level of transcription initiation by facilitating pre-initiation complex loading onto promoters. Therefore, it is proposed that the NSL complex is a key trans-acting factor that bridges the promoter architecture, defined by the DNA sequence, histone marks and higher chromatin structures with transcription regulation of constitutive genes in Drosophila (see Summary model: NSL-dependent Pol II recruitment to promoters of housekeeping genes) (Lam, 2012).
Excitingly, the enrichment of DNA motifs on NSL target gene promoters in combination with the genome-wide Pol II binding data has established functional links between the motifs enriched on housekeeping genes and the NSL-dependent Pol II binding to promoters. The abundance of DRE motifs, for example, was found to be positively associated with the magnitude of Pol II loss upon NSL knockdowns. The DRE binding factor (DREF) interacts tightly with TRF2 to modulate the transcription of DRE-containing promoters in a TATA-box-independent fashion (Hochheimer, 2002). It is tempting to speculate that the NSL complex might also cooperate with the TRF2 complex to facilitate transcription in a specific manner, rendering DRE-containing promoters more sensitive to NSL depletions. As the NSL-bound promoters are associated with a large variety of transcription factors, it will be of great interest to study whether the NSL complex communicates with different transcription regulators, perhaps making use of distinct mechanisms (Lam, 2012).
In contrast to DRE, motif 1 showed an opposing effect on Pol II recruitment to NSL-complex-bound genes as the presence of strong motif 1 sequences was associated with decreased Pol II loss upon NSL depletion. The mechanistic reasons for this remain unclear. However, one can envisage several possible scenarios. It is possible that motif 1 may recruit another transcription factor, which can also function to recruit the transcription machinery. Alternatively, the turnover of the transcription machinery might be slower on promoters containing strong motif 1 sequences. There is precedent for the transcription machinery having various turnover rates on different promoters. For example, in yeast, it has been shown that TBP turnover is faster on TATA-containing than on TATA-less promoters. It is therefore possible that certain levels of the initiation complexes may still be maintained on motif-1-containing promoters, even though the recruitment of the transcription machinery will be compromised in the absence of NSL complex. Further work is required to understand the importance of sequence determinants for NSL complex recruitment and the analysis sets the grounds for targeted experiments in the future (Lam, 2012).
Taking MOF-mediated H4K16 acetylation into consideration, a putative role of the NSL complex might be to coordinate the opening of promoter architecture by histone acetylation and the assembly of PIC. Coupling of histone acetylation and PIC formation has been described before. For example, TAF1, a component of TFIID, is a histone aceyltransferase. The SAGA complex, which contains Gcn5 and can acetylate H3K9, is reported to interact with TBP and other PIC components to regulate tissue-specific genes and the recruitment of P300 to the promoter and H3 acetylation have been shown to proceed binding of TFIID in a coordinated manner. H4K16ac is also well-known for its role in transcription regulation of the male X chromosome, yet how H4K16 acetylation and PIC assembly are coordinated remains elusive. Interestingly, absence of the NSL complex does not severely abolish H4K16ac from target genes. Since the turnover of H4K16ac on target promoter is unknown, it remains possible that H4K16ac could remain for some time at the promoter after the NSL complex is depleted. Further studies will be crucial in unraveling the functional relevance of H4K16 acetylation and NSL complex function on housekeeping genes (Lam, 2012).
Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3-Par-6-aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs-Stardust (Sdt)-Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. By overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones is demonstrated. A novel physical interaction occurs between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and Crumbs is identified as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3-Par-6-aPKC and Crumbs-Sdt-Patj complexes based in the posttranslational modification of Crb by DaPKC (Sotillos, 2004; full text of article ).
The C tail of Fz receptors regulates their localization and signaling activity. A short Fz Cterm governs apical localization, which is critical for effective Fz-PCP signaling. In contrast, a long Cterm (like that of Fz2) governs baso-lateral localization, promoting β-catenin signaling and preventing PCP activity. Thus, a striking feature of all core PCP proteins, including Fz1, is their apical localization within imaginal disc cells. Fz1 colocalizes partially with several components that regulate A/B polarity such as the Crumbs/Sdt/Patj and Baz/aPKC/Par-6 complexes within the marginal domain, even though it is also present more basally relative to these complexes (Djiane, 2005).
Detailed sequence analysis of the Fz1 Cterm has revealed the presence of two clustered conserved PKC phosphorylation sites (Ser554 and Ser560 in Fz1). Given that aPKC expression in the apical domain overlaps with Fz1, a test was performed to see if aPKC can phosphorylate the Fz1 Cterm on the two conserved PKC sites in an in vitro kinase assay. Purified human aPKC protein phosphorylates in vitro a GST::Fz1 Cterm fusion protein. Furthermore, mutations of the two PKC consensus sites (Ser to Ala) prevent aPKC-mediated Fz1 phosphorylation, confirming that these sites are targets of aPKC (Djiane, 2005).
To investigate the importance of these phosphorylation sites in vivo, flies were generated carrying UAS-inducible transgenes of Fz1 mutant derivatives with either both serines mutated to alanine (Fz1-AA), inactivating the two prospective PKC sites, or both Serines mutated to Glutamic acid (Fz1-EE), mimicking phosphorylation. These transgenes were analyzed under sevenless (sev)-Gal4 control, which is expressed specifically in R3/R4 precursor cells just posterior to the MF during PCP establishment. Overexpression of wild-type Fz1 provides too much activity and interferes with the balance of Fz1 regulation within the R3/R4 pair, resulting in ommatidia with random R3/R4 cell fate decision and chirality, as well as symmetrical R3/R3 type ommatidia. Similarly, overexpression of Fz1-AA (with both aPKC sites inactivated; sev>Fz1-AA) induces ommatidia with random chirality and symmetrical clusters. In contrast, the phosphomimetic Fz1-EE (sev>Fz1-EE) shows hardly any effect. These data suggest that aPKC-mediated Fz1 phosphorylation inhibits Fz-PCP signaling activity (Djiane, 2005).
Since apical Fz1 localization is critical for its proper PCP signaling activity (Wu, 2004), it was hypothesized that the Fz1-EE mutation could affect the localization of the receptor. To investigate this possibility, the expression of the different myc tagged Fz1 transgenes was examined in imaginal discs (under en-Gal4 or dpp-Gal4 control). No difference between the expression of either Fz1-AA or Fz1-EE with that of wild-type Fz1 was found. These mutant Fz1 isoforms were expressed at similar levels and colocalized apically with aPKC, indicating that phosphorylation of Fz1 by aPKC does not affect Fz1's localization (Djiane, 2005).
A second feature critical to Fz1 signaling activity is its ability to recruit Dsh to the membrane. Interestingly, the aPKC sites partly overlap with the region of the Fz Cterm known to bind Dsh, raising the possibility that phosphorylation by aPKC could interfere with Dsh recruitment. Thus tests were performed to see whether Dsh recruitment is affected by phosphorylation of the Fz1 aPKC sites. S2 cells, which have no endogenous Fz, were transfected with Dsh-GFP and the different Fz1 mutants. In this assay, wild-type and both mutant forms of Fz1 recruit Dsh-GFP efficiently to the membrane. Then, whether overexpression of Fz1-AA and Fz1-EE can recruit Dsh-GFP to apical membranes like wild-type Fz1 in vivo (expressed with en-Gal4 in the posterior compartment of wing discs, where there is a sharp boundary between expressing and nonexpressing cells) was examined. Both Fz1-AA and Fz1-EE behave like wild-type Fz1, sequestering Dsh to the apical cell membrane in imaginal disc cells (Djiane, 2005).
In summary, it was shown that Fz1 can be phosphorylated in vitro by aPKC. Together with the in vitro results, the in vivo analysis of a phosphomimetic Fz1 mutant suggests that aPKC phosphorylation regulates Fz1 activity negatively and that this effect is not mediated by affecting Fz1 localization or Fz1-mediated Dsh membrane recruitment (Djiane, 2005).
In light of the importance of the aPKC sites in Fz1, it was of interest to determine how aPKC is recruited to the receptor. This could be mediated by direct binding or through a bridging factor, the most likely candidates being the A/B determinants that bind aPKC. Thus a two-hybrid interaction screen was conducted using the Fz1 Cterm as bait and components of different A/B protein complexes as prey. The closely related Fz2 Cterm was included as well as the Stbm Cterm as control baits. All components of the aPKC/Par-6/Bazooka apical complex were tested. For Baz, three different fragments were used: an N-terminal fragment involved in Baz dimerization (BazA), a central fragment with three PDZ domains involved in Par-6 binding (BazB), and a C-terminal fragment that binds aPKC (BazC). Similarly, all components of the Crb/Sdt/Patj apical complex were tested except Crb (since Crb is a transmembrane protein) and the components of the more baso-lateral Scrib/Dlg/Lgl complex were analyzed. No direct interaction was detected between the Fz1 Cterm and aPKC, but, interestingly, Patj was found to be a specific binding partner of the Fz1 Cterm. No other protein was found to interact with the Fz1 Cterm, and in turn Patj did not interact with the Fz2 or Stbm Cterms. The interaction between the Stbm Cterm and Dlg was confirmed as was the interaction of aPKC with Par-6 and BazC (Djiane, 2005)
To confirm the Patj-Fz1 interaction in vivo, coimmunoprecipitation (CoIP) experiments were performed from Drosophila S2 cell extracts transfected with GFP fusion proteins with the Fz1 or Fz2 Cterms (GFP::Fz1 and GFP::Fz2, respectively). Patj could be co-immunoprecipitated from cells transfected with GFP::Fz1 but importantly not with GFP::Fz2 or GFP alone, demonstrating that Fz1 and Patj interact in Drosophila cells. A weak interaction was found between Fz1 and endogenous Baz and aPKC, suggesting the existence of one or several multiprotein complexes among Fz1, Patj, Baz, and aPKC. In contrast, other components of A/B protein complexes, such as Par-6 or Dlg, did not CoIP with either GFP::Fz1 or GFP::Fz2 (Djiane, 2005).
To map the Patj interaction domain with the Fz1 Cterm, GST pull-down experiments were performed. Patj is a modular protein containing a N-terminal L27 domain (previously referred to as MRE), mediating its interaction with Sdt, and four PDZ domains. Consistent with the yeast two-hybrid and CoIP results, in vitro translated full-length Patj bind the GST-Fz1 Cterm protein. The fourth PDZ domain of Patj is sufficient for direct binding to the Fz1 Cterm (Djiane, 2005).
Using the CoIP approach, the residues in the Fz1 Cterm required for Patj interaction were also mapped. Whereas the full-length Fz1 Cterm interacts with Patj, removing the last three residues (Fz1ΔBS) abolishes this interaction. The removal of an internal Cterm motif, encompassing the tryptophan critical for Dsh binding, retains Fz1 ability to bind Patj albeit to a lesser extent (Djiane, 2005).
These results support a direct interaction between the apical determinant Patj and the Fz1 Cterm and suggest that Patj could provide a link between Fz1 and aPKC, since Patj was shown in vitro to bind to aPKC either directly or indirectly through Par-6. The Fz1/Patj interaction is mediated by the fourth PDZ domain of Patj, requires the last three residues of Fz1, and is largely independent of the Fz motif that mediates Dsh binding (Djiane, 2005).
A prediction from these results that 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).
In the CoIP experiments in S2 cells, Baz was recovered together with Fz1, Patj, and aPKC (although at lower levels than Patj), raising the possibility that Baz might be in a complex with aPKC and Patj to regulate Fz1 signaling. In order to investigate this, endogenous Baz expression was examined in third instar eye discs. Although expressed apically in all cells anterior and posterior to the MF, Baz becomes enriched in R3/R4 cells during PCP establishment, showing a complementary pattern to Patj and aPKC. Subsequently, Baz is enriched in the polar R4 cell, similar to core PCP determinants such as Fz1, Dsh, and Fmi. Next, Baz expression and localization was examined in PCP mutants to determine whether this pattern is under the control of PCP signaling. Interestingly, the only significant difference was found in fz mutant clones, where the Baz pattern was less resolved. In particular, the late accumulation in only one of the two cells of the R3/R4 pair, reflecting the R4 cell fate, was missing. Thus, Baz seems to have two expression phases in the PCP context; an initial accumulation in both R3 and R4 that is likely independent of PCP establishment, and a later enrichment in R4 which is downstream of Fz1 signaling (Djiane, 2005).
The reciprocal expression pattern of aPKC/Patj and Baz and the evidence that Par-3, the vertebrate Baz homolog, can act as an inhibitor of aPKC kinase activity suggested that Baz could play an antagonizing role to the negative regulation of Fz1 by aPKC in cells where Fz1 should be active. Therefore a baz requirement in eye PCP establishment was tested by performing loss-of-function (LOF) and GOF experiments. Although baz mutant clones are very disorganized in adult eye sections, probably due to a general Baz requirement for photoreceptor differentiation, they can be analyzed for PCP defects in eye imaginal discs where the integrity of cells is maintained and large clones could be obtained. Interestingly, baz mutant clones have ommatidial clusters with inverted chirality and some that show symmetrical features, as shown with the R4 specific mδ-lacZ marker that highlights ommatidial chirality in the eye disc. Furthermore, it was found that removal of one copy of baz suppressed the PCP GOF phenotype of sev-Fz1, but not sev-Dsh or sev-Fmi, suggesting that Baz acts specifically on Fz1 as a positive regulator. Finally, sev-Gal4-driven overexpression of Baz (sev>Baz) induces eye PCP defects, with misrotated and symmetric clusters where the R3 and R4 fates have not been resolved. This effect of Baz is unlikely to be a direct effect on Patj localization, since Baz overexpression (sev>Baz or GMR>Baz; GMR-Gal4 drives expression in all cells of the eye disc posterior to MF) does not change the Patj expression pattern (Djiane, 2005).
Taken together, these results suggest that Baz-aPKC-Patj and Fz1 form a complex before the MF, but during PCP establishment, Baz localizes in a complementary pattern to Patj/aPKC and antagonizes the negative regulation of Fz1 by aPKC (Djiane, 2005).
Essential for proper function of small GTPases of the Rho family, which control many aspects of cytoskeletal and membrane dynamics, is their temporal and spatial control by activating GDP exchange factors (GEFs) and deactivating GTPase-activating-proteins (GAPs). The regulatory mechanisms controlling these factors are not well understood, especially during development, when the organization and behaviour of cells change in a stage dependent manner. During Drosophila cellularization Rho signalling and RhoGEF2 are involved in furrow canal formation and the organization of actin and myosin. This study analyzed, how RhoGEF2 is localized at the sites of membrane invagination.The PDZ domain is necessary for localization and function of RhoGEF2, and Slam was identified as a factor that is necessary for RhoGEF2 localization. It was also demonstrated that Slam can recruit RhoGEF2 to ectopic sites. Furthermore the PDZ domain of RhoGEF2 can form a complex with Slam in vivo, and Slam transcripts and protein colocalize at the furrow canal and in basal particles. Based on these findings, it is proposed that accumulation of slam mRNA and protein at the presumptive invagination site provides a spatial and temporal trigger for RhoGEF2-Rho1 signalling (Wenzl, 2010).
RhoGEF2 is an essential regulator of Rho1 activity during many different stages of Drosophila development including cellularization. However, little has been known about the events and factors that control RhoGEF2 localization and subsequent Rho1 activation at the furrow canal. This study assigned a new function to the PDZ domain of RhoGEF2 in being sufficient and required for furrow canal localization. The pattern and the dynamics of furrow canal localization of different PDZRG2 containing constructs are very similar to that of endogenous RhoGEF2 thereby reflecting the behaviour of the full-length protein during cellularization. The domain could be used to effectively target other proteins like RFP, Myc or GST to the furrow canal. Thus despite being a multidomain protein, furrow canal localization depends ultimately only on residues that assure the structural integrity of the ligand recognition site of the PDZ domain. It was reported previously that the RhoGEF2 PDZ domain is involved in the subcellular localization of RhoGEF2 during apical constriction of mesodermal cells in gastrulation. It has been suggested that a direct interaction between the PDZ domain and the PDZ binding motive at the C-terminus of the apically localized transmembrane protein T48 is involved in the recruitment of RhoGEF2 to the apical site of the cells. However, it is clear that this interaction is not essential for apical RhoGEF2 localization, since this localization is lost only in T48/cta double mutants (Wenzl, 2010).
By using immunoprecipitations from staged embryonic extracts it was possible to show that a transgenic 4xPDZRG2-myc6 construct can physically interact with Slam in vivo. Of course this does not directly proof that Slam also interacts with full-length endogenous RhoGEF2. Nevertheless different arguments are presented that support the assumption that a physical interaction between Slam and RhoGEF2 underlies the observed functional relationship between these two factors in cellularizing embryos. The PDZ domain is the critical element that mediates the localization of RhoGEF2 at the furrow canal where it colocalizes with Slam. It was shown that this PDZ domain can form a complex with Slam in vivo. Further in vivo experiments confirmed that furrow canal localization of RhoGEF2 depends on slam in a dosage dependent manner which supports the biochemical findings. Moreover Slam can recruit RhoGEF2 to ectopic sites in embryos as well as in S2 cells and aspects of the RhoGEF2 mutant phenotype can be observed in slam deficient embryos. Overall it is reasonable to conclude that there may be a direct or indirect interaction between Slam and RhoGEF2 during formation of the cellular blastoderm. This interaction would be mediated by the PDZ domain of RhoGEF2. The data also demonstrate that slam acts upstream of RhoGEF2 (Wenzl, 2010).
The molecular function of slam has remained unknown, although the essential role of this gene in cellularization is well established (Merrill, 1988). It has been proposed that Slam is involved in membrane traffic, since in slam mutants the polarized insertion of membrane is disturbed. This study describes an additional cell biological function of slam in being a developmental switch that temporally and spatially controls Rho activity in blastoderm embryos by regulating the subcellular localization of the Rho1 activator RhoGEF2. Thus by proposing the existence of a protein complex containing RhoGEF2 and Slam, physiological and molecular function of Slam can be linked (Wenzl, 2010).
PDZ domains often interact with the C-termini of transmembrane proteins. There are different classes of PDZ binding motifs that can be classified according to their amino acid composition. Although not being a transmembrane but a membrane associated protein, Slam possesses a potential class II PDZ binding motif at its C-terminus. However, this motif seems to be dispensable for the recruitment of RhoGEF2 by Slam to ectopic sites. This is consistent with the fact that a slam allele with a mutated C-terminus rescues the cellularization phenotype of slam deficient embryos. In addition this allele is able to recruit RhoGEF2 to the furrow canal membrane. Furthermore RhoGEF2 to be still present although with reduced levels at the furrow canal in germline clones of a C-terminally truncated slam allele slamwaldo1 (Wenzl, 2010).
Besides the interaction between Slam and the PDZ domain of RhoGEF2, an interaction between Slam and Patj was observed in co-IPs from staged embryonic extracts. This is consistent with the fact that both proteins almost perfectly colocalize during cellularization at the furrow canal as well as in basal particles. Furthermore a functional relation between Slam and Patj is seen, since Patj levels at the furrow canal are reduced in embryos that are zygotically deficient for slam. Patj is a conserved protein that contains 4 PDZ domains and was previously reported to be able to interact with Crumbs in vitro and in vivo during epithelial polarity establishment later in development. However, the importance of this interaction remains unclear, since embryos that are maternally and zygotically mutant for Patj have been reported to develop until adulthood without obvious phenotypes. This would argue against an essential role of Patj during cellularization. As shown by another report, the mutants used in the study still expressed a truncated Patj protein that contained the first PDZ domain thus it is likely that residual Patj function was still present. Zygotic Patj null mutants, in which the coding sequence of Patj was removed completely, died during second instar larval stage, indicating that Patj is an essential gene. Therefore it would be worth to generate maternal Patj null mutants to investigate the role of this protein during cellularization in more detail. Nevertheless the interaction between Patj and Slam seems to depend mainly on the C-terminus of Slam, since in slamwaldo1 mutants Patj levels at the furrow canal are strongly reduced. Thus it is possible that the putative PDZ binding motif at the C-terminus of Slam is important for a direct interaction with one of the PDZ domains of Patj. The Slam Patj interaction also shows that besides controlling RhoGEF2 localization Slam has other independent functions, which could account for the strikingly stronger cellularization phenotype of slam mutants compared to the weaker phenotype of RhoGEF2 deficient embryos (Wenzl, 2010).
RhoGEF2 also functions in different epithelial invagination processes like salivary gland formation or in the establishment of the epithelium in the wing imaginal disc of Drosophila L3 larvae. It appears likely that the subcellular localization of the protein is controlled by genes encoding different receptors that are expressed during different developmental stages in a tissue specific manner like slam or T48 which would allow a very precise temporal and spatial regulation of Rho activity by employing the same ubiquitously expressed activating factor. RhoGEF2 also has a function in the maternally controlled formation of the metaphase furrows during the cleavage divisions 10-13 of the syncytial blastoderm stage and it was shown that localization of the protein to these furrows depends on maternal components of the recycling endosome. The start of zygotic slam expression at the onset of cellularization thus could assure that sufficient levels of RhoGEF2 and thus Rho activity become associated with the membrane tip during invagination. At the same time the metaphase furrows that have recently been shown to be rather active endocytic membrane domains are transformed into a domain forming the furrow canal, which were reported to be much more inactive and stable (Wenzl, 2010).
This study also shows that slam transcripts exhibit a new and unique mRNA localization pattern. A significant portion of slam mRNA is associated with the furrow canal membrane domain. Surprisingly the initial processes that ensure a local restriction of Rho activity would be the proper localization of the slam RNA/protein particles. The asymmetrical localization of transcripts within a cell often linked with localized translation is an important mechanism for the spatial regulation of gene activity. Apical localization of transcripts during cellularization has been described for a number of genes including wg, run and ftz. This study showed that the transcripts are transported to localize to the apical cytoplasm of the cells of the cellular blastoderm. However, little is known about the functional importance of this transcript localization. The localization of slam transcripts might also include a basal to apical transport step, since large basal particles were seen containing slam mRNA and protein in cellularizing embryos. It has been reported previously that apical Rho activity during posterior spiracle formation is mediated in part by RhoGEF64C. The transcript of this gene does localize to the apical membrane of the epithelial cells which undergo apical constriction and subsequent invagination. The mechanisms that ensure the association of transcripts with a specific membrane domain remain to be solved and slam would offer a good system to study this question. Future studies will show whether and how the localization of slam mRNA is involved in defining the sites for membrane invagination and what other functions are served by slam besides initiating Rho signalling (Wenzl, 2010).
Taken together, a model is proposed for the developmental control of Rho1 signalling at the furrow canal, in that the slam RNA-protein particles are targeted to the prospective site of membrane invagination at the onset of cellularization. Slam would have several functions, mainly initiating the formation of the furrow canal as a distinct membrane domain by regulating membrane traffic and at the same time it would recruit and restrict RhoGEF2 and maybe other factors to this domain. After reaching a critical concentration the GEF activity would be activated by a yet unknown mechanism. Rho1 would be converted into its GTP-bound form and downstream targets like Dia or Rho-kinase would be activated. Consistent with this model is the observation that the dose-dependent activity of Slam, both higher or lower than normal levels, directly corresponds to the amount of RhoGEF2 protein and the speed of cellularization as for example shown by the local injection of slam RNA (Wenzl, 2010).
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