BIOLOGICAL OVERVIEW

Mammalian protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. Protein 4.1 functions to link transmembrane proteins with the underlying spectrin/actin cytoskeleton. The Drosophila homolog (termed Coracle) of protein 4.1 has been identified and characterized: such work has advanced an understanding of the developmental role and a genetic analysis of the cellular functions involving this membrane-skeletal protein (Fehon, 1994).

Coracle is a component of septate junctions, structures that serve as selective-permeability barriers, separating the apical from the basal regions in sheets of epithelial cells. For more information about the Drosophila septate boundary see Neurexin. Another component of Drosophila septate junctions is the protein Discs large. The region of a human erythroid Discs large protein (p55) that interacts with mammalian protein 4.1 has also been identified. A novel sequence located between the SH3 motif and the guanylate kinase domain of p55 is the binding site for the 30kDa domain of protein 4.1. A characteristic of this 39-amino acid sequence is the presence of a cluster of lysine residues located in the C-terminal half of the protein. This sequence is conserved in the human Discs large protein (hDlg) and in Drosophila Discs large. Protein 4.1, and its Drosophila homolog Coracle may provide a membrane localization site for Discs large homologs (Marfatia, 1995).

Mammalian protein 4.1 is known to interact with the Drosophila cytoskeletal proteins actin and spectrin). The cytoskeletal interaction domain of Mammalian protein 4.1 is not conserved in Coracle; neither actin nor spectrin associate with septate junctions in Drosophila. Thus, while the presumed function of mammalian erythrocyte protein 4.1 may be to mediate interactions between the transmembrane protein glycophorin and the cytoskeletal proteins actin and spectrin, this function is not conserved in Drosophila. Nevertheless, the N-terminal 350 amino acids of the protein 4.1 homolog (Coracle) is highly conserved in both Drosophila and mammalian proteins. This domain serves to localize these proteins to their respective membranes. This implies that Coracle may be important for junctional structure in Drosophila, a role that is clearly not relevant for erythrocytes. Studies of human 4.1 in non-erythoid cells have reported that 4.1 is assocated with membranes in regions of cell-cell contacts, indicating that Coracle function in junctions could be conserved in vertebrates (Fehon, 1994).

Coracle is required for Dorsal closure, one of the more dramatic of all the morphogenetic movements taking place during Drosophila embryogenesis. The closure event occurs late in embryogenesis, and results in the establishment of the dorsal epidermis. Cells in the two lateral epidermal primordia change shape and spread over the amnioserosa, a membranous structure covering the dorsal side of the embryo. Epithelial cells elongate in this spreading, covering process. Neither cell proliferation nor cell rearrangement occurs. Included among the Drosophila genes known to be involved in dorsal closure are Rac1, a low molecular weight GTPase; basket, also known a Jun N-terminal kinase, and zipper, Drosophila's non-muscle myosin. A role for Coracle in dorsal closure is unknown, although it is known that human protein 4.1 can interact with muscle and nonmuscle myosin (Fehon, 1994 and references).

The original coracle mutations were identified based on their ability to dominantly suppress the Ellipse gain of function hypermorphic allele of the Drosophila Epidermal growth factor receptor homolog. The Ellipse mutation produces a rough eye phenotype in Drosophila. It is not known whether this genetic interaction represents a direct interaction between these two genes. However, it is known the mammalian EGF receptor tyrosine kinase can phosphorylate protein 4.1 and that phosphorylation affects the functions of 4.1 family proteins. Thus EgfR regulation of Coracle could play a role in cell dynamics during Drosophila eye morphogenesis (Fehon, 1994).

The N-terminal 383 amino acids of Coracle define an essential functional domain possessing membrane-organizing properties. The full range of functions provided by this highly conserved domain has been investigated and the domain has been found to be sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles (Ward, 2001).

Three coracle transcripts have been precisely mapped, and Northern blot analysis indicates the presence of several others. coracle cDNA 1 (isoform 1) encodes a protein that is predicted to be 1698 amino acids (aa) in length, while cDNAs 2 and 3 (isoforms 2 and 3) encode much shorter products of 889 and 703 aa, respectively. Isoforms 2 and 3 differ primarily from isoform 1 in that they lack coding region E, a large (2427 bp), alternatively spliced exonic region. To determine the number of introns and the precise intron/exon boundaries of the coracle gene, genomic DNA from the coracle locus was PCR amplified and sequenced and this genomic sequence was compared to the known cDNA isoforms. On the basis of these comparisons, coracle is composed of 17 exons and 16 introns. In addition, this analysis reveals a complex pattern of splicing that is due to the use of alternative splice acceptor sequences that in some cases are not separated by intervening intronic sequences. Three introns are flanked by alternate splice acceptor sites that result in mRNAs with different coding sequences. The first of these regions, at intron 8, results in alternative splicing involving regions B and C. Region B contains an alternative splice acceptor that inserts a 12-bp coding region and is used in isoform 3. Region C, which is contiguous with region B in the genomic DNA, is present in isoforms 2 and 3, while in isoform 1, splicing spans from the 5' end of intron 8, across regions B and C to the 3' end of the 711-bp intron 9, thereby excising these coding regions. A similar behavior is observed around intron 11, which falls in between regions D and E in the coding sequence. Surprisingly, region E, the largest of the alternatively spliced regions in coracle (2427 bp), is not immediately bounded by introns on both ends. Rather, the 3' end of region E is defined by another alternative splice acceptor site within the large exon that encodes regions E and F. Thus, splicing in this area can span just intron 11 (353 bp; isoform 1), intron 11 plus region E (2780 bp; isoform 2), or intron 11, regions E and F, plus intron 12 (3717 bp; isoform 3). Similar behavior is observed around exon H, although in this case there does not appear to be any alternative splice acceptor within the exon, thereby simplifying the splicing pattern (Ward, 2001).

The observed complex pattern of alternative splicing raises the possibility that different isoforms may have different functions during development. This notion is supported by the observation that the cor¹ and cor² alleles are associated with nonsense mutations that should affect only isoform 1. The observation that these mutations display a fully penetrant, embryonic lethal phenotype implies that isoform 1 encodes functions that are not present in the other isoforms and, therefore, that region E is likely to contain an essential functional domain. Alternatively, it is possible that while functionally equivalent, the expression level of the other isoforms is too low to provide sufficient coracle function for viability, consistent with previous observations that the alternative mRNA splice forms are expressed at lower levels than isoform 1 (Ward, 2001).

To distinguish between qualitative and quantitative functional differences between the three Coracle isoforms, P-element-based transgenes encoding isoforms 1, 2, and 3 under the control of the Drosophila Ubiquitin promoter were constructed. Use of the Ubiquitin promoter ensures that all three isoforms are expressed at roughly equivalent levels and that they are expressed in all tissues throughout embryonic development. To test the ability of these isoforms to rescue coracle lethal mutations, independent insertion lines of each were crossed into the appropriate coracle mutant background, and viability of the homozygous mutant class was scored. All three of the tested isoforms provided sufficient coracle function to rescue >67% of the expected coracle mutant offspring bearing null alleles, indicating that all encode the essential coracle functions when expressed ubiquitously and at sufficient levels. In all cases both males and females were fertile, allowing the maintainance of stocks of rescued homozygous mutant animals. In comparison to isoform 1, isoform 2 lacks coding sequences within regions E (2427 bp) and H (102 bp), and isoform 3 lacks, in addition, region F (570 bp). In comparison to isoform 3, isoform 1 lacks coding sequences within regions B (12 bp) and C (102 bp). Thus, the observation that isoforms lacking these regions can rescue severe loss-of-function coracle mutations indicates that, although in combination regions B, C, E, F, and H encode 1071 amino acid residues, they do not contain any essential functional domains. Likewise, the observation that each of these isoforms rescues cor⁵, a null coracle allele, indicates that each encodes all essential coracle functions and thus that these functions are restricted to coding regions A, D, G, and I (Ward, 2001).

Interestingly, all coracle mutations that are predicted to affect the N-terminal functional domain are embryonic lethal, whereas cor¹⁴, which truncates the C-terminal domain, shows no embryonic lethality. This observation raises the possibility that the N-terminal functional domain is required to complete embryonic development, whereas the C-terminal domain is required at a later stage. Ectopic expression experiments using just the FERM domain strongly support this supposition. Embryos completely lacking coracle function display a range of defects including failure in dorsal closure, thinning of the cuticle, necrosis of the salivary glands, and an inability to inflate the trachea at the end of embryogenesis. Expression of the N-terminal 383 amino acids fully rescues all of these defects. Additionally, the cor⁸ and cor¹⁰ mutations, which affect the FERM domain and cause embryonic lethality, can be rescued to viable, fertile adults by the ubiquitous expression of just the FERM domain. In contrast, cor⁴ and cor⁶ mutant animals, which also have molecular lesions within the FERM domain, are not rescued by ubiquitous expression of COR^1-383. Interestingly, the cor⁴ and cor⁶ mutant proteins display abnormal subcellular localizations, raising the possibility that correct subcellular localization is as crucial for the function of the C-terminal domain as it is for the function of the FERM domain. Also, cor¹ and cor², which have an intact FERM domain display embryonic defects, but like most nonsense mutations these alleles show reduced protein expression (Ward, 2001).

These experiments strongly suggest the existence of an essential functional domain within the C-terminal region of Coracle. Even though cor¹⁴ is one of the weakest alleles tested in these experiments, ectopic expression of the FERM domain is incapable of rescuing cor¹⁴ animals. In contrast, cor¹⁴ is rescued by expression of a full-length coracle transgene, indicating that a region outside the FERM domain is necessary for viability. cor¹⁴ results from a nonsense mutation at Arg¹⁶⁰⁷, suggesting that the C-terminal functional domain includes sequences within the highly conserved C-terminal 100 amino acids (Ward, 2001).

The proposed modular organization of the functional domains within Coracle leads to the following prediction: alleles that specifically alter only one functional domain should complement alleles that affect only the other functional domain. Specifically, combinations involving cor⁸ and cor¹⁰ (specifically affecting the N-terminal domain) and cor¹⁴ (specifically affecting the C-terminal domain) support this hypothesis. Although all three of these alleles are recessive lethal (either homozygous or over a deficiency), cor¹⁰ is >75% viable when heterozygous with cor¹⁴, and cor⁸ weakly complements cor¹⁴. This result is in agreement with the results using molecular genetic approaches and strongly supports the conclusions on the modular nature of the functional domains within Coracle (Ward, 2001).

Most, if not all, of the embryonic defects associated with loss of coracle function are due to an inability to maintain a physiologically 'tight' epithelium. Ectopic expression of the FERM domain in coracle mutant embryos is sufficient to rescue all of the described embryonic defects, raising the possibility that this rescue is accomplished by restoring the integrity of the septate junction. Ultrastructural and physiological analyses confirm this hypothesis, demonstrating that this domain provides an essential structural function at the septate junction in embryonic epithelia (Ward, 2001).

Although these results indicate that the FERM domain provides an essential structural function during embryonic development, it is less clear what role this domain plays in post-embryonic development. Loss of coracle function in imaginal epithelia results in a proliferative disadvantage that is ameliorated by expression of just the FERM domain, but does not alter overall epithelial integrity or polarity. A number of cell signaling pathways have been implicated in controlling cell proliferation and growth in imaginal epithelia. Included among these are the epidermal growth factor, Wingless, Notch, and the Dpp pathways. Mutations that perturb the proper transmission of these signals produce imaginal defects similar to those reported for coracle. Additionally, coracle was originally identified as a dominant suppressor of Egfr^Ellipse, a hypermorphic allele of the epidermal growth factor receptor homolog. It is possible that the FERM domain facilitates the transduction of one or more of these signaling cascades by binding to and thereby localizing an important intracellular factor or factors. Continuing efforts to identify additional genes that interact with coracle will undoubtedly help resolve the function of this domain in regulating proliferation (Ward, 2001).

What, then, is the role of the C-terminal domain? By analogy with Protein 4.1 and the ERM proteins, it is suspected that this region in Coracle contains a protein-binding domain. In the ERM proteins, the C-terminal domain is thought to regulate the function of the FERM domain via an intramolecular interaction. It is not known yet if similar interactions may occur in Coracle or Protein 4.1. In addition, recent experiments have identified several potential protein-protein interactions mediated by this domain. For example, the immunophilin FKBP13 interacts with the C-terminal region of Protein 4.1G, one of several Protein 4.1 paralogs in mammals. If such interactions occur via the Coracle C-terminal domain, they could function to anchor additional proteins to the region of the septate junction. In contrast, two recent studies have suggested interactions between this same domain of Protein 4.1 and proteins known to function in the nucleus. These proteins are NuMA, the nuclear mitotic apparatus protein, and PIKE, a putative regulator of PI₃ kinase activity in the nucleus. At the moment the functional significance of these interactions is not known, nor is it known if Coracle has similar interactions. Further experiments, particularly using genetic approaches, will be required to determine their functional significance. However, both these data and data regarding putative protein-protein interactions are consistent with a function for the C-terminal domain that is distinct from that of the FERM domain and may differ significantly from current ideas about Protein 4.1/Coracle functions (Ward, 2001).

Occluding junctions maintain stem cell niche homeostasis in the fly testes

Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).

The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).

During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).

Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).

Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w¹¹¹⁸ progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).

Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).

To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).

Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).

In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).

One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).

Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).

Select septate junction proteins direct ROS-mediated paracrine regulation of Drosophila cardiac function

Septate junction (SJ) complex proteins act in unison to provide a paracellular barrier and maintain structural integrity. This study has identified a non-barrier role of two individual SJ proteins, Coracle (Cora) and Kune-kune (Kune). Reactive oxygen species (ROS)-p38 MAPK signaling in non-myocytic pericardial cells (PCs) is important for maintaining normal cardiac physiology in Drosophila. However, the underlying mechanisms remain unknown. This study has found that in PCs, Cora and Kune are altered in abundance in response to manipulations of ROS-p38 signaling. Genetic analyses establish Cora and Kune as key effectors of ROS-p38 signaling in PCs on proper heart function. It was further determined that Cora regulates normal Kune levels in PCs, which in turn modulates normal Kune levels in the cardiomyocytes essential for proper heart function. These results thereby reveal select SJ proteins Cora and Kune as signaling mediators of the PC-derived ROS regulation of cardiac physiology (Lim, 2019).

Cell-cell interaction is typically maintained and regulated by various multi-protein complexes such as tight junctions, adherens junctions (AJs), and gap junctions. Invertebrate septate junctions (SJs), which have functional and molecular similarity to vertebrate tight junctions (TJs), are specialized, multi-protein junctional complexes that reside between the apposed plasma membranes of adjacent epithelial cells. In Drosophila, more than 20 molecular constituents of the SJ have been identified, and characterization of these proteins reveals their canonical role in in sealing neighboring cells and restricting the free diffusion of solutes between adjacent cells, thereby providing a paracellular permeability barrier. The SJ protein complex is also involved in the coordinated changes in cell shape and rearrangement during tissue morphogenesis at a stage when the SJ structure has not yet formed or matured to become optically visible. For instance, mutations in all tested SJ genes cause defects in head involution, dorsal closure, and salivary gland elongation during early embryonic development before a clear SJ structure has been formed. Mutations in all tested SJ genes also cause cell-cell dissociation in the Drosophila embryonic heart, a tissue that seemingly lacks discernable SJs. Although most studies on the SJ proteins are focused on their canonical barrier function, it has been known that subsets of SJ proteins may have a different, non-barrier role. For instance, the SJ proteins Neurexin-IV (Nrx-IV), the Na+K+ATPase β subunit Nervana 2 (Nrv2), Coracle (Cora), and Yurt form a group with a distinct role in promoting epithelial apical-basal polarity. SJ components have also recently been found to play a role in regulating Hippo signaling to control intestinal stem cell activity and hematopoiesis. Together, these findings support the emerging notion that SJ proteins could serve important roles beyond their canonical barrier function. However, the non-barrier functions of the SJ proteins and the individual SJ proteins that could be involved remain poorly understood (Lim, 2019).

The heart is a heterogeneous organ comprising the contractile cardiomyocytes (CMs) and non-myocytes, such as the epicardial cells and endocardial cells. The non-myocytes have important signaling roles that contribute to CM development, growth, and function. The Drosophila heart is a linear tube comprising two inner rows of contractile CMs closely flanked by two outer rows of non-myocytic pericardial cells (PCs). PCs have been characterized as nephrocytes that are analogous to the mammalian podocytes that function to filter toxins and proteins from the hemolymph, the equivalent of mammalian blood. The PC nephrocytes are characterized by an intricate cell shape that includes elongated infoldings of the plasma membrane to form foot processes and labyrinthine channels. The labyrinthine channels are sealed by the slit diaphragm, which is a highly organized structure composed of similar proteins as the slit diaphragm in mammals. The slit diaphragm serves as a filtration barrier to control the inflow of certain substances into the labyrinthine channels from the hemolymph. In addition, vesicular invaginations of the plasma membrane occur along the labyrinthine channels that are indicative of endocytosis of the sequestered materials from the hemolymph. Materials endocytosed into the nephrocytes, presumably toxic molecules from the hemolymph, are targeted for either degradation in the lysosome or recycling back to the hemolymph. Moreover, the CMs and PCs are separated by a basement membrane composed of extracellular matrix (ECM), which could serve as a filtration system for hemolymph content (Lim, 2019).

On the other hand, accumulating evidence is indicating an important secretory function of PC nephrocytes. An early observation of an increased synthesis of the bactericidal enzyme lysozyme in PCs following the experimental infection of the insect Calliphora erythrocephala with bacteria provided the first indication that PCs could manufacture proteins for release into the hemolymph. More recently, Drosophila PCs have been reported to secrete factors, such as the ECM components and hemolymph proteins that could directly control neighboring CM function. In addition, PCs have been reported to produce reactive oxygen species (ROS) under normal, non-stressed conditions. ROS belong to a group of reactive chemical species produced by the incomplete reduction of molecular oxygen and are now recognized to serve an important role in the regulation of various cardiac physiological processes. Physiological ROS produced in the PCs of the Drosophila heart control the production of downstream signals such as D-p38 MAPK in PCs that then act in a paracrine manner to regulate CM function and morphology. The phenomenon is apparently conserved, as a study on the zebrafish heart reported that injury-induced H2O2 in the epicardial cells promotes the regeneration of the neighboring myocardium through the activation of ERK1/2 MAPK signaling and likely the generation of soluble factors from the epicardial cells. Together, these findings support the notion that a conserved ROS-MAPK signaling axis operates in the epi- or pericardium to influence myocardial function. However, the molecular mechanisms underlying ROS-MAPK-mediated paracrine interactions are currently unknown (Lim, 2019).

This study found that among the SJ proteins tested in adult PCs, only Cora and Kune-kune (Kune) are altered in abundance by ROS-D-p38 signaling in PCs. The results further showed that pericardial ROS-D-p38 signaling regulates CM function and structure through Cora and Kune. It was also found that Cora controls Kune amount in PCs and that pericardial Kune in turn modulates myocardial Kune expression that is essential for normal cardiac physiology. This study thereby unravels an unexpected function of the select SJ proteins Cora and Kune as physiological signaling mediators in PCs, a role that is distinct from their common primary barrier function (Lim, 2019).

On the basis of the results of this study, a model is proposed for the ROS-mediated paracrine regulation of cardiac physiology. In PCs, physiological ROS-p38 level governs Cora amount, which in turn regulates the level of Kune in the cellular surface. Peripheral Kune then directs the abundance of Kune in the CMs, which is essential for proper myocardial function and morphology. As a result, lowering of ROS-p38 signaling to sub-physiological level in PCs reduces pericardial Cora level and heightens pericardial Kune level, thereby raising Kune in CMs to a level that is detrimental to normal cardiac function. Conversely, elevating ROS-p38 signaling to supra-physiological level in PCs increases pericardial Cora quantity and diminishes pericardial Kune content, thereby suppressing Kune in the CMs to a level that perturbs normal heart function (Lim, 2019).

The findings suggest that Cora and/or Kune serve dual roles as structural elements of the SJ complex and as downstream effectors of ROS signaling. Such a dual function of Cora or Kune is unexpected but perhaps not unprecedented. The signaling role of Cora and Kune as core SJ components appears analogous to that of Arm as a core AJ component. Within the AJ, Arm mediates cell-cell adhesion and anchoring of the actin cytoskeleton. However, upon activation by Wingless, the Drosophila homolog of Wnts, Arm accumulates in the cell and serves as a key effector of Wingless signal transduction. In the case of Cora and Kune in the PCs, in response to the ROS signal, p38 is activated which then regulates the abundance and/or activity of these two individual SJ proteins. It is therefore proposed that Cora and/or Kune in the SJ have parallel functions as Arm in the AJ in that they serve as a structural component of the junctional complex and as downstream effector of signaling pathways (Lim, 2019).

The results indicate that Kune level in the PC affects Kune level in the CM; however, the underlying mechanism is unclear. One possibility is that pericardial Kune and cardiomyocyte Kune homotypically interact. In this scenario, one would predict that Kune is likely localized at the cell-cell interface. This was not observed; however, it does not necessarily rule out the homotypic interaction hypothesis. It is possible that in addition to engaging in homotypic interaction to mediate ROS signaling, other obligations of Kune may cause Kune to become more evenly distributed across the cells. For instance, Kune might be involved in the nephrocytic activity of PCs, and hence localization of Kune all over the cell surface is essential to promote the uptake of materials from the hemolymph into PCs. In the myocardium, Kune might be involved in the synchronous contraction of the CMs, a process that could be facilitated by the uniform localization of Kune across the entire CM surface. Alternatively, Kune interaction between the pericardial and cardiac cells might not involve their direct homotypic interaction but rather be mediated by the basement membrane that resides between PCs and CMs, at least in certain regions of the fly heart. In addition, an aberrant change in the pericardial nephrocyte morphology caused by loss of pericardial Kune might also alter Kune level in the CM. Last but not least, paracrine factors could be released from the PC in a Kune-controlled manner, which then influence Kune level in the CM. Regardless of whether intercellular Kune interaction occurs via direct cell-cell contact or indirect mechanisms, the results have demonstrated an interesting phenomenon by which the maintenance of normal Kune abundance in CMs by its pericardial counterpart is essential for proper adult cardiac morphology and physiology. This further raises the question as to how Kune acts in CMs to control proper cardiac performance and morphology. One possibility is that Kune regulates ion channel level and/or activity in the CM plasma membrane, such as the transient receptor potential (TRP) family of Ca2+ channels. As such, alterations in the CM Kune level could perturb intracellular Ca2+ homeostasis, thereby disrupting proper cardiac contractility and structure. These possibilities remain to be investigated in future studies (Lim, 2019).

In summary, these findings reveal that select SJ proteins can act as signaling effectors and suggest that the SJ, like the AJ, could serve to organize signaling centers. This work also provides important insights into the essential mechanisms of ROS-mediated non-myocyte-myocyte signaling interactions, a process that appears to be conserved between invertebrates and vertebrates (Lim, 2019).

RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development

Both mRNA-binding Fragile X Mental Retardation Protein (FMRP) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. This study tested whether these RNA-binding proteins (RBPs) act jointly in a common mechanism. Both dfmr1 and staufen mutants, and trans-heterozygous double mutants, were shown to display increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNAi, a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, this study showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and this study confirmed that Staufen binds to coracle mRNA. FMRP and Staufen were shown to act sequentially to co-regulate postsynaptic Coracle expression, and show Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, this study found dfmr1, staufen and coracle mutants elevate neurotransmission strength. FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development (Song, 2022).

This study reveals the mechanism of the established FMRP negative regulation of postsynaptic GluRIIA receptors and presynaptic bouton formation in the Drosophila FXS disease model. Specifically, the mRNA-binding FMRP-positive translational regulator binds to staufen mRNA as predicted, within the postsynaptic cell. Consequently, both dfmr1 and staufen mutants share the elevated GluRIIA level and bouton number phenotypes based on a common postsynaptic pathway function, and genetically interact as trans-heterozygotes to reproduce these phenotypes. Staufen acts as a dsRBP to bind coracle mRNA as predicted; both dfmr1 and staufen mutants exhibit elevated postsynaptic Coracle levels, and genetically interact as trans-heterozygotes to reproduce this phenotype. Coracle acts as a GluRIIA-binding anchoring scaffold within the postsynaptic domain to regulate local receptor accumulation (Chen, 2005). Consequently, dfmr1, staufen and coracle mutants all increase NMJ synaptic functional differentiation to elevate neurotransmission strength. Finally, the elevated postsynaptic GluRIIA levels mediate retrograde BMP receptor trans-synaptic signaling that induces pMad to drive new presynaptic bouton development. dfmr1, staufen and coracle mutants all exhibit elevated presynaptic pMad levels, thereby linking the postsynaptic GluRIIA accumulation and presynaptic supernumerary bouton formation defects shared by all of these mutants (Song, 2022).

The staufen mutant increased synaptic Coracle levels, GluRIIA levels and bouton number are all internally consistent. In a previous study, opposite phenotypes were measured in staufenHL/Df(2R)Pcl7B, which reduces another 14 genes in heterozygous deficiency, including loci involved in neuronal development (e.g. grh, nopo). Importantly, this study similarly found reduced synaptic protein levels and bouton number in staufenHL/Df(2R)Pcl7B, suggesting that heterozygosity of one or more of the neighboring genes impairs synaptic development. However, this study showed that a staufen RNAi that reduces transcript levels by ~90% replicates the staufen mutant NMJ phenotypes of increased GluRIIA levels and synaptic bouton numbers. This was also replicated with a second, independent staufen RNAi line. Moreover, this study showed that the effect is entirely restricted to postsynaptic muscle RNAi, with no effect from presynaptic neuron RNAi, consistent with restricted postsynaptic Staufen function. In addition, postsynaptic staufen rescue of the staufen mutant restored normal synaptic bouton formation, with OE reducing GluRIIA levels in staufen mutants and rescuing GluRIIA levels in dfmr1 mutants. Both staufen mutants and postsynaptic staufen RNAi also share the arrested supernumerary satellite bouton development characterizing dfmr1 null mutants. These many independent lines of evidence confirm the results, and are consistent with the known parallel FMRP role in restricting GluRIIA levels and synaptic bouton formation (Song, 2022).

To regulate Staufen, FMRP binds staufen mRNA and protects targeted staufen transcripts from degradation. FMRP contains at least three distinct RNA-binding domains (RBDs), and Staufen has five RBDs. Staufen reportedly binds a specific RNA hairpin structure formed by long 3' UTRs, but RIP shows that Staufen also binds mRNAs that are not predicted to generate this secondary structure. Although the decreased staufen mRNA levels in both dfmr1 mutants and muscle-targeted dfmr1 RNAi are predicted to be due to the lack of FMRP binding, it is also possible that other unregulated interactors cause the downregulated staufen mRNA expression (Shah et al., 2020). Localized labeling with an anti-Staufen antibody has been reported in the postsynaptic NMJ, which can be confirmed, but it was not possible to reduce labeling in staufen hypomorphic mutants. Therefore Staufen labeling was not shown in the current study. Moreover, western blots have been reported with the same anti-Staufen antibody; however, attempts were unsuccessful. Therefore qPCR was used to measure staufen mRNA levels. Staufen binds to coracle mRNA, but does so in a non-selective manner. This result is consistent with Staufen acting as a very broad spectrum dsRBP, and suggests that Staufen likely acts with a translational regulator partner to generate specificity. FMRP is very well established to partner with other RBPs to mediate the translational regulation of its target transcripts (Song, 2022). ------

The postsynaptic Coracle scaffold acts in a GluRIIA local anchoring mechanism, presumably to link the receptors to the underlying actin cytoskeleton (Chen, 2005). The jointly elevated Coracle and GluRIIA levels in both dfmr1 and staufen mutants are consistent with this scaffold function. Because the dfmr1/+; staufen/+ trans-heterozygotes share this correlated Coracle and GluRIIA upregulation in the postsynaptic domain, a single common signaling pathway is indicated. Coracle also restricts terminal branching development in peripheral sensory neurons. Both coracle mutants and sensory neuron-targeted coracle RNAi also display increased dendritic branch and termini numbers. These phenotypes are similar to the expanded NMJ terminals and increased synaptic bouton development reported in this study. Importantly, both coracle loss of function (mutants and muscle-targeted RNAi) and gain of function (muscle-targeted OE) increase postsynaptic GluRIIA levels and generate supernumerary boutons. Likewise, the knockdown and OE of many other similar scaffolds are known to cause phenocopying defects. Some examples include the muscle chaperone UNC-45, the tight junction scaffold zonula occludens-1 (ZO-1) and synaptic UNC-13. Indeed, both coracle loss and OE similarly cause increased dendritic crossing in Drosophila sensory neurons, similar to the phenocopy of developmental defects reported in this study. Combining the roles of postsynaptic FMRP-Staufen-Coracle in GluRIIA clustering, it was reasoned that this pathway must be a regulatory determinant of synaptic functional development (Song, 2022).

Removing FMRP, Staufen and Coracle strongly enhances functional synaptic differentiation and NMJ neurotransmission strength. This is consistent with expectations from the postsynaptic GluRIIA accumulation in all of these mutants. Elevated GluRIIA levels are well known to be associated with increased evoked functional responses and prolonged channel open times. A GluRIIA pore sequence (MQQ) critically required for the Drosophila channel Ca2+ permeability is conserved in mammalian receptors. This selectivity allows Ca2+-dependent participation in spontaneous (mEJC) and evoked (EJC) neurotransmission. Although enhanced evoked EJC amplitudes are typically accompanied by mEJC alterations, this study found that mEJC amplitude and frequency are unchanged in both the staufen and coracle mutants, and show only minimal changes in the dfmr1 mutants. Classically, both evoked and spontaneous neurotransmission were thought to be mediated by the same vesicles; however, more recent evidence has indicated that spontaneous and evoked neurotransmission have distinct machinery and vesicle pools. Postsynaptic receptors can be segregated into different compartments that are activated by either spontaneous or evoked release. This work supports this growing body of evidence for differential regulation. Importantly, GluRIIA has unique functions, modulating both presynaptic glutamate release and presynaptic bouton development (Song, 2022).

The dfmr1, staufen and coracle mutants all showed upregulated presynaptic pMad correlated with postsynaptic activated GluRIIA accumulation. GluRIIA activation triggers presynaptic pMad signaling via BMP receptors surrounding active zones, which, in turn, stabilizes GluRIIA receptors in the postsynaptic domains. This trans-synaptic signaling mechanism induces new presynaptic bouton development. The targeted postsynaptic RNAi for all three genes confirms this intercellular link. Synaptic BMP signaling involves both the type I serine/threonine kinase receptors and the type II receptor Wit. Although BMP ligand Glass bottom boat (Gbb) signaling via Wit presynaptic receptors is well established at the NMJ to modulate synaptogenesis, the mechanism of presynaptic bouton formation induced by activated GluRIIA signaling does not involve canonical BMP signaling via Gbb. In the dfmr1 mutants, it is suggested that postsynaptic GluRIIA accumulation induces presynaptic bouton development via non-canonical GluRIIA-Wit trans-synaptic retrograde signaling. Similarly, the muscle postsynaptic glypican Dally-like protein (Dlp) negatively regulates NMJ synaptic development by inhibiting this same non-canonical BMP pathway through decreased activated GluRIIA expression. Postsynaptic GluRIIA clustering can thus trigger presynaptic bouton formation, although supernumerary boutons do not always induce reciprocal GluRIIA changes. It is concluded that an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulates intertwined structural and functional glutamatergic synapse development (Song, 2022).

GENE STRUCTURE

In addition to the 5.9 kb cDNA, two other cDNA clones have been analyzed. These other cDNAs are identical for 1596 bp at the 5' end but diverge by alternative splicing 3' to this point. One cDNA contains a 102 bp insert, and a second contains the same insert, plus a second one of 12 bp just 5' to the first. In both cases, a continuous open reading frame is maintained through these inserts to 3' sequences that are shared with the 5.9 kb cDNA. The sizes of the three cDNAs (5.9, 3.5 and 2.9 kb) determined by sequence analyis correlate well with the measured sizes of the mRNAs in Northern blots (Fehon, 1994).

PROTEIN STRUCTURE

Amino Acids - 1698

Structural Domains

The Drosophila, Xenopus and human sequences are 54% identical over a stretch of 350 aa in the N-terminal region. Within this region there is a significant but lower identity with other members of the protein 4.1 gene family, including ezrin (31% over 229 amino acids), talin (22% over 194 amino acids), moesin A and B (30% over 229 amino acids) and merlin (27% over 229 amino acids) and the two tyrosine phosphatase genes (37% over 287 amino acids). In addition, Coracle shares sequence similarity with the human and Xenopus 4.1 genes in a smaller region at the C terminus that is not shared with any other members of the 4.1 gene family. The intervening 1200 amino acids of Coracle sequence shows no similarity to any sequences in the GenBank database. Protein 4.1 homologs exhibit a predicted alpha-helical structure that extends for approximately 400 amino acids immediately C terminal to the highly conserved domain of Coracle. The additional 800 amino acids of sequence in Coracle that extend beyond this putative alpha-helical domain are predicted to be composed of beta-sheet structures. No such extended beta-sheet domain has been proposed for any of the other protein 4.1 family members. The N-terminal conserved region in Coracle appears to have several hydrophobic domains that could interact with the cell membrane. The region corresponding to the spectrin/actin binding domain in the vertebrate erythroid 4.1 protein is not conserved in any of the Coracle splice forms that have been characterized, although this region is highly conserved between the human and Xenopus genes (Fehon, 1994).

date revised: 2 December 2001  

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