Unique cell biological profiles of retinal disease-causing missense mutations in the polarity protein Crumbs.

Mutations in human crumbs 1 (CRB1) are a major cause of retinal diseases that lead to blindness. CRB1 is a transmembrane protein found in the inner segment of photoreceptor cells (PRCs) and the apical membrane of Müller glia. The function of the extracellular region of CRB1 is poorly understood, although more than 80 disease-causing missense mutations have been mapped to it. We have recreated four of these mutations, affecting different extracellular domains, in Drosophila Crumbs (Crb). Crb regulates epithelial polarity and growth, and contributes to PRC differentiation and survival. The mutant Crb isoforms showed a remarkable diversity in protein abundance, subcellular distribution and ability to rescue the lack of endogenous Crb, elicit a gain-of-function phenotype or promote PRC degeneration. Interestingly, although expression of mutant isoforms led to a substantial rescue of the developmental defects seen in crb mutants, they accelerated PRC degeneration compared to that seen in retinas that lacked Crb, indicating that the function of Crb in cellular differentiation and cell survival depends on distinct molecular pathways. Several Crb mutant proteins accumulated abnormally in the rhabdomere and affected rhodopsin trafficking, suggesting that abnormal rhodopsin physiology contributes to Crb/CRB1-associated retinal degeneration.

Mutational analysis supports a core role for Drosophila α-catenin in adherens junction function.

α-catenin associates the cadherin-catenin complex with the actin cytoskeleton. α-catenin binds to ß-catenin, which links it to the cadherin cytoplasmic tail, and F-actin, but also to a multitude of actin-associated proteins. These interactions suggest a highly complex cadherin-actin interface. Moreover, mammalian αE-catenin has been implicated in a cadherin-independent cytoplasmic function in Arp2/3-dependent actin regulation, and in cell signaling. The function and regulation of individual molecular interactions of α-catenin, in particular during development, are not well understood. We have generated mutations in Drosophila α-Catenin (α-Cat) to investigate α-Catenin function in this model, and to establish a setup for testing α-Catenin-related constructs in α-Cat-null mutant cells in vivo. Our analysis of α-Cat mutants in embryogenesis, imaginal discs and oogenesis reveals defects consistent with a loss of cadherin function. Compromising components of the Arp2/3 complex or its regulator SCAR ameliorate the α-Cat loss-of-function phenotype in embryos but not in ovaries, suggesting negative regulatory interactions between α-Catenin and the Arp2/3 complex in some tissues. We also show that the α-Cat mutant phenotype can be rescued by the expression of a DE-cadherin::α-Catenin fusion protein, which argues against an essential cytosolic, cadherin-independent role of Drosophila α-Catenin.

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression.

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. Here, we show that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. Our findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.

The FERM protein Yurt is a negative regulatory component of the Crumbs complex that controls epithelial polarity and apical membrane size.

The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation.

The agrin/perlecan-related protein eyes shut is essential for epithelial lumen formation in the Drosophila retina.

The formation of epithelial lumina is a fundamental process in animal development. Each ommatidium of the Drosophila retina forms an epithelial lumen, the interrhabdomeral space, which has a critical function in vision as it optically isolates individual photoreceptor cells. Ommatidia containing an interrhabdomeral space have evolved from ancestral insect eyes that lack this lumen, as seen, for example, in bees. In a genetic screen, we identified eyes shut (eys) as a gene that is essential for the formation of matrix-filled interrhabdomeral space. Eys is closely related to the proteoglycans agrin and perlecan and secreted by photoreceptor cells into the interrhabdomeral space. The honeybee ortholog of eys is not expressed in photoreceptors, raising the possibility that recruitment of eys expression has made an important contribution to insect eye evolution. Our findings show that the secretion of a proteoglycan into the apical matrix is critical for the formation of epithelial lumina in the fly retina.

Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells.

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.

Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly.

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity.

Monomeric α-catenin links cadherin to the actin cytoskeleton.

The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin-catenin complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the function of α-catenin, which binds to cadherin through ß-catenin and can bind F-actin directly or indirectly. Here, we delineate regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic morphogenetic processes. Moreover, we address whether persistent α-catenin-mediated physical linkage between cadherin and F-actin is crucial for cell adhesion and characterize the functions of α-catenin monomers and dimers at adherens junctions. Our data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin-ß-catenin complex and the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin.

The germline stem cells of Drosophila melanogaster partition DNA non-randomly.

The Immortal Strand Hypothesis proposes that asymmetrically dividing stem cells cosegregate chromatids to retain ancestral DNA templates. Using both pulse-chase and label retention assays, we show that non-random partitioning of DNA occurs in germline stem cells (GSCs) in the Drosophila ovary as these divide asymmetrically to generate a new GSC and a differentiating cystoblast. This process is disrupted when GSCs are forced to differentiate through the overexpression of Bag of Marbles, a factor that impels the terminal differentiation of cystoblasts. When Decapentaplegic, a ligand which maintains the undifferentiated state of GSCs, is expressed ectopically the non-random partitioning of DNA is similarly disrupted. Our data suggest asymmetric chromatid segregation is coupled to mechanisms specifying cellular differentiation via asymmetric stem cell division.

Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis.

The apical transmembrane protein Crumbs is a central regulator of epithelial apical-basal polarity in Drosophila. Loss-of-function mutations in the human homologue of Crumbs, CRB1 (RP12), cause recessive retinal dystrophies, including retinitis pigmentosa. Here we show that Crumbs and CRB1 localize to corresponding subdomains of the photoreceptor apical plasma membrane: the stalk of the Drosophila photoreceptor and the inner segment of mammalian photoreceptors. These subdomains support the morphogenesis and orientation of the photosensitive membrane organelles: rhabdomeres and outer segments, respectively. Drosophila Crumbs is required to maintain zonula adherens integrity during the rapid apical membrane expansion that builds the rhabdomere. Crumbs also regulates stalk development by stabilizing the membrane-associated spectrin cytoskeleton, a function mechanistically distinct from its role in epithelial apical-basal polarity. We propose that Crumbs is a central component of a molecular scaffold that controls zonula adherens assembly and defines the stalk as an apical membrane subdomain. Defects in such scaffolds may contribute to human CRB1-related retinal dystrophies.