The Spectrin cytoskeleton regulates the Hippo signalling pathway

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta‐heavy dimers) produces tissue overgrowth and mis‐regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM‐domain protein Expanded (Ex). Apical beta‐heavy Spectrin binds to Ex and co‐localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.     

Genetic interaction of centrosomin and bazooka in apical domain regulation in Drosophila photoreceptor

Background

Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes'' role in photoreceptor morphogenesis.

Methodology/Principal Findings

Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn''s gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs).

Conclusions/Significance

These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor.     

Role of Tau,a microtubule associated protein,in Drosophila photoreceptor morphogenesis

Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Here, Tau, a microtubule associated protein, was analyzed to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins' localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par‐1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. It is found that Tau has a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation.     

Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.

Methodology/Principal Findings

Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc''s gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.

Conclusions/Significance

In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development.     

Use of gain-of-function study to delineate the roles of<Emphasis Type="Italic">crumbs</Emphasis> in<Emphasis Type="Italic">Drosophila</Emphasis> eye development

The cell polarity gene,crumbs (crb), has been shown to participate in the development and degeneration of theDrosophila retina. Mutations inCRB1, the human homologue ofDrosophila crb, also result in retinitis pigmentosa and Leber congential amaurosis. In this study, we used the gain-of-function approach to delineate the roles ofcrb in developingDrosophila eye. In the third-instar larval stage, eye development is initiated with photoreceptor differentiation and positioning of photoreceptor nuclei in the apical cellular compartment of retinal epithelium. In the pupal stage, differentiated photoreceptors begin to form the photosensitive structures, the rhabdomeres, at their apical surface. UsingGMR-Gal4 to drive overexpression of the Crb protein at the third-instar eye disc, we found that differentiation of photoreceptors was disrupted and the nuclei of differentiated photoreceptors failed to occupy the apical compartment. Usinghs-Gal4 to drive Crb overexpression in pupal eyes resulted in interference with extension of the adherens junctions and construction of the rhabdomeres, and these defects were stage-dependent. This gain-of-function study has enabled us to delineate the roles of Crb at selective stages of eye development inDrosophila.     

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background  

Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.     

Role of spectraplakin in Drosophila photoreceptor morphogenesis

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors.

Methodology/Principal Findings

The localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin''s gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test.

Conclusions/Significance

In summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis.     

Role of Spastin in Apical Domain Control along the Rhabdomere Elongation in Drosophila Photoreceptor

Background

Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.

Methodology/Principal Findings

The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin''s gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.

Conclusions/Significance

These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.     

Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.     

Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila

Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. We show that the Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. We also show that the absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. We further demonstrate that Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors.     

Dynactin affects extension and assembly of adherens junctions in<Emphasis Type="Italic">Drosophila</Emphasis> photoreceptor development

Drosophila eye development is a progressive process including cell fate determination, pattern formation, and rhabdomere morphogenesis. During eye development, a dramatic change in cell shape, which involves turning and extension of the photoreceptor apical surface, occurs in the early pupal stages. It is known that assembly and extension of adherens junctions (AJs) play an important role in this process. In the present study, I show that mutation of the largest subunit of dynactin complexes encoded byGlued (GI) affects the extension and assembly of Ajs in developing photoreceptors. InGl ¹/+ mutants and transgenic flies expressing the dominant negative form of Glued, the AJs failed to properly assemble and extend. In addition, the morphogenesis of rhabdomeres was also affected in these flies. Taken together, these results suggest that the extension and assembly of AJs as well as determination of the rhabdomere domain in photoreceptor development areGl dependent.     

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1‐integrin and Na⁺/K⁺ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na⁺/K⁺ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na⁺/K⁺ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical–basal polarity in an EspF‐dependent manner, which would contribute to EPEC‐associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.     

Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.     

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.     

Crumbs3 Is Essential for Proper Epithelial Development and Viability

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.     

Retromer regulates apical-basal polarity through recycling Crumbs

Epithelial cells are characterized by an “apical–basal” polarization. The transmembrane protein Crumbs (Crb) is an essential apical determinant which confers apical membrane identity. Previous studies indicated that Crb did not constantly reside on the apical membrane, but was actively recycled. However, the cellular mechanism(s) underlying this process was unclear. Here we showed that in Drosophila, retromer, which was a retrograde complex recycling certain transmembrane proteins from endosomes to trans-Golgi network (TGN), regulated Crb in epithelial cells. In the absence of retromer, Crb was mis-targeted into lysosomes and degraded, causing a disruption of the apical–basal polarity. We further showed that Crb co-localized and interacted with retromer, suggesting that retromer regulated the retrograde recycling of Crb. Our data presented here uncover the role of retromer in regulating apical–basal polarity in epithelial cells and identify retromer as a novel regulator of Crb recycling.     

Function of <Emphasis Type="Italic">Drosophila mob2</Emphasis> in photoreceptor morphogenesis

The Drosophila photoreceptor is a highly polarized cell; a mature photoreceptor cell in Drosophila contains a photosensitive structure (the rhabdomere) and a supporting membrane (stalk) at its apical membrane. In a screen to isolate genes involved in determining stalk and rhabdomere formation, this study has identified the Drosophila mob2 (Dmob2) gene. Dmob2 belongs to a Mob1/phocein domain protein family whose functions are involved in polarized cell growth and asymmetric cell fate determination in yeast. To study the role of Dmob2 in photoreceptor development, we have raised an antibody against the Dmob2 protein. An immunocytochemical study has shown that Dmob2 is mainly localized in the apical membrane of photoreceptor cells during early development. As development proceeds, Dmob2 is gradually confined to the rhabdomere base of the photoreceptor cells. RNA interference (RNAi) for knockdown Dmob2 expression during eye development impairs rhabdomere formation. Our study further shows that the subcellular localization of phosphorylated Moesin and Crumbs in the developing photoreceptor cell is disrupted in Dmob2 RNAi flies. This work thus reports a novel function of Dmob2 in photoreceptor cell development.     

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, we studied the effects of Par-1 on Baz localization in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. We also show that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, we propose that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis.     

Crb apical polarity proteins maintain zebrafish retinal cone mosaics via intercellular binding of their extracellular domains

Cone photoreceptors are assembled by unknown mechanisms into geometrically regular mosaics in many vertebrate species. The formation and maintenance of photoreceptor mosaics are speculated to require differential cell-cell adhesion. However, the molecular basis for this theory has yet to be identified. The retina and many other tissues express Crumbs (Crb) polarity proteins. The functions of the?extracellular domains of Crb proteins remain to be understood. Here we report cell-type-specific expression of the crb2a and crb2b genes at the cell membranes of photoreceptor inner segments and Müller cell apical processes in the zebrafish retina. We demonstrate that the extracellular domains of Crb2a and Crb2b mediate a cell-cell adhesion function, which plays an essential role in maintaining the integrity of photoreceptor layer and cone mosaics. Because Crb proteins are expressed in many types of epithelia, the Crb-based cell-cell adhesion may underlie cellular patterning in other epithelium-derived tissues as well.