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.     

Localization of apical epithelial determinants by the basolateral PDZ protein Scribble

The generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.     

Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.

The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.     

Adherens junctions inhibit asymmetric division in the Drosophila epithelium

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. Here we show that by disrupting adherens junctions we can convert the symmetric epithelial division into asymmetric division. We further confirm that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrate that both APC and microtubule-associated EB1 homologues are required for the symmetric epithelial division along the planar axis. Our results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions.     

Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation

Shaping a developing organ or embryo relies on the spatial regulation of cell division and shape. However, morphogenesis also occurs through changes in cell-neighbourhood relationships produced by intercalation. Intercalation poses a special problem in epithelia because of the adherens junctions, which maintain the integrity of the tissue. Here we address the mechanism by which an ordered process of cell intercalation directs polarized epithelial morphogenesis during germ-band elongation, the developmental elongation of the Drosophila embryo. Intercalation progresses because junctions are spatially reorganized in the plane of the epithelium following an ordered pattern of disassembly and reassembly. The planar remodelling of junctions is not driven by external forces at the tissue boundaries but depends on local forces at cell boundaries. Myosin II is specifically enriched in disassembling junctions, and its planar polarized localization and activity are required for planar junction remodelling and cell intercalation. This simple cellular mechanism provides a general model for polarized morphogenesis in epithelial organs.     

Differential positioning of adherens junctions is associated with initiation of epithelial folding

During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. The prevailing model underlying epithelial folding involves cell shape changes driven by myosin-dependent apical constriction. Here we describe an alternative mechanism that requires differential positioning of adherens junctions controlled by modulation of epithelial apical-basal polarity. Using live embryo imaging, we show that before the initiation of dorsal transverse folds during Drosophila gastrulation, adherens junctions shift basally in the initiating cells, but maintain their original subapical positioning in the neighbouring cells. Junctional positioning in the dorsal epithelium depends on the polarity proteins Bazooka and Par-1. In particular, the basal shift that occurs in the initiating cells is associated with a progressive decrease in Par-1 levels. We show that uniform reduction of the activity of Bazooka or Par-1 results in uniform apical or lateral positioning of junctions and in each case dorsal fold initiation is abolished. In addition, an increase in the Bazooka/Par-1 ratio causes formation of ectopic dorsal folds. The basal shift of junctions not only alters the apical shape of the initiating cells, but also forces the lateral membrane of the adjacent cells to bend towards the initiating cells, thereby facilitating tissue deformation. Our data thus establish a direct link between modification of epithelial polarity and initiation of epithelial folding.     

Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz

Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. Here we identify an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. We identify two polarized cues that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. We show that Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, our data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia.     

Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds)

Mutant mice homozygous for 'retinal degeneration slow' (rds/rds) are characterized phenotypically by abnormal development of photoreceptor outer segments in the retina, followed by gradual degeneration of photoreceptors. This process of degeneration is complete by one year, with preservation of all other retinal cells. The biochemical defect that leads to the mutant phenotype is not known. Our strategy for cloning the rds gene was based upon three previously reported observations. First, the rds locus maps to chromosome 17. Second, experimental rds/rds----+/+ and rds/+----+/+ tetra-parental mice manifest patchy photoreceptor changes in the retina, suggesting that the wild-type rds locus is expressed within cells of the photoreceptor lineage. Finally, the process of degeneration is specific to photoreceptors. On the basis of these observations, we predicted that the rds mRNA is encoded by a gene on chromosome 17 and is normally expressed exclusively within photoreceptors in the retina. We here present evidence that this is the case.     

ON and OFF pathways in Drosophila motion vision

Motion vision is a major function of all visual systems, yet the underlying neural mechanisms and circuits are still elusive. In the lamina, the first optic neuropile of Drosophila melanogaster, photoreceptor signals split into five parallel pathways, L1-L5. Here we examine how these pathways contribute to visual motion detection by combining genetic block and reconstitution of neural activity in different lamina cell types with whole-cell recordings from downstream motion-sensitive neurons. We find reduced responses to moving gratings if L1 or L2 is blocked; however, reconstitution of photoreceptor input to only L1 or L2 results in wild-type responses. Thus, the first experiment indicates the necessity of both pathways, whereas the second indicates sufficiency of each single pathway. This contradiction can be explained by electrical coupling between L1 and L2, allowing for activation of both pathways even when only one of them receives photoreceptor input. A fundamental difference between the L1 pathway and the L2 pathway is uncovered when blocking L1 or L2 output while presenting moving edges of positive (ON) or negative (OFF) contrast polarity: blocking L1 eliminates the response to moving ON edges, whereas blocking L2 eliminates the response to moving OFF edges. Thus, similar to the segregation of photoreceptor signals in ON and OFF bipolar cell pathways in the vertebrate retina, photoreceptor signals segregate into ON-L1 and OFF-L2 channels in the lamina of Drosophila.     

Adherens junctions and beta-catenin-mediated cell signalling in a non-metazoan organism

Mechanical forces between cells have a principal role in the organization of animal tissues. Adherens junctions are an important component of these tissues, connecting cells through their actin cytoskeleton and allowing the assembly of tensile structures. At least one adherens junction protein, beta-catenin, also acts as a signalling molecule, directly regulating gene expression. To date, adherens junctions have only been detected in metazoa, and therefore we looked for them outside the animal kingdom to examine their evolutionary origins. The non-metazoan Dictyostelium discoideum forms a multicellular, differentiated structure. Here we describe the discovery of actin-associated intercellular junctions in Dictyostelium. We have isolated a gene encoding a beta-catenin homologue, aardvark, which is a component of the junctional complex, and, independently, is required for cell signalling. Our discovery of adherens junctions outside the animal kingdom shows that the dual role of beta-catenin in cell-cell adhesion and cell signalling evolved before the origins of metazoa.     

Targeted misexpression of a Drosophila opsin gene leads to altered visual function

Drosophila mutants transformed with a chimaeric gene that expresses the ocellar visual pigment in the major class of photoreceptor cells of the retina were used to investigate the properties of this minor pigment. The photoreceptor cells in which this opsin was misexpressed showed new spectral characteristics and physiology.     

Transforming the architecture of compound eyes

Eyes differ markedly in the animal kingdom, and are an extreme example of the evolution of multiple anatomical solutions to light detection and image formation. A salient feature of all photoreceptor cells is the presence of a specialized compartment (disc outer segments in vertebrates, and microvillar rhabdomeres in insects), whose primary role is to accommodate the millions of light receptor molecules required for efficient photon collection. In insects, compound eyes can have very different inner architectures. Fruitflies and houseflies have an open rhabdom system, in which the seven rhabdomeres of each ommatidium are separated from each other and function as independent light guides. In contrast, bees and various mosquitoes and beetle species have a closed system, in which rhabdomeres within each ommatidium are fused to each other, thus sharing the same visual axis. To understand the transition between open and closed rhabdom systems, we isolated and characterized the role of Drosophila genes involved in rhabdomere assembly. Here we show that Spacemaker, a secreted protein expressed only in the eyes of insects with open rhabdom systems, acts together with Prominin and the cell adhesion molecule Chaoptin to choreograph the partitioning of rhabdomeres into an open system. Furthermore, the complete loss of spacemaker (spam) converts an open rhabdom system to a closed one, whereas its targeted expression to photoreceptors of a closed system markedly reorganizes the architecture of the compound eyes to resemble an open system. Our results provide a molecular atlas for the construction of microvillar assemblies and illustrate the critical effect of differences in a single structural protein in morphogenesis.     

Vinculin activation by talin through helical bundle conversion

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.     

Restoration of vision after transplantation of photoreceptors

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1?/? mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1?/? mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.     

Basement membrane polarizes lectin binding sites of Drosophila larval fat body cells

Vertebrate epithelial cells in monolayers are asymmetrical in that their apical and basal membranes differ in morphology and function. That this cell polarity depends on the presence of tight junctions can be demonstrated by labelling one surface of a cell monolayer in culture with fluorescent lectins and lipid probes, and subsequently observing whether the labels disperse to the opposite cell surfaces. Here we report on a differential distribution of binding sites for the lectin wheat germ agglutinin (WGA) on the cells of Drosophila melanogaster larval fat body, and show that the pattern is correlated with the structural association between the cell surfaces and their overlying basement membrane.     

Nir2碳端结构域的晶体结构

Nir2蛋白具有多个结构域，在生物体中可能发挥多种生理功能。本实验用MAD软件对大肠杆菌BL21（plys）原核表达的Nir2蛋白碳端进行了晶体结构分析。结果显示，硒蛋氨酸取代蛋白晶体及未取代蛋白晶体同属空间群P212121,晶胞参数a=109.468 Å, b=120.621 Å, c=70.315 Å，分辨率分别为2.2 Å和2.7 Å。一个不对称单位含有三个分子，含水量为48%。Nir2蛋白碳端由C片层和N片层构成，其中C片层与钙ATPase的催化区域P相似，N片层为许多不相关的功能性蛋白所共有。研究证明二者之间的静电作用是影响Nir2蛋白碳端与Pyk2 FERM区域结合的主要因素。该晶体结构的分析结果为进一步研究Nir2蛋白碳端的潜在功能提供了结构基础。     

SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans

Muscular dystrophies are among the most common human genetic diseases and are characterized by progressive muscle degeneration. Muscular dystrophies result from genetic defects in components of the dystrophin-glycoprotein complex (DGC), a multimeric complex found in the muscle cell plasma membrane. The DGC links the intracellular cytoskeleton to the extracellular matrix and is thought to be important for maintaining the mechanical integrity of muscles and organizing signalling molecules. The exact role of the DGC in the pathogenesis of disease has, however, remained uncertain. Mutations in Caenorhabditis elegans DGC genes lead to specific defects in coordinated movement and can also cause muscle degeneration. Here we show that mutations in the gene snf-6 result in phenotypes indistinguishable from those of the DGC mutants, and that snf-6 encodes a novel acetylcholine/choline transporter. SNF-6 mediates the uptake of acetylcholine at neuromuscular junctions during periods of increased synaptic activity. SNF-6 also interacts with the DGC, and mutations in DGC genes cause a loss of SNF-6 at neuromuscular junctions. Improper clearing of acetylcholine and prolonged excitation of muscles might contribute to the pathogenesis of muscular dystrophies.     

Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase

Mice homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa. In affected animals the retinal rod photoreceptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left. Degeneration is preceded by accumulation of cyclic GMP in the retina and is correlated with deficient activity of the rod photoreceptor cGMP-phosphodiesterase. We have recently isolated a candidate complementary DNA for the rd gene from a mouse retinal library and completed the characterization of cDNAs encoding all subunits of bovine photoreceptor phosphodiesterase. The candidate cDNA shows strong homology with a cDNA encoding the bovine phosphodiesterase beta subunit. Here we present evidence that the candidate cDNA is the murine homologue of bovine phosphodiesterase beta cDNA. We conclude that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterase beta subunit.