Ectopic expression of cadherin 8 is sufficient to cause cyst formation in a novel 3D collagen matrix renal tubule culture

While a variety of genetic mutations have been shown to be associated with renal cyst formation, mechanisms of renal cyst formation are largely unknown. In prior communications we described alterations in E-cadherin assembly in cultured cystic epithelial cells (Charron AJ, Nakamura S, Bacallao R, Wandinger-Ness A. J Cell Biol 149: 111-124, 2000). Using the same cell line we assayed cadherin expression by RT-PCR using primer pairs that anneal to highly conserved sequences of cadherin genes but flank informative regions of cadherins. Using this approach we found that autosomal dominant polycystic kidney disease (ADPKD) cells express cadherin 8, a neuronal cadherin with limited expression in the kidney. Immunohistochemistry confirmed cadherin 8 expression in cystic epithelia. To test the functional significance of cadherin 8 expression in renal epithelial cells, we adapted a three-dimensional collagen culture method in which HK-2 cells form tubule structures and microinjected adenovirus into the matrix space surrounding tubule structures. Adenovirus expressing cadherin 8 under the control of a tet promoter caused cyst structures to grow out of the tubules when coinjected with adenovirus expressing a tet transactivator. Microinjection of single adenovirus expressing either tet transactivator or cadherin 8 failed to cause cyst formation. When doxycycline was added to the culture, following coinjection of adenovirus, there was a dose-response reduction in cadherin 8 expression and cyst formation. Similarly, HK-2 cells transfected with Flag-tagged cadherin 8 form cysts in addition to tubular structures. HK-2 cells transfected with Flag-tagged N-cadherin do not form cysts. These data suggest that ectopic expression of cadherin 8 in renal epithelial cells is sufficient to cause the morphogenic pattern of cyst formation.     

FAT1 cadherin is multiply phosphorylated on its ectodomain but phosphorylation is not catalysed by the four-jointed homologue

The interaction between the Drosophila cadherins fat and dachsous is regulated by phosphorylation of their respective ectodomains, a process catalysed by the atypical kinase four-jointed. Given that many signalling functions are conserved between Drosophila and vertebrate Fat cadherins, we sought to determine whether ectodomain phosphorylation is conserved in FAT1 cadherin, and also whether FJX1, the vertebrate orthologue of four-jointed, was involved in such phosphorylation events. Potential Fj consensus phosphorylation motifs were identified in FAT1 and biochemical experiments revealed the presence of phosphoserine and phosphothreonine residues in its extracellular domain. However, silencing FJX1 did not influence the levels of FAT1 ectodomain phosphorylation, indicating that other mechanisms are likely responsible.     

Dengue 3 virus distribution in the mosquito Aedes aegyptl: an immunocytochemical study

Abstract. The dissemination of dengue (DEN) 3 virus in parenterally infected female Aedes aegypti mosquitoes was studied imrnunocytochemically. Antigen was first detected in fat body cells near the thoracic site of virus inoculation. The intussuscepted foregut, salivary glands and nervous tissue were the first major tissues infected. Nervous tissue appeared to be the primary site of amplification. Muscles, tracheae, Malphigian tubules and the posterior midgut did not become infected. The only part of the reproductive system to be infected was the calyx (71% of specimens 16–22 days post-infection) consistent with low rates of vertical transmission. After 7 days post-inoculation the salivary glands of 100% of the specimens examined were infected. Virus dissemination was slow and the most common sequence of infection following intrathoracic inoculation was as follows: thoracic fat body, intussuscepted foregut, salivary glands, cardial epithelium, thoracic ganglion, brain, compound eye, anterior midgut, intermediate midgut/anterior abdominal ganglia, and calyx/hindgut/posterior abdominal ganglia. Fat body and intussuscepted foregut tissues lost infections after 16 days post-inoculation.     

Mammalian Fat1 cadherin regulates actin dynamics and cell-cell contact

Fat cadherins form a distinct subfamily of the cadherin gene superfamily, and are featured by their unusually large extracellular domain. In this work, we investigated the function of a mammalian Fat cadherin. Fat1 was localized at filopodial tips, lamellipodial edges, and cell-cell boundaries, overlapping with dynamic actin structures. RNA interference-mediated knockdown of Fat1 resulted in disorganization of cell junction-associated F-actin and other actin fibers/cables, disturbance of cell-cell contacts, and also inhibition of cell polarity formation at wound margins. Furthermore, we identified Ena/vasodilator-stimulated phosphoproteins as a potential downstream effector of Fat1. These results suggest that Fat1 regulates actin cytoskeletal organization at cell peripheries, thereby modulating cell contacts and polarity.     

Diversification of epithelial adherens junctions with independent reductive changes in cadherin form: identification of potential molecular synapomorphies among bilaterians

The adherens junction (AJ) is the most universal junction found in bilaterian epithelia and may represent one of the earliest types of cell-cell junctions. The adhesion molecules responsible for forming AJs are the classic cadherins (referred to simply as cadherins), whose extracellular domain organization displays marked variety among species examined so far. In this study, we attempted to reconstruct the evolution of cadherin by analyzing new data from several arthropods (two insects, one non-insect hexapod, three crustaceans, and one chelicerate) and previously published sequences for Drosophila melanogaster and other animals. The results of comparative analyses using the BLAST tool and immunohistochemical analyses revealed that the extracellular domain organizations of a decapod, an isopod, a spider, and a starfish cadherin, which are present at AJs in the embryonic epithelia are homologous. Independent reductive changes from the ancestral state were evident in the epithelia of hexapods+branchiopod, vertebrates+urochordates, and a cephalochordate. The form of cadherins in hexapods is more closely related to that of a branchiopod than to that of malacostracan crustaceans, and one of those of vertebrates is more closely related to that of urochordates than to that of a cephalochordate. Although the sampling of taxa is limited at this stage of research, we hypothesize that the reductive events in cadherin structure related to AJ formation in the epithelia may possess information about bilaterian relationships as molecular synapomorphies.     

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.     

Phenotypic and developmental analysis of mutations at thecrumbs locus,a gene required for the development of epithelia inDrosophila melanogaster

Summary The genecrumbs (crb) ofDrosophila melanogaster provides an essential function for the embryonic development of ectodermally derived epithelia. Complete loss of function alleles of thecrb gene are recessive embryonic lethals and lead to a disorganization of the primordia of these epithelia, followed by cell death in some tissues. Incrb mutant embryos, different organs are affected to a different extent. Some tissues die almost completely (as the epidermis, the atrium and the pharynx) while others partially survive and conserve their basic epithelial structure (as the tracheal system, the oesophagus, the proventriculus, the salivary glands, the hindgut and the Malpighian tubules). Degeneration is first visible at stage 11 and continues successively throughout development. There is evidence that the loss of epithelial cell polarity may be the cause for the degeneration of these tissues, suggesting that thecrb gene product is involved in stabilizing the apico-basal polarity of epithelial cells. As previously shown, thecrb protein is specifically expressed on the apical side of embryonic epithelia in a reticular pattern outlining the borders of the cells. Here we demonstrate that thecrb protein shows the same subcellular localization in epithelial cells of imaginal discs and in follicle cells, indicating a similar function ofcrb during the development of embryonic, imaginal and follicle epithelia. Clonal analysis experiments indicate that the genecrb is not cell-autonomous in its expression, suggesting that the gene product may act as a diffusible factor and may serve as a signal in a cell-cell communication process. This signal is thought to be required for the formation and/or maintenance of the cell and tissue structure of the respective epithelia.     

Noncoordinate expression of Drosophila glue genes: Sgs-4 is expressed at many stages and in two different tissues.

The glue genes of Drosophila melanogaster comprise a family of genes expressed at high levels in the salivary glands of late third instar larvae in response to the insect hormone ecdysone. We present evidence that, in contrast to the other glue genes, Sgs-4 is turned on throughout Drosophila development and is not expressed exclusively in the larval salivary glands. Larvae transformed with an Sgs-4/Adh (alcohol dehydrogenase) hybrid gene exhibit Sgs-4-directed Adh expression in the larval proventriculus as well as in the salivary glands as early as the first instar. Sgs-4-specific RNA can be detected at very low levels during all stages of development. During late third instar, levels of Sgs-4 RNA in the salivary glands increase several-thousand-fold, thereby accounting for the large amounts of Sgs-4 protein present in the glue produced by the salivary glands. This pattern of expression is unique to the Sgs-4 gene. While expression of several of the other glue genes can be detected in embryos and early larvae, they appear to be expressed neither throughout development nor in the larval proventriculus. Appearance of the glue gene RNAs in mid third instar salivary glands is noncoordinate, even for the chromosomally clustered genes Sgs-3, Sgs-7, and Sgs-8.     

Nonchordate classic cadherins have a structurally and functionally unique domain that is absent from chordate classic cadherins

Classic cadherins, which are adhesion molecules in cell-cell adherens junctions, have a large contribution to the construction of the animal body. Their molecular structures show clear differences between chordate and nonchordate metazoans. Although nonchordate classic cadherins have cadherin superfamily-specific extracellular repeats (CRs) and a highly conserved cytoplasmic domain (CP), these cadherins have a unique extracellular domain that is absent from vertebrate and ascidian classic cadherins. We called this the primitive classic cadherin domain (PCCD). To understand the roles of the PCCD, we constructed and characterized a series of mutant forms of the Drosophila classic cadherin DE-cadherin. Biochemical analyses indicated that the last two CRs and PCCD form a special structure with proteolytic cleavage. Mutations in the PCCD did not eliminate the cell-cell-binding function of DE-cadherin in cultured cells, but prevented the cadherin from efficiently translocating to the plasma membrane in epithelial cells of the developing embryo. In addition, genetic rescue assays suggested that although CP-mediated control plays a central role in tracheal fusion, the role of the PCCD in efficient recruitment of DE-cadherin to apical areas of the plasma membranes is also important for dynamic epithelial morphogenesis. We propose that there is a fundamental difference in the mode of classic cadherin-mediated cell-cell adhesion between chordate and nonchordate metazoans.     

Nucleation and growth of cadherin adhesions

Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.     

Protocadherins branch out: Multiple roles in dendrite development

The proper formation of dendritic arbors is a critical step in neural circuit formation, and as such defects in arborization are associated with a variety of neurodevelopmental disorders. Among the best gene candidates are those encoding cell adhesion molecules, including members of the diverse cadherin superfamily characterized by distinctive, repeated adhesive domains in their extracellular regions. Protocadherins (Pcdhs) make up the largest group within this superfamily, encompassing over 80 genes, including the ～60 genes of the α-, β-, and γ-Pcdh gene clusters and the non-clustered δ-Pcdh genes. An additional group includes the atypical cadherin genes encoding the giant Fat and Dachsous proteins and the 7-transmembrane cadherins. In this review we highlight the many roles that Pcdhs and atypical cadherins have been demonstrated to play in dendritogenesis, dendrite arborization, and dendritic spine regulation. Together, the published studies we discuss implicate these members of the cadherin superfamily as key regulators of dendrite development and function, and as potential therapeutic targets for future interventions in neurodevelopmental disorders.     

黑水虻消化道形态及组织结构的观察

本文比较了不同发育阶段黑水虻Hermetia illucens消化道的形态学差异,掌握了幼虫消化系统的组织学特征。利用体视镜观察黑水虻5龄幼虫、预蛹及成虫的消化道形态,利用光学显微镜和扫描电镜观察幼虫消化道各段(前肠、中肠、后肠)的显微及超微结构。结果表明：黑水虻幼虫及预蛹的消化道均由前肠(食道和前胃)、中肠及后肠组成,从幼虫到成虫,消化道的长度不断缩短。与幼虫和预蛹相比,成虫消化道形态变化明显,前胃消失,出现了嗉囊及胃盲囊,中肠进一步缩短,后肠分化为回肠、结肠和直肠。组织学观察结果显示,幼虫的唾液腺开口于口腔,由膨大的管状腺体和腺管组成。食道由特化为角质刺突的内膜层及发达的肌层组成,其末端延伸至前胃。前胃膨大为球状,包括三层组织结构。根据上皮细胞形态的差异,中肠可分为四个区段。后肠薄,肠腔内褶丰富,肠壁可见数量较多的杆状细菌。马氏管开口于中、后肠交界处,包括4支盲管,管内壁密布微绒毛。黑水虻消化道形态随发育阶段的变化,反映了各阶段摄食及消化生理的差异。幼虫消化道各段具有各自典型的组织学特征,其前、中、后肠可能分别承担了食物接纳与初步消化、消化与吸收以及重吸收功能。本研究结果为进一步了...     

Diversity of the cadherin family: evidence for eight new cadherins in nervous tissue.

To examine the diversity of the cadherin family, we isolated cDNAs from brain and retina cDNA preparations with the aid of polymerase chain reaction. The products obtained included cDNAs for two of three known cadherins as well as eight distinct cDNAs, of which deduced amino acid sequences show significant similarity with the known cadherin sequences. Larger cDNA clones were isolated from human cDNA libraries for six of the eight new molecules. The deduced amino acid sequences show that the overall structure of these molecules is very similar to that of the known cadherins, indicating that these molecules are new members of the cadherin family. We have tentatively designated these cadherins as cadherin-4 through -11. The new molecules, with the exception of cadherin-4, exhibit features that distinguish them as a group from previously cloned cadherins; they may belong to a new subfamily of cadherins. Northern blot analysis showed that most of these cadherins are expressed mainly in brain, although some are expressed in other tissues as well. These findings show that the cadherin family of adhesion molecules is much larger than previously thought, and suggest that the new cadherins may play an important role in cell-cell interactions within the central nervous system.     

普通齿蛉幼虫的消化系统结构和取食分析

研究了普通齿蛉Neoneuromus ignobilis Navás幼虫消化系统的解剖和电镜扫描结构,并结合肠道内食物残渣的成分分析了其取食特性.结果表明,普通齿蛉幼虫消化系统较为简单,无特殊的变异,消化道细长,由前肠、中肠和后肠构成,前后肠较长,分别占体长的42%和47%,中肠最短,仅占总长度的11%.前肠由食道、嗉...     

Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell-cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell-cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin-actin cooperation that supports productive contact formation.     

The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover

Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.