Ultrastructure of the surface of the epithelial cells in the rat retina

Summary The epithelial cells in the retina of rats were examined by scanning and transmission electron microscopy.The apical surface of the epithelial cells is covered by a large number of microvilli, which are embedded in an extracellular material. Distinct holes remaining after detached photoreceptor cell segments are numerous. The cells are polyhedronal, usually hexagonal and holes are scattered in their plasma membrane. Many of the epithelial cells are binucleated. The borders between adjacent cells are always close to each other. Numerous cytoplasmic folds or foot processes occur at the base of the cells as well as an extracellular space.The characteristic surface structures are discussed in relation to the function of the retinal epithelial cells.Supported by grants from the Swedish Medical Research Council (B70-12X-2543-02) and Riksföreningen mot Cancer (69171, 265-B69-01X).     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

mTOR信号通路在iPS定向分化RPE细胞中的调控机制研究

目的：探讨mTOR信号通路在iPS定向分化RPE细胞中的调控机制。方法培养iPS细胞,悬浮培养后形成拟胚体EB,诱导分化为RPE细胞。通过免疫细胞化学的方法,观察iPS-RPE细胞分化一个月后特异性蛋白（RPE65、LRAT、zo-1）的表达。同时通过Q-PCR,Western Blotting的方法检测iPS-RPE在不同分化时间点（分化1个月、2个月、3个月）上,iPS-RPE细胞中特异性基因、蛋白的表达变化以及mTOR信号通路的活性。最后应用雷帕霉素抑制iPS-RPE细胞中mTOR的通路,进一步观察iPS-RPE细胞中的蛋白表达变化。Q-PCR采用单因素方差分析（One-Way ANOVA）进行组间比较。结果荧光显微镜观察到iPS-RPE细胞在分化1个月时,表达RPE65、LRAT、zo-1蛋白。与对照组iPS比较,Q-PCR结果显示,实验组RPE65在分化1个月,2个月,3个月时的表达量分别为0.84±0.13,4.8±1.1,20.3±4.9（P=0.000）;Best1分化1个月,2个月,3个月时的表达量分别为1.5±0.16,2.3±0.68,11.78±1.57（P=0.000）;MerTK在分化1个月,2个月,3个月时的表达量分别为4.12±1.94,1.87±0.76,15.53±1.33（P=0.000）,CK18分化1个月,2个月,3个月时的表达量分别为2.4±0.63,2.3±0.37,9.67±1.44（P=0.000）,Western Blotting结果也表明,随着分化时间延长,iPS-RPE细胞中特异性蛋白（BEST1、catenin、MerTK）表达量有显著提高,而mTOR信号通路的活性受到抑制。与对照组（加DMSO）比较,雷帕霉素处理获得的iPS-RPE细胞中BEST1、MerTK、ck18蛋白表达量上升不是很明显,catenin蛋白显著提高。结论建立了体外高等分化、功能高效的iPS-RPE细胞,随iPS-RPE细胞分化时间延长,mTOR信号通路的活性是逐步抑制的。     

Store-operated Ca channels in prostate cancer epithelial cells: function, regulation, and role in carcinogenesis

Ca²⁺ homeostasis mechanisms, in which the Ca²⁺ entry pathways play a key role, are critically involved in both normal function and cancerous transformation of prostate epithelial cells. Here, using the lymph node carcinoma of the prostate (LNCaP) cell line as a major experimental model, we characterize prostate-specific store-operated Ca²⁺ channels (SOCs)—a primary Ca²⁺ entry pathway for non-excitable cells—for the first time. We show that prostate-specific SOCs share major store-dependent, kinetic, permeation, inwardly rectifying, and pharmacological (including dual, potentiation/inhibition concentration-dependent sensitivity to 2-APB) properties with “classical” Ca²⁺ release-activated Ca²⁺ channels (CRAC), but have a higher single channel conductance (3.2 and 12 pS in Ca²⁺- and Na⁺-permeable modes, respectively). They are subject to feedback inhibition via Ca²⁺-dependent PKC, CaMK-II and CaM regulatory pathways and are functionally dependent on caveolae integrity. Caveolae also provide a scaffold for spatial co-localization of SOCs with volume-regulated anion channels (VRAC) and their Ca²⁺-mediated interaction. The TRPC1 and TRPV6 members of the transient receptor potential (TRP) channel family are the most likely molecular candidates for the formation of prostate-specific endogenous SOCs. Differentiation of LNCaP cells to an androgen-insensitive, apoptotic-resistant neuroendocrine phenotype downregulates SOC current. We conclude that prostate-specific SOCs are important determinants in the transition to androgen-independent prostate cancer.     

Independent roles of Drosophila Moesin in imaginal disc morphogenesis and hedgehog signalling

The three ERM proteins (Ezrin, Radixin and Moesin) form a conserved family required in many developmental processes involving regulation of the cytoskeleton. In general, the molecular function of ERM proteins is to link specific membrane proteins to the actin cytoskeleton. In Drosophila, loss of moesin (moe) activity causes incorrect localisation of maternal determinants during oogenesis, failures in rhabdomere differentiation in the eye and alterations of epithelial integrity in the wing imaginal disc. Some aspects of Drosophila Moe are related to the activity of the small GTPase RhoA, because the reduction of RhoA activity corrects many phenotypes of moe mutant embryos and imaginal discs. We have analysed the phenotype of moesin loss-of-function alleles in the wing disc and adult wing, and studied the effects of reduced Moesin activity on signalling mediated by the Notch, Decapentaplegic, Wingless and Hedgehog pathways. We found that reductions in Moesin levels in the wing disc cause the formation of wing-tissue vesicles and large thickenings of the vein L3, corresponding to breakdowns of epithelial continuity in the wing base and modifications of Hedgehog signalling in the wing blade, respectively. We did not observe any effect on signalling pathways other than Hedgehog, indicating that the moe defects in epithelial integrity have not generalised effects on cell signalling. The effects of moe mutants on Hedgehog signalling depend on the correct gene-dose of rhoA, suggesting that the requirements for Moesin in disc morphogenesis and Hh signalling in the wing disc are mediated by its regulation of RhoA activity. The mechanism linking Moesin activity with RhoA function and Hedgehog signalling remains to be elucidated.     

Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila

We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The “Tomato/GFP-FLP/FRT” method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.     

角膜上皮细胞代谢的研究进展

角膜上皮层位于角膜表面,外邻泪膜,内与角膜前弹力层相连。角膜上皮细胞代谢所需营养及氧分主要通过泪膜、房水和角膜缘毛细血管运送。正常的角膜上皮细胞代谢是维持角膜上皮细胞正常增殖与分化状态的关键。角膜上皮细胞代谢异常可导致上皮损伤或变性,是多种角膜疾病的病理基础。本文就近年来关于角膜上皮细胞代谢相关的组织结构、营养来源、细胞增殖分化以及相关疾病的研究进展进行综述。     

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.     

Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.     

Gap junction formation between normal and reaggregated endoderm cells ofXenopus laevis neurulae

Summary Using freeze-fracture electron microscopy and fluorescent dye injection we have analysed the contacts between cells of the deeper endoderm taken from neurulae ofXenopus laevis. Endodermal cells in situ have large 1.5 m diameter gap junctions composed of 8 nm P-face particles and corresponding E-face pits. Beside gap junctions, particle aggregates typical of desmosomal plaques are present but there are no tight junctions. The dissociation of endoderm into single cells involves profound structural alterations in the surface membrane including the complete disappearance of junctional structures among them gap junctions. The reaggregation of endoderm cells leads to the restoration of the surface membrane IMP (Intra Membrane Particle) pattern and, after ca. 30 min, to the establishment of functional pathways allowing for the intercellular transfer of fluorescent dye. Concomitantly gap junctions reappear. The observation that the dissociation and reaggregation of endodermal cells involves IMP alterations which go beyond the cell junctions themselves is discussed as an adaptation of the plasma membrane to changing environmental conditions.     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Differentiation of epithelial cells in the mouse thymus

Summary The foetal and post-natal development of the mouse thymus was studied with the electron microscope paying particular attention to the differentiation of the epithelial cells. At about 13 days' gestation, the thymus was composed principally of undifferentiated epithelial cells and some lymphoblasts. The latter accumulated rapidly but did not show much evidence of mitotic activity until after the development of differentiated cortical epithelial cells which appeared during the 15th day of gestation. Further differentiation of epithelial cells did not occur until near term when medullary cystic epithelial cells appeared, and post-natally when small Hassall's corpuscles were developed. Undifferentiated and dividing epithelial cells were seen in the medulla and were present in all postnatal animals examined.This is publication number 1400 from the Walter & Eliza Hall Institute of Medical Research.The author is grateful to Prof. G. J. V. Nossal, Dr. J. F. A. P. Miller and Dr. P. J. Russell for their interest and assistance with various aspects of this study. Special thanks are due to Miss Mary Bravington for her skilled technical assistance. This investigation was supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and the National Health and Medical Research Council of Australia. The Electron Microscope Laboratory was equipped and supported by grants from the Australian Research Grants Committee, J. B. Were and Sons and the Potter Foundation.     

Tight junction formation in cultured epithelial cells (MDCK)

Summary Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca²⁺, 20 hr later, while monolayers with Ca²⁺ have fully developed junctions that confer an electrical resistance across of 346±51 cm², those without Ca²⁺ have a negligible resistance. If at this time Ca²⁺ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca²⁺ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca²⁺ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25°C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains.     

Structural Basis for the Phosphorylation-regulated Interaction between the Cytoplasmic Tail of Cell Polarity Protein Crumbs and the Actin-binding Protein Moesin

The type I transmembrane protein crumbs (Crb) plays critical roles in the establishment and maintenance of cell polarities in diverse tissues. As such, mutations of Crb can cause different forms of cancers. The cell intrinsic role of Crb in cell polarity is governed by its conserved, 37-residue cytoplasmic tail (Crb-CT) via binding to moesin and protein associated with Lin7–1 (PALS1). However, the detailed mechanism governing the Crb·moesin interaction and the balance of Crb in binding to moesin and PALS1 are not well understood. Here we report the 1.5 Å resolution crystal structure of the moesin protein 4.1/ezrin/radixin/moesin (FERM)·Crb-CT complex, revealing that both the canonical FERM binding motif and the postsynaptic density protein-95/Disc large-1/Zonula occludens-1 (PDZ) binding motif of Crb contribute to the Crb·moesin interaction. We further demonstrate that phosphorylation of Crb-CT by atypical protein kinase C (aPKC) disrupts the Crb·moesin association but has no impact on the Crb·PALS1 interaction. The above results indicate that, upon the establishment of the apical-basal polarity in epithelia, apical-localized aPKC can actively prevent the Crb·moesin complex formation and thereby shift Crb to form complex with PALS1 at apical junctions. Therefore, Crb may serve as an aPKC-mediated sensor in coordinating contact-dependent cell growth inhibition in epithelial tissues.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

Lipid polarity is maintained in absence of tight junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.     

Phosphatidylinositol-4-phosphate 5-kinase gamma is associated with cell-cell junction in A431 epithelial cells

Cell to cell contact in epithelial cells is crucial for tissue integrity and is maintained by junctional complexes, such as the adherens junction (AJ). Actin polymerization has been shown to be important for AJ formation; however, the molecular mechanisms have yet to be clarified. It has been shown that increased phosphatidylinositol-4,5-bisphosphate (PIP2) induces actin polymerization. It is thus of interest to know more about the production of PIP2 during cell-cell adhesion formation in epithelial cells. The distribution of phosphatidylinositol-4-phosphate 5-kinase gamma635 (PIP5Kgamma635), an isoform of the PIP2 synthesizing enzymes, was examined in epithelial cell line A431. It was found that, in non-contact cells, PIP5Kgamma635 was not concentrated at the plasma membrane. However, in cells that were in contact, PIP5Kgamma635 localized to the intercellular contact sites and colocalized with E-cadherin and beta-catenin, two components of AJ, and with polymerized actin, but did not colocalize with focal adhesion, integrin-mediated cell-substratum complex. Decreasing calcium ion concentration induced both disruption of intercellular adhesion and the dissociation of both PIP5Kgamma635 and actin from the contact site. These results suggest that PIP5K has an important role in actin polymerization in epithelial cell-cell adhesion.