Assembly of the CD8α/p56
                            lck
                        
 protein complex in stably expressing rat epithelial cells

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

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Intestinal absorption of intrinsic factor and B12-intrinsic factor complex

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1964.207.1.27 ◽

1964 ◽

Vol 207 (1) ◽

pp. 27-32 ◽

Cited By ~ 13

Author(s):

Agna Boass ◽

T. Hastings Wilson

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Protein Complex ◽

Soluble Fraction ◽

Intrinsic Factor ◽

Vitamin B ◽

Intestinal Epithelial ◽

Basal Surface ◽

Blood Capillaries ◽

Intestinal Lymphatics

Evidence is presented which suggests that the vitamin B12-intrinsic factor (B12-IF) complex is absorbed into the intestinal epithelial cell. Following incubation of sacs of hamster ileum with B12-IF complex, the tissue was washed, the mucosa was homogenized and assayed for the complex. The soluble fraction from such a mucosal homogenate was found to possess significant amounts of the B12-IF complex. The exit of vitamin B12 from the basal surface of the epithelial cells was investigated by studying its appearance in the intestinal lymphatics. In a series of rats it was found that 3–9% of the absorbed B12 entered the lymphatic capillaries while the remaining fraction (91–97%) passed into the blood capillaries. Vitamin B12 present in lymph, although not dialyzable, was not bound to intrinsic factor. It is inferred from these studies that B12-IF complex enters the intestinal epithelial cells where it is converted into some other B12-protein complex. Following the exit of this B12-protein complex from the epithelial cell, it enters both lymphatic and blood capillaries, the latter being a quantitatively more important route.

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Monoclonal Antibodies to Different Components of the Human Cytomegalovirus (HCMV) Pentamer gH/gL/pUL128L and Trimer gH/gL/gO as well as Antibodies Elicited during Primary HCMV Infection Prevent Epithelial Cell Syncytium Formation

Journal of Virology ◽

10.1128/jvi.00121-16 ◽

2016 ◽

Vol 90 (14) ◽

pp. 6216-6223 ◽

Cited By ~ 31

Author(s):

Giuseppe Gerna ◽

Elena Percivalle ◽

Laurent Perez ◽

Antonio Lanzavecchia ◽

Daniele Lilleri

Keyword(s):

Endothelial Cells ◽

Monoclonal Antibodies ◽

Epithelial Cell ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Protein Complex ◽

Syncytium Formation ◽

Large Set ◽

Viral Fusion Protein ◽

Virus Dissemination

ABSTRACTHuman cytomegalovirus (HCMV) may cause disseminated/end-organ disease in congenitally infected newborns and immunosuppressed transplant recipients. Two glycoprotein complexes, gH/gL/gO and gH/gL/pUL128/pUL130/pUL131 (gH/gL/pUL128L; referred to as the pentamer), are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively, in the presence of the viral fusion protein gB. In addition, gH/gL/gO was recently reported to also be required for infection of endothelial/epithelial cells. Virus entry into human fibroblasts involves fusion of the virus envelope with the plasma membrane, whereas entry into endothelial/epithelial cells involves macropinocytosis or endocytosis and low-pH-dependent fusion with endosomes. A large set of neutralizing monoclonal antibodies (MAbs), directed to gH, gB, and multiple components of the pentamer, was developed. In addition, novel anti-gO human monoclonal antibodies were recently isolated. It is known that epithelial cell infection with a wild HCMV strain at a high multiplicity of infection produces a large number of syncytia. Incubation of heavily HCMV VR1814-infected ARPE-19 epithelial cells with neutralizing MAbs to one, two, or three components of the pUL128L portion of the pentamer blocked syncytium formation at an antibody concentration of 10 μg/ml, whereas only a partial inhibitory effect was displayed for MAbs to gO, gH, or gB at the same concentration. A blocking effect was also exhibited by convalescent-phase sera from primary HCMV infections. These findings indicate that the pentamer is required for syncytium formation in epithelial cells.IMPORTANCEHuman cytomegalovirus (HCMV) mostly infects epithelial and endothelial cellsin vivo. Recently, the pentamer protein complex (gH/gL/pUL128L) was identified as being required for infection of these cells, in association with the other protein complex, gH/gL/gO. In primary infections, HCMV migrates to endothelial cells and then to leukocytes, which disseminate the infection throughout the body. The virus then spreads to organs and tissues, mostly infecting either single cells or multinucleated epithelial giant cells (syncytia), depending on the viral load. Potent neutralizing human MAbs directed to distinct binding sites of the pUL128L portion of the pentamer were shown in the past to block virus dissemination. In the present study, MAbs to pUL128L were shown to block syncytium formation with a higher potency than that of MAbs to gO, gH, or gB, thus suggesting their role in limiting virus dissemination. This finding provides additional information useful for the development of anti-HCMV therapeutic antibodies and subunit vaccines.

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446. Ezrin and EBP50 relocate apically in rat uterine epithelial cells during contact with opposing cells, except cells contacting the blastocyst

Reproduction Fertility and Development ◽

10.1071/srb08abs446 ◽

2008 ◽

Vol 20 (9) ◽

pp. 126

Author(s):

L. Venuto ◽

L. A. Lindsay ◽

C. R. Murphy

Keyword(s):

Epithelial Cells ◽

Protein Complex ◽

Apical Membrane ◽

Cell Ultrastructure ◽

Cell Fractionation ◽

Uterine Epithelial Cells ◽

Pregnant Rat ◽

Signalling Molecules ◽

Indirect Immunofluorescence Microscopy ◽

Membrane Transformation

Uterine epithelial cells are important in constantly maintaining a tissue protective barrier, and only under the specific hormonal conditioning of pregnancy which involves a remodelling of the cell ultrastructure termed the 'plasma membrane transformation'. This allows for the successful invasion of the blastocyst. Indirect immunofluorescence microscopy in rat uterine epithelial cells during pregnancy shows that ezrin and EBP50 are relocated to the apical membrane upon lumen closure at the time of implantation, and on average results in 90% colocalisation. Ezrin and EBP50 function as a linked protein complex at the time of implantation seen through immunoprecipitation results from day 6 of pregnancy. The ezrin-EBP50 complex is also associated with the membrane, shown using cell fractionation and western blotting analysis in which ezrin increased dramatically in the membrane concentrated fraction, and correspondingly decreased in the cytosolic fraction leading up to implantation. At the apical membrane these proteins are likely associating with intra-membranous signalling molecules which allow communication between contacting cells. The same protein complex is also relocated to the apical membrane when cells contact an inanimate filament inserted into the uterus of a non-pregnant rat. The only unique contacting circumstance in which these proteins are not seen at the apical membrane is within the implantation chamber itself, more specifically the cells in direct contact with the implanting blastocyst. These results highlight the unique situation that is implantation, which involves specific blastocyst signalling and influence upon the uterine epithelial cells lining the implantation chamber. It may be that the ezrin-EBP50 protein complex is critical at the earlier stage of apposition, and through blastocyst influence, are subsequently lost from the apical membrane to allow for successful invasion.

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Inhibition of Human Papillomavirus Type 16 E7 Phosphorylation by the S100 MRP-8/14 Protein Complex

Journal of Virology ◽

10.1128/jvi.79.2.1099-1112.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 1099-1112 ◽

Cited By ~ 43

Author(s):

Sharof Tugizov ◽

Jennifer Berline ◽

Rossana Herrera ◽

Maria Elena Penaranda ◽

Mayumi Nakagawa ◽

...

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Protein Complex ◽

Calcium Binding ◽

Inhibitory Effect ◽

Related Factor ◽

Primary Keratinocytes ◽

Viral Oncoprotein ◽

Human Papillomavirus Type 16 ◽

Oncogenic Activity

ABSTRACT The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.

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Structure of the major membrane protein complex from urinary bladder epithelial cells by cryo-electron crystallography

Journal of Molecular Biology ◽

10.1006/jmbi.2001.5128 ◽

2001 ◽

Vol 314 (2) ◽

pp. 245-252 ◽

Cited By ~ 16

Author(s):

Gert T Oostergetel ◽

Wilko Keegstra ◽

Alain Brisson

Keyword(s):

Epithelial Cells ◽

Urinary Bladder ◽

Membrane Protein ◽

Protein Complex ◽

Electron Crystallography ◽

Membrane Protein Complex

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Gliotactin and Discs large form a protein complex at the tricellular junction of polarized epithelial cells in Drosophila

Journal of Cell Science ◽

10.1242/jcs.03208 ◽

2006 ◽

Vol 119 (21) ◽

pp. 4391-4401 ◽

Cited By ~ 25

Author(s):

J. Schulte ◽

K. Charish ◽

J. Que ◽

S. Ravn ◽

C. MacKinnon ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Complex ◽

Large Form ◽

Discs Large ◽

Polarized Epithelial Cells

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An Atypical PKC Directly Associates and Colocalizes at the Epithelial Tight Junction with ASIP, a Mammalian Homologue of Caenorhabditis elegans Polarity Protein PAR-3

The Journal of Cell Biology ◽

10.1083/jcb.143.1.95 ◽

1998 ◽

Vol 143 (1) ◽

pp. 95-106 ◽

Cited By ~ 364

Author(s):

Yasushi Izumi ◽

Tomonori Hirose ◽

Yoko Tamai ◽

Syu-ichi Hirai ◽

Yoji Nagashima ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Epithelial Cells ◽

Cell Polarity ◽

Protein Complex ◽

Protein Transport ◽

Sequence Similarity ◽

Physiological Role ◽

Junctional Complex ◽

Asymmetric Distribution ◽

Atypical Pkc

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

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Separable mechanisms drive local and global polarity establishment in the C. elegans intestinal epithelium

10.1101/2021.11.01.466827 ◽

2021 ◽

Author(s):

Melissa A Pickett ◽

Maria D. Sallee ◽

Victor F. Naturale ◽

Deniz Akpinaroglu ◽

Joo Lee ◽

...

Keyword(s):

Epithelial Cells ◽

Small Groups ◽

Intestinal Epithelium ◽

Protein Complex ◽

Specific Protein ◽

Dependent Manner ◽

Epithelial Polarity ◽

C Elegans ◽

Polarity Establishment

Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resiliency to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional, and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue specific protein depletion in the C. elegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but is not required for local polarity establishment as small groups of cells are able to correctly establish polarized domains in PAR-3 depleted intestines in an HMR-1/E-cadherin dependent manner. Despite belonging to the same apical protein complex, we additionally find that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.

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Electron Microscopic Study of the Epithelium of the Seminal Vesicle of Aging Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050706 ◽

1974 ◽

Vol 32 ◽

pp. 138-139

Author(s):

V. F. Allison ◽

G. C. Fink ◽

G. W. Cearley

Keyword(s):

Cell Growth ◽

Epithelial Cell ◽

Epithelial Cells ◽

Seminal Vesicle ◽

Seminal Vesicles ◽

Epithelial Layer ◽

Epithelial Hyperplasia ◽

Electron Microscopic ◽

Aging Rats ◽

Pseudostratified Epithelium

It is well known that epithelial hyperplasia (benign hypertrophy) is common in the aging prostate of dogs and man. In contrast, little evidence is available for abnormal epithelial cell growth in seminal vesicles of aging animals. Recently, enlarged seminal vesicles were reported in senescent mice, however, that enlargement resulted from increased storage of secretion in the lumen and occurred concomitant to epithelial hypoplasia in that species.The present study is concerned with electron microscopic observations of changes occurring in the pseudostratified epithelium of the seminal vescles of aging rats. Special attention is given to certain non-epithelial cells which have entered the epithelial layer.

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