scholarly journals Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

1999 ◽  
Vol 10 (12) ◽  
pp. 4247-4261 ◽  
Author(s):  
Peter M. Steinert ◽  
Lyuben N. Marekov
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Cell Envelope ◽  
Immunogold Electron Microscopy ◽  
Epidermal Keratinocytes ◽  
Cross Linking ◽  
Cell Periphery ◽  
Human Epidermal Keratinocytes ◽  
Squamous Epithelia ◽  
D Cells

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

scholarly journals MEMBRANE-COATING GRANULES OF KERATINIZING EPITHELIA

1965 ◽  
Vol 24 (2) ◽  
pp. 297-307 ◽  
Author(s):  
A. Gedeon Matoltsy ◽  
Paul F. Parakkal
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Envelope ◽  
Cell Periphery ◽  
Cell Surfaces ◽  
Intercellular Spaces ◽  
Cytoplasmic Granules ◽  
Coated Cell ◽  
Membrane Coating

The purpose of this study has been to obtain information on the development of the envelop of horny cells that resists the action of keratinolytic agents. Toward this end the epidermis, oral mucosa, and tongue epithelium of various vertebrates, as well as the isolated envelopes of horny cells, were examined by electron microscopy. It was found that small cytoplasmic granules (1,000 to 5,000 A) that develop within differentiating epithelial cells move toward the cell periphery, and after fusion with the plasma membrane, empty their contents into the intercellular spaces. The content of the granules spreads over the cell surfaces, and subsequently a thickened and coated cell envelope is formed that resists the action of keratinolytic agent. The membrane-coating granule is regarded as a specific differentiation product of the keratinizing epithelium. It contains numerous inner membranes and is assumed to engage in synthetic activities such as, perhaps, the formation of polysaccharides.

The Functional Role of TLR9 in Human Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 366-366
Author(s):  
Jonathan Noah Thon ◽  
Christian G. Peters ◽  
Rukhsana Aslam ◽  
Jesse Rowley ◽  
Andrew S. Weyrich ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Type Iv Collagen ◽  
Surface Expression ◽  
Dense Granule ◽  
Immunogold Electron Microscopy ◽  
Cell Periphery ◽  
Severe Thrombocytopenia ◽  
Viral Dna ◽  
Type Iv ◽  
Type C

Abstract Abstract 366 Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. This is perhaps best exemplified by the observation that severe thrombocytopenia is associated with sepsis. While recent phenotypic and functional studies have addressed the roles of TLRs 2 and 4 in human/murine PLTs, which are primarily extracellular receptors, very little is known about the cellular localization, expression, and physiological significance of intracellular TLR9. Here we show that TLR9 localizes to unique granular compartments along the cell periphery (underlying the plasma membrane) in human PLTs. While thought to reside exclusively in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells, TLR9 expression is significantly enhanced in PLTs following incubation with thombin, ADP, PMA, CRP and type IV collagen, suggesting activation-mediated granule release. Surprisingly, TLR9 surface expression did not coincide with CD62P and CD61 expression levels upon PLT activation (which were not increased following type IV collagen incubation), and TLR9 was shown not to co-localize with known alpha-granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, CD42b), dense granule (serotonin), endosomal (M6P, syntaxin-13), or lysosomal (LAMP-1) proteins. Immunogold electron microscopy revealed that TLR9 was expressed instead in a novel intracellular electron-dense granule (T-granule) that is mostly distributed along the plasma membrane. Incubation of resting PLTs with synthetic unmethylated Type C CpG dinucleotides (characteristic of bacterial/viral DNA) resulted in increased TLR9 surface expression, followed by increased Type C CpG sequestration and CD62P surface expression over 20 minutes. Interestingly, when TLR9 surface expression was specifically upregulated by pre-incubating PLTs with type IV collagen, subsequent incubation with Type C CpG resulted in considerably increased Type C CpG sequestration, CD62P surface expression, and PLT aggregation within 30 seconds of addition. These results imply that PLTs must be primed to express TLR9 on their surface prior to signal transduction through TLR9, and provide a mechanism for PLT regulation of the immune response to infection in human whole blood by sequestering bacterial/viral DNA and marking themselves for clearance. Taken together, this paper; (1) tracks TLR9 to a new intracellular compartment in PLTs, (2) describes a novel mechanism of TLR9 signaling, and (3) reveals a unique role for human PLTs as mediators of innate immunity at sites of vascular damage. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes.

1986 ◽  
Vol 103 (1) ◽  
pp. 41-48 ◽  
Author(s):  
A S Ma ◽  
T T Sun
Keyword(s):  
Human Keratinocytes ◽  
Epidermal Differentiation ◽  
Immunofluorescent Staining ◽  
Soluble Form ◽  
Epidermal Keratinocytes ◽  
Cell Periphery ◽  
Human Epidermal Keratinocytes ◽  
Intracellular Location ◽  
Differentiated Cells ◽  
Insoluble Form

We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation.

scholarly journals Periplakin, a Novel Component of Cornified Envelopes and Desmosomes That Belongs to the Plakin Family and Forms Complexes with Envoplakin

1997 ◽  
Vol 139 (7) ◽  
pp. 1835-1849 ◽  
Author(s):  
Christiana Ruhrberg ◽  
M.A. Nasser Hajibagheri ◽  
David A.D. Parry ◽  
Fiona M. Watt
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Sequence Analysis ◽  
Cdna Sequence ◽  
Terminal Differentiation ◽  
Immunogold Electron Microscopy ◽  
Epidermal Keratinocytes ◽  
Cornified Envelope ◽  
Keratin Filaments

The cornified envelope is a layer of transglutaminase cross-linked protein that is assembled under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We have determined the cDNA sequence of one of the proteins that becomes incorporated into the cornified envelope of cultured epidermal keratinocytes, a protein with an apparent molecular mass of 195 kD that is encoded by a mRNA with an estimated size of 6.3 kb. The protein is expressed in keratinizing and nonkeratinizing stratified squamous epithelia and in a number of other epithelia. Expression of the protein is upregulated during the terminal differentiation of epidermal keratinocytes in vivo and in culture. Immunogold electron microscopy was used to demonstrate an association of the 195-kD protein with the desmosomal plaque and with keratin filaments in the differentiated layers of the epidermis. Sequence analysis showed that the 195-kD protein is a member of the plakin family of proteins, to which envoplakin, desmoplakin, bullous pemphigoid antigen 1, and plectin belong. Envoplakin and the 195-kD protein coimmunoprecipitate. Analysis of their rod domain sequences suggests that the formation of both homodimers and heterodimers would be energetically favorable. Confocal immunofluorescent microscopy of cultured epidermal keratinocytes revealed that envoplakin and the 195-kD protein form a network radiating from desmosomes, and we speculate that the two proteins may provide a scaffolding onto which the cornified envelope is assembled. We propose to name the 195-kD protein periplakin.

scholarly journals Calcium pump of the plasma membrane is localized in caveolae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1147-1157 ◽  
Author(s):  
T Fujimoto
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Epidermal Keratinocytes ◽  
Cardiac Muscle Cells ◽  
Mouse Tissues ◽  
Visceral Smooth Muscle

The Ca2+ pump in the plasma membrane plays a key role in the fine control of the cytoplasmic free Ca2+ concentration. In the present study, its subcellular localization was examined with immunocytochemical techniques using a specific antibody generated against the erythrocyte membrane Ca2+ pump ATPase. By immunofluorescence microscopy of cultured cells, the labeling with the antibody was seen as numerous small dots, often distributed in linear arrays or along cell edges. Immunogold EM of cryosections revealed that the dots correspond to caveolae, or smooth invaginations of the plasma membrane. The same technique applied to mouse tissues in vivo showed that the Ca2+ pump is similarly localized in caveolae of endothelial cells, smooth muscle cells, cardiac muscle cells, epidermal keratinocytes and mesothelial cells. By quantitative analysis of the immunogold labeling, the Ca2+ pump in capillary endothelial cells and visceral smooth muscle cells was found to be concentrated 18-25-fold in the caveolar membrane compared with the noncaveolar portion of the plasma membrane. In renal tubular and small intestinal epithelial cells, which have been known to contain the Ca2+ pump but do not have many caveolae, most of the labeling was randomly distributed in the basolateral plasma membrane, although caveolae were also positively labeled. The results demonstrate that the caveolae in various cells has the plasmalemmal Ca2+ pump as a common constituent. In conjunction with our recent finding that an inositol 1,4,5-trisphosphate receptor-like protein exists in the caveolae (Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. 1992. J. Cell Biol. 119:1507-1513), it is inferred that the smooth plasmalemmal invagination is an apparatus specialized for Ca2+ intake and extrusion from the cytoplasm.

scholarly journals Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2

2001 ◽  
Vol 12 (10) ◽  
pp. 3031-3045 ◽  
Author(s):  
Claudia A.O. Stuermer ◽  
Dirk M. Lang ◽  
Friederike Kirsch ◽  
Marianne Wiechers ◽  
Sören-Oliver Deininger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Cross Linking ◽  
Fyn Kinase ◽  
Glycosylphosphatidyl Inositol ◽  
Plasma Membrane Microdomains

Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in ≤0.1-μm plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after cross-linking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.

scholarly journals Relation of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte

1978 ◽  
Vol 76 (3) ◽  
pp. 705-711 ◽  
Author(s):  
Rh Rice ◽  
H Green
Keyword(s):  
Protein Synthesis ◽  
Suspension Culture ◽  
Average Rate ◽  
Large Fraction ◽  
Epidermal Keratinocytes ◽  
Cross Linking ◽  
Human Epidermal Keratinocytes ◽  
The Cross ◽  
Human Epidermal Keratinocyte ◽  
Cellular Ca

When serially cultivated human epidermal keratinocytes are placed in suspension culture they stop growing and form, beneath the plasma membrane, an insoluble envelope consisting of protein cross-linked by ε- (γ-glutamyl)lysine. The formation of envelopes in suspended cells is preceded by a sharp decline in the rate of protein synthesis, and most envelopes appear only after the average rate of protein synthesis has fallen to a very low level. If protein synthesis is reduced over 98 percent with cycloheximide or emetine at the time that surface-grown cells are placed in suspension culture, cross-linked envelopes form in most of the cells. This shows that the precursor of the envelope and the cross-linking enzyme are already in the cytoplasm in most cells of growing surface cultures. The process of envelope formation by suspension cultures is actually accelerated by the inhibitors of protein synthesis; an increased number of cells with cross-linked envelopes is observable within 4-6 h after the addition of cycloheximide. The inhibitor also induces a large fraction of the cells of surface cultures to form enveloped within a few days. These findings suggest that arrest of protein synthesis leads to activation of the cross-linking process. Agents known to inhibit transglutaminase-mediated protein cross-linking-putrescine, iodoacetamide, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA)- also prevent envelope formation. Though the activity of the cross-linking transglutaminase depends on the presence of cellular Ca++, we have not been able to activate the cross-linking process by high external Ca++ concentration or ionophores.

Patterns of spontaneous motility in videomicrographs of human epidermal keratinocytes (HEK)

1995 ◽  
Vol 73 (7-8) ◽  
pp. 441-459 ◽  
Author(s):  
Wolfgang Alt ◽  
Oana Brosteanu ◽  
Boris Hinz ◽  
Hans Wilhelm Kaiser
Keyword(s):  
Correlation Analysis ◽  
Statistical Correlation ◽  
Epidermal Keratinocytes ◽  
Cell Periphery ◽  
Actin Network ◽  
Human Epidermal Keratinocytes ◽  
Spontaneous Motility ◽  
Associated Proteins ◽  
Regulatory Effects ◽  
Kinetics Of

The subject of our observations was the spontaneous behaviour of normal and transfected human epidermal keratinocytes. Cell movements were recorded on video micrographs and analyzed by a mathematical approach, using new methods of image processing and statistical correlation analysis. Protrusive activity of single lamellae was examined using one-dimensional analysis of phase-contrast image sequences along section lines transversal to the cell edge. This method revealed high periodicity and correlation in the motility patterns of lamellae and ruffles. Two-dimensional correlation analysis of automatically digitized cell outlines was applied to detect spatiotemporal patterns and coordination of lamellar extension and retraction. Most cells showed regularly alternating pulsations of lamellar protrusions. In some extreme cases, extension waves rotating around the cell periphery were observed. The results were compared with computer simulations of two simple models for lamellar dynamics and shape deformation, based on few assumptions about chemical kinetics of F-actin and cytomechanical properties of the actin network, neglecting regulatory effects of actin-associated proteins or extracellular-stimulations. The simulation results reproduced the main dynamical features of the observed real cells, indicating the possibility that the basic universal mechanism for lateral coordination of lamellipodial protrusion is the interplay between hydrostatic pressure and viscocontractile tension in the cortical F-actin – plasma membrane complex.Key words: keratinocytes, cytomechanics, actin network, statistical correlation analysis, mathematical models.

SPRR4, a novel cornified envelope precursor: UV-dependent epidermal expression and selective incorporation into fragile envelopes

2001 ◽  
Vol 114 (21) ◽  
pp. 3837-3843
Author(s):  
Adriana Cabral ◽  
Arzu Sayin ◽  
Sandrine de Winter ◽  
David F. Fischer ◽  
Stan Pavel ◽  
...  
Keyword(s):  
Uv Light ◽  
Cell Envelope ◽  
Tissue Response ◽  
Cell Periphery ◽  
Normal Human Skin ◽  
Squamous Epithelia ◽  
Selective Incorporation ◽  
Epidermal Expression

The cornified cell envelope (CE), a structure formed in the outermost layers of stratified squamous epithelia, provides a physical barrier against environmental insults. It is composed of several structural proteins, which are irreversibly crosslinked by calcium-activated transglutaminases. The small proline rich proteins (SPRRs) are one set of CE precursors. SPRR4, a novel member of this gene family, displayed very low or undetectable expression levels in normal human skin or other stratified squamous epithelia, but was clearly induced by UV light both in vivo and in vitro. High epidermal expression of SPRR4 was monitored only after chronic UV exposure and was concomitant with a thickening of the stratum corneum, which is believed to provide protection against subsequent damage. The calcium-dependent translocation of an SPRR4-GFP fusion protein to the cell periphery in living keratinocytes and its integration into both rigid and fragile cornified envelopes proved that SPRR4 is a novel CE precursor. Interestingly, after UV irradiation, SPRR4 was selectively incorporated into fragile CEs. Our results show for the first time that UV-induced cornification is accompanied by qualitative changes in CE precursor assembly. SPRR4 is part of an adaptive tissue response to environmental stress, which is likely to compensate for UV induced impairment of the epidermal barrier function.

Sign in / Sign up

Export Citation Format

Share Document