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 Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin.

1996 ◽  
Vol 134 (3) ◽  
pp. 715-729 ◽  
Author(s):  
C Ruhrberg ◽  
M A Hajibagheri ◽  
M Simon ◽  
T P Dooley ◽  
F M Watt
Keyword(s):  
Plasma Membrane ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Single Copy ◽  
Epidermal Keratinocytes ◽  
Single Copy Gene ◽  
Cornified Envelope ◽  
Keratin Filaments ◽  
Copy Gene

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."

Species-specific difference in distribution of voltage-gated L-type Ca2+ channels of cardiac myocytes

2000 ◽  
Vol 279 (6) ◽  
pp. C1963-C1969 ◽  
Author(s):  
Yoshiko Takagishi ◽  
Kenji Yasui ◽  
Nicholas J. Severs ◽  
Yoshiharu Murata
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cardiac Myocytes ◽  
Immunogold Electron Microscopy ◽  
Surface Plasma ◽  
Gold Particles ◽  
Intracellular Ca ◽  
Rat Myocytes ◽  
T Tubules ◽  
Species Specific

Ca2+influx via sarcolemmal voltage-dependent Ca2+ channels (L-type Ca2+ channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca2+ influx and intracellular Ca2+ release to activation of contraction. We investigated the distribution of L-type Ca2+ channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca2+ via surface sarcolemmal Ca2+ channels contributes to intracellular Ca2+ to a greater degree in rabbit than in rat myocytes.

scholarly journals Laccase Expression in Murine Pulmonary Cryptococcus neoformans Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 3124-3127 ◽  
Author(s):  
Javier Garcia-Rivera ◽  
Stephanie C. Tucker ◽  
Marta Feldmesser ◽  
Peter R. Williamson ◽  
Arturo Casadevall
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Lung Tissue ◽  
Immunogold Electron Microscopy ◽  
Cell Cytoplasm ◽  
Fungal Cell ◽  
Time Of Infection

ABSTRACT Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation

2007 ◽  
Vol 56 (3) ◽  
pp. 253-265 ◽  
Author(s):  
Miho Oyasu ◽  
Mineko Fujimiya ◽  
Kaori Kashiwagi ◽  
Shiho Ohmori ◽  
Hirotsugu Imaeda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Subcellular Targeting ◽  
Electron Microscopic ◽  
Proper Distance

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation.

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 Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy.

1990 ◽  
Vol 111 (5) ◽  
pp. 1895-1904 ◽  
Author(s):  
I C Baines ◽  
E D Korn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Indirect Immunofluorescence ◽  
Synthetic Peptide ◽  
Myosin Ii ◽  
Phosphorylation Site ◽  
Acanthamoeba Castellanii ◽  
Immunogold Electron Microscopy ◽  
Membrane Phospholipids ◽  
Immunogold Cytochemistry

Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytoplasm and appeared to be concentrated in the cortex. Immunogold cytochemistry revealed at high resolution that myosin II is organized into rodlike filaments approximately 200 nm long. The antibody raised against the myosin IC synthetic peptide recognized both the plasma membrane and the membrane of the contractile vacuole. The plasma membrane staining was labile to treatment with saponin suggesting an intimate association of the myosin IC with membrane phospholipids. Immunogold cytochemistry with the antimyosin IC synthetic peptide showed that the myosin IC is closely associated with the membrane bilayer.

Immunoelectronmicroscopic imaging of spermadhesin AWN epitopes on boar spermatozoa bound in vivo to the zona pellucida

1998 ◽  
Vol 10 (6) ◽  
pp. 491 ◽  
Author(s):  
H. Rodríguez-Martínez ◽  
A. Iborra ◽  
P. Martínez ◽  
J. J. Calvete
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Plasma Membrane ◽  
Zona Pellucida ◽  
Genital Tract ◽  
Major Protein ◽  
Sperm Surface ◽  
Boar Spermatozoa ◽  
Scanning Electron

Spermadhesin AWN is a major protein of boar seminal plasma and a sperm surface-associated lectin. AWN binds to β-galactosides and to porcine zona pellucida glycoproteins, suggesting a role for this protein in primary gamete interaction. However, because capacitation induces remodelling of the sperm surface and AWN is peripherally bound to the plasma membrane, the present study sought to investigate whether AWN is present or absent in the subpopulation of spermatozoa that reaches the ovulated oocyte at the period of fertilization in vivo. Therefore, tubal tissues and oocytes from sows mated with a fertile boar were collected 6–8 h after ovulation. Tissues and oocyte–sperm complexes were fixed, immunolabelled with anti-AWN monoclonal antibodies, and examined by means of light and scanning electron microscopy. The results show that spermadhesin AWN is present in spermatozoa seen along the genital tract of the natural mated sow as well as on plasmalemmal remnants of spermatozoa bound to the zona pellucida in vivo.

scholarly journals Correlative Light-Electron Microscopy Reveals the Tubular-Saccular Ultrastructure of Carriers Operating between Golgi Apparatus and Plasma Membrane

2000 ◽  
Vol 148 (1) ◽  
pp. 45-58 ◽  
Author(s):  
Roman S. Polishchuk ◽  
Elena V. Polishchuk ◽  
Pierfrancesco Marra ◽  
Saverio Alberti ◽  
Roberto Buccione ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Molecular Organization ◽  
Maximum Diameter ◽  
Intracellular Traffic ◽  
Fusion Site ◽  
Green Fluorescent

Transport intermediates (TIs) have a central role in intracellular traffic, and much effort has been directed towards defining their molecular organization. Unfortunately, major uncertainties remain regarding their true structure in living cells. To address this question, we have developed an approach based on the combination of the green fluorescent protein technology and correlative light-electron microscopy, by which it is possible to monitor an individual carrier in vivo and then take a picture of its ultrastructure at any moment of its lifecycle. We have applied this technique to define the structure of TIs operating from the Golgi apparatus to the plasma membrane, whose in vivo dynamics have been characterized recently by light microscopy. We find that these carriers are large (ranging from 0.3–1.7 μm in maximum diameter, nearly half the size of a Golgi cisterna), comprise almost exclusively tubular-saccular structures, and fuse directly with the plasma membrane, sometimes minutes after docking to the fusion site.

scholarly journals Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae.

1992 ◽  
Vol 119 (6) ◽  
pp. 1507-1513 ◽  
Author(s):  
T Fujimoto ◽  
S Nakade ◽  
A Miyawaki ◽  
K Mikoshiba ◽  
K Ogawa
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Immunogold Electron Microscopy ◽  
Type I ◽  
Agarose Beads ◽  
Inositol 1,4,5 Trisphosphate ◽  
Tissue Specimens ◽  
Cerebellar Type ◽  
Insp3 Receptor

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.

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