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.

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 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 Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex.

1982 ◽  
Vol 2 (11) ◽  
pp. 1444-1458 ◽  
Author(s):  
E Kuismanen ◽  
K Hedman ◽  
J Saraste ◽  
R F Pettersson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Indirect Immunofluorescence ◽  
Golgi Complex ◽  
Nucleocapsid Protein ◽  
Double Staining ◽  
Virus Maturation ◽  
Virus Particles ◽  
Infected Cells ◽  
N Proteins

We studied the maturation of Uukuniemi virus and the localization of the viral surface glycoproteins and nucleocapsid protein in infected cells by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy with specific antisera prepared in rabbits against the two glycoproteins G1 and G2 and the nucleocapsid protein N. Electron microscopy of thin sections from infected cells showed virus particles maturing at smooth-surfaced membranes close to the nucleus. Localization of the G1/G2 and N proteins by indirect immunofluorescence at different stages after infection showed the antigens to be present throughout the cell interior but concentrated in the juxtanuclear region. The G1/G2 antiserum also appeared to stain the nuclear and plasma membranes. Double staining with tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, which preferentially stains the Golgi complex, and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, which stained the G1/G2 or N proteins, showed that the staining of the juxtanuclear region coincided. Similarly, double staining for thiamine pyrophosphatase, an enzyme activity specific for the Golgi complex, showed the fluorescence and the cytochemical stain to coincide in the juxtanuclear region. Immunoperoxidase electron microscopy of cells permeabilized with saponin revealed that the viral glycoproteins were present in the rough endoplasmic reticulum and the nuclear and Golgi membranes; the latter was heavily stained. With this method, the N protein was localized to the cytoplasm, especially around smooth-surfaced vesicles in the Golgi region. Taken together, the results indicate that Uukuniemi virus and its structural proteins accumulate in the Golgi complex, supporting the idea that this compartment rather than the plasma membrane is the site of virus maturation. This raises the interesting possibility that deficient transport of the glycoproteins to the plasma membrane and hence their accumulation in the Golgi complex determines the site of virus 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 Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail.

1995 ◽  
Vol 129 (3) ◽  
pp. 641-658 ◽  
Author(s):  
P M Haney ◽  
M A Levy ◽  
M S Strube ◽  
M Mueckler
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Glucose Transporter ◽  
Cytoplasmic Tail ◽  
Structural Basis ◽  
Immunogold Electron Microscopy ◽  
Subcellular Targeting ◽  
Intracellular Targeting ◽  
Necessary And Sufficient

The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2-6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH-terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin-sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin-sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.

scholarly journals Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p

2004 ◽  
Vol 167 (5) ◽  
pp. 889-901 ◽  
Author(s):  
Charles Boyd ◽  
Thom Hughes ◽  
Marc Pypaert ◽  
Peter Novick
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Immunogold Electron Microscopy ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Yeast Saccharomyces Cerevisiae ◽  
Family Protein ◽  
Photobleaching Recovery ◽  
Discrete Domains

Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex

1982 ◽  
Vol 2 (11) ◽  
pp. 1444-1458
Author(s):  
E Kuismanen ◽  
K Hedman ◽  
J Saraste ◽  
R F Pettersson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Indirect Immunofluorescence ◽  
Golgi Complex ◽  
Nucleocapsid Protein ◽  
Double Staining ◽  
Virus Maturation ◽  
Virus Particles ◽  
Infected Cells ◽  
N Proteins

We studied the maturation of Uukuniemi virus and the localization of the viral surface glycoproteins and nucleocapsid protein in infected cells by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy with specific antisera prepared in rabbits against the two glycoproteins G1 and G2 and the nucleocapsid protein N. Electron microscopy of thin sections from infected cells showed virus particles maturing at smooth-surfaced membranes close to the nucleus. Localization of the G1/G2 and N proteins by indirect immunofluorescence at different stages after infection showed the antigens to be present throughout the cell interior but concentrated in the juxtanuclear region. The G1/G2 antiserum also appeared to stain the nuclear and plasma membranes. Double staining with tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, which preferentially stains the Golgi complex, and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, which stained the G1/G2 or N proteins, showed that the staining of the juxtanuclear region coincided. Similarly, double staining for thiamine pyrophosphatase, an enzyme activity specific for the Golgi complex, showed the fluorescence and the cytochemical stain to coincide in the juxtanuclear region. Immunoperoxidase electron microscopy of cells permeabilized with saponin revealed that the viral glycoproteins were present in the rough endoplasmic reticulum and the nuclear and Golgi membranes; the latter was heavily stained. With this method, the N protein was localized to the cytoplasm, especially around smooth-surfaced vesicles in the Golgi region. Taken together, the results indicate that Uukuniemi virus and its structural proteins accumulate in the Golgi complex, supporting the idea that this compartment rather than the plasma membrane is the site of virus maturation. This raises the interesting possibility that deficient transport of the glycoproteins to the plasma membrane and hence their accumulation in the Golgi complex determines the site of virus maturation.

Uneven Distribution of Surface Antigens During Antigenic Variation in Paramecium Primaurelia

1989 ◽  
Vol 92 (2) ◽  
pp. 205-215
Author(s):  
CLAUDE ANTONY ◽  
YVONNE CAPDEVILLE
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Antigenic Variation ◽  
Temperature Shift ◽  
Surface Antigens ◽  
Immunogold Electron Microscopy ◽  
Membrane Domains ◽  
Phosphatidylinositol Phospholipase C

In Paramecium primaurelia surface antigen (SAg) expression can be experimentally controlled by temperature-shift-induced antigenic variation. As only one SAg is usually expressed at the cell surface under stable environmental conditions, we used the temperature-shift-induced change in SAg to follow the newly expressed antigen and the disappearing one, by both immunofluorescence and immunogold electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary and interciliary membrane domains, without any readily identifiable specific site of insertion into the plasma membrane. The concentration of the newly incorporated molecules then increased gradually on the plasma membrane. In contrast, the surface of the previously expressed SAg was not complementary to the pattern of the appearing SAg. The loss of the old SAg is delayed after the temperature shift and seems to occur more suddenly the appearance of new SAg. This loss is characterized by a subpopulation of cilia bearing old SAg coexisting with other cilia and a pellicle almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is discussed in relation to the lipidic nature of the SAg anchor and to a possible role of an Paramecium phosphatidylinositol phospholipase C.

scholarly journals Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii.

1995 ◽  
Vol 130 (3) ◽  
pp. 591-603 ◽  
Author(s):  
I C Baines ◽  
A Corigliano-Murphy ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Plasma Membranes ◽  
Phosphorylation Site ◽  
Acanthamoeba Castellanii ◽  
Single Site ◽  
Specific Antibodies ◽  
Large Vacuole ◽  
Myosin I ◽  
Phagocytic Cups

The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.

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