Ultrastructure of the membrane attachment sites of the extrusomes of Ciliophrys marina and Heterophrys marina (Actinopoda)

1976 ◽  
Vol 170 (3) ◽  
pp. 353-365 ◽  
Author(s):  
Lloyd A. Davidson
Keyword(s):  
Attachment Sites ◽  
Membrane Attachment

scholarly journals Membrane Specializations in the Form of Regular Membrane-to-Membrane Attachment Sites in Paramecium. A Correlated Freeze-Etching and Ultrathin-Sectioning Analysis

1973 ◽  
Vol 13 (3) ◽  
pp. 687-719
Author(s):  
H. PLATTNER ◽  
F. MILLER ◽  
L. BACHMANN
Keyword(s):  
Membrane Complex ◽  
Type D ◽  
Attachment Sites ◽  
Morphometric Analyses ◽  
Concentric Zone ◽  
Membrane Attachment ◽  
Ciliary Necklaces ◽  
Freeze Etching ◽  
Membrane Bound ◽  
Membrane Type

The outer membrane complex of Paramecium was investigated by ultrathin-sectioning techniques and by freeze-etching of unfixed cells without cryoprotectants. Granules were found in the freeze-etched cortical membrane complex in a highly ordered arrangement; morphometric analyses and partial disruption of this membrane complex showed that some of these granular membrane specializations (which we call types a, b, e, f, g) represent membrane-to-membrane attachment sites. Type a: granules arranged in rings (single or double), 300 nm in diameter, connect plasmalemma and alveolar membranes around trichocysts. Type b: within these a-type rings, a concentric zone (180 nm diameter) of granules connects plasma membrane and trichocyst membrane. Type c: the trichocyst membrane further contains a central ring or patch, 80 nm in diameter, of rather large granules where it is in contact with the crystalline trichocyst matrix. Type d: ‘ciliary necklaces’ are formed by groups of triple rows of 3-6, most frequently 5 granules, with 21-nm periodicity. Rows of granules also connect the alveolar membranes to the apical portion of the trichocyst membrane (type e) and the alveolar membrane to the plasmalemma around cilia (type f). Type g: the inner alveolar membrane displays an intense granularity and contains double rows of granules along alveolar septa at attachment sites between 2 alveolar membranes. Upon experimental discharge of the majority of trichocysts only the innermost concentric circles of membrane-bound granules of this region (b- and c-type) disappeared from the plasmalemma, while the outermost a-type rings of granules persisted for a longer time. Among other possible functions, these regular membrane-to-membrane attachments are likely to maintain the specific cellular shape.

scholarly journals An integral glycoprotein associated with the membrane attachment sites of actin microfilaments.

1985 ◽  
Vol 101 (3) ◽  
pp. 785-801 ◽  
Author(s):  
A A Rogalski ◽  
S J Singer
Keyword(s):  
Smooth Muscle ◽  
Chick Embryo ◽  
Molecular Mass ◽  
Apparent Molecular Mass ◽  
Reduced Form ◽  
Actin Microfilaments ◽  
Attachment Sites ◽  
Sds Page ◽  
Membrane Attachment ◽  
Embryo Fibroblasts

An integral membrane protein associated with sites of microfilament-membrane attachment has been identified by a newly developed IgG1 monoclonal antibody. This antibody, MAb 30B6, was derived from hybridoma fusion experiments using intact mitotic cells of chick embryo fibroblasts as the immunization vehicle as well as the screening probe for cell surface antigens. In immunofluorescent experiments with fixed cells, MAb 30B6 surface labeling is uniquely correlated with microfilament distributions in the cleavage furrow region of dividing chick embryo fibroblasts and cardiac myocytes in culture. The MAb 30B6 antigen in addition is associated with microfilament-membrane attachment sites in interphase fibroblasts at the dorsal surface, the adhesion plaque region at the ventral surface, and at junction-like regions of cell-cell contact. It is also found co-localized with the membrane-dense plaques of smooth muscle. The MAb 30B6 antigen is expressed in a wide number of chicken cell types (particularly smooth muscle cells, platelets, and endothelial cells), but not in erythrocytes. Some of the molecular characteristics of the MAb 30B6 antigen have been determined from immunoblotting, immunoaffinity chromatography, immunoprecipitation, cell extraction, and charge shift electrophoresis experiments. It is an integral sialoglycoprotein with an apparent molecular mass of 130 kD (reduced form)/107 kD (nonreduced form) in SDS PAGE. Another prominent glycoprotein species with an apparent molecular mass of 175 kD (reduced form)/165 kD (nonreduced form) in SDS PAGE is co-isolated on MAb 30B6 affinity columns, but appears to be antigenically distinct since it is not recognized by MAb 30B6 in immunoblotting or immunoprecipitation experiments. By virtue of its surface distributions relative to actin microfilaments and its integral protein character, we propose that the MAb 30B6 antigen is an excellent candidate for the function of directly or indirectly anchoring microfilaments to the membrane.

An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain

1988 ◽  
Vol 8 (2) ◽  
pp. 564-570
Author(s):  
P A Maher ◽  
S J Singer
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Protein Complex ◽  
Apparent Molecular Weight ◽  
Integral Membrane Protein ◽  
Beta Chain ◽  
Attachment Sites ◽  
Wide Range ◽  
Membrane Attachment

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

scholarly journals Quantitation of the number of molecules of glycophorins C and D on normal red blood cells using radioiodinated Fab fragments of monoclonal antibodies

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1668-1672 ◽  
Author(s):  
J Smythe ◽  
B Gardner ◽  
DJ Anstee
Keyword(s):  
Monoclonal Antibodies ◽  
Blood Cells ◽  
Binding Assays ◽  
Membrane Glycoproteins ◽  
Human Erythrocyte Membrane ◽  
Protein 4.1 ◽  
Fab Fragments ◽  
Attachment Sites ◽  
Membrane Attachment ◽  
Glycophorin C

Abstract Two rat monoclonal antibodies (BRAC 1 and BRAC 11) have been produced. BRAC 1 recognizes an epitope common to the human erythrocyte membrane glycoproteins glycophorin C (GPC) and glycophorin D (GPD). BRAC 11 is specific for GPC. Fab fragments of these antibodies and BRIC 10, a murine monoclonal anti-GPC, were radioiodinated and used in quantitative binding assays to measure the number of GPC and GPD molecules on normal erythrocytes. Fab fragments of BRAC 11 and BRIC 10 gave values of 143,000 molecules GPC per red blood cell (RBC). Fab fragments of BRAC 1 gave 225,000 molecules of GPC and GPD per RBC. These results indicate that GPC and GPD together are sufficiently abundant to provide membrane attachment sites for all of the protein 4.1 in normal RBCs.

scholarly journals Structure of myofibrils at extra-junctional membrane attachment sites in cultured cardiac muscle cells

1988 ◽  
Vol 89 (1) ◽  
pp. 97-106
Author(s):  
B.T. Atherton ◽  
M.M. Behnke
Keyword(s):  
Cell Membrane ◽  
Cardiac Muscle ◽  
Muscle Cells ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Cardiac Muscle Cells ◽  
Lateral Contact ◽  
Attachment Sites ◽  
Membrane Attachment ◽  
Actin Cables

The composition and organization of myofibrils at extra-junctional membrane attachment sites in cultured neonatal rat cardiac muscle cells were analysed by immunofluorescence and electron microscopy. When myofibril terminals attached to the cell membrane via focal contacts at regions of the sarcolemma that lacked intercalated discs, they appeared to be non-striated and resembled thick actin cables. Although the non-striated terminals contained actin, myosin and alpha-actinin, the proteins were not organized into recognizable sarcomeres at the light microscopic level. Analysis of the structure of the terminals in the electron microscope confirmed that the usual sarcomeric organization and attachments to the sarcolemma were markedly modified. The non-striated myofibril terminals differed in structure from both stress fibres in non-muscle cells and stress fibre-like structures present in embryonic heart cells in culture. Non-striated myofibril terminals attached to the cell membrane by lateral contact with extra-junctional electron-dense membrane plaques rather than by insertion by their ends into the fascia adherens. It is proposed that the structure and composition of membrane-attachment points for myofibrils may have an influence on the structure, organization or stability of contractile elements in cardiac muscle.

scholarly journals A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment.

1987 ◽  
Vol 105 (2) ◽  
pp. 819-831 ◽  
Author(s):  
A A Rogalski
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cardiac Muscle ◽  
Cultured Cells ◽  
Molecular Forms ◽  
Cell Coupling ◽  
Membrane Structures ◽  
Adult Chicken ◽  
Attachment Sites ◽  
Membrane Attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.

scholarly journals Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches.

1988 ◽  
Vol 36 (3) ◽  
pp. 297-306 ◽  
Author(s):  
S C Mueller ◽  
T Hasegawa ◽  
S S Yamada ◽  
K M Yamada ◽  
W T Chen
Keyword(s):  
Plasma Membrane ◽  
Band 3 ◽  
Cell Contact ◽  
Positive Staining ◽  
Double Labeling ◽  
Intact Cells ◽  
Fibronectin Receptor ◽  
Contact Sites ◽  
Attachment Sites ◽  
Membrane Attachment

The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co-localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti-fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.

ALTERED ABUNDANCE OF MESSENGER RNA TRANSCRIPTS WHOSE PRODUCTS HAVE MEMBRANE ATTACHMENT SITES IN PREGNANCY-INDUCED HYPERTENSION

2002 ◽  
Vol 10 (04) ◽  
pp. 447-461 ◽  
Author(s):  
GREGORY I. C. SIMPSON ◽  
LESLIE C. SHARKEY ◽  
JOHN FRAY
Keyword(s):  
Gene Expression ◽  
Oral Administration ◽  
Messenger Rna ◽  
Gene Products ◽  
Microarray Technology ◽  
Rna Transcripts ◽  
Attachment Sites ◽  
Glycosylation Sites ◽  
Induced Hypertension ◽  
Membrane Attachment

Pregnancy-induced hypertensive disorders (PIH) are leading causes of maternal mortality. Although the mechanism responsible for initiating and maintaining the disorder is unproven, physiologic molecular attachments in kidney and placenta play a role. The SHHF/Mcc-facp (SHHF) rat has features of the disorder, including abnormal placenta gene expression. To gain a molecular understanding of the gene expression profile associated with PIH, kidneys and placentas of SHHF rats at gestation day 20 were compared to WKY controls using microarray technology. We report that SHHF rats have spontaneous PIH, elevated total placenta weights, and reduced total pup weights than WKY controls and that they also have greater total number of mRNA transcripts expressed in placenta. Kidneys of SHHF rats, on the other hand, not only expressed disproportionately more predicted gene products with attachment sites such as RGD motifs, N-glycosylation sites, and N-myristoylation sites they also responded more profoundly to oral administration of L-arginine. We conclude that the increased abundance of transcripts whose products engage in posttranslational attachments using RGD motifs, N-glycosylation sites, and N-myristoylation sites and the reversal of these increases by oral administration of L-arginine suggests that NO may be of importance in PIH at the level of molecular attachments.

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