scholarly journals Co-segregation of AMPA receptors with GM1 ganglioside in synaptosomal membrane subfractions

2010 ◽  
Vol 427 (3) ◽  
pp. 535-540 ◽  
Author(s):  
Andy A. Cole ◽  
Ayse Dosemeci ◽  
Thomas S. Reese
Keyword(s):  
Electron Microscopy ◽  
Lipid Rafts ◽  
Ampa Receptors ◽  
Synaptic Proteins ◽  
Immunogold Electron Microscopy ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Similar Distribution ◽  
Gm1 Ganglioside ◽  
Synaptosomal Membrane

Biochemical studies have suggested that certain synaptic proteins associate with lipid rafts to perform key functions within the synapse. However, variability in biochemical preparations raises questions as to which synaptic proteins actually associate with lipid rafts. In the present study, we use both electron microscopy and biochemistry to investigate AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor localization in synaptic membrane subfractions prepared in two different ways, by Triton X-100 detergent treatment or without detergent by sonication at high pH. Immunogold electron microscopy shows that a detergent-resistant synaptosomal membrane subfraction consists of empty vesicles 0.1–1.0 μm in diameter. A subpopulation of these vesicles labelled for glycosphingolipid GM1 ganglioside, a marker of lipid rafts, and 46% of the labelled vesicles also labelled for the AMPA receptor subunit GluR2. This co-segregation into specific vesicles does not depend on effects of detergent because a similar distribution of label was found in vesicles isolated without the use of detergent. Our results suggest that AMPA receptors localize within specific regions of synaptic membranes rich in GM1 ganglioside.

scholarly journals Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses

2020 ◽  
Vol 21 (18) ◽  
pp. 6737
Author(s):  
David Kleindienst ◽  
Jacqueline Montanaro ◽  
Pradeep Bhandari ◽  
Matthew J. Case ◽  
Yugo Fukazawa ◽  
...  
Keyword(s):  
Deep Learning ◽  
Calcium Channels ◽  
Calcium Channel ◽  
Nmda Receptors ◽  
Ampa Receptors ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Synaptic Proteins ◽  
Immunogold Electron Microscopy ◽  
Zone Model

The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography.

scholarly journals The GM1 Ganglioside Forms GM1-Rich Gel Phase Microdomains within Lipid Rafts

Coatings ◽  
2014 ◽  
Vol 4 (3) ◽  
pp. 450-464 ◽  
Author(s):  
Lucia Becucci ◽  
Francesco Vizza ◽  
Yolanda Duarte ◽  
Rolando Guidelli
Keyword(s):  
Lipid Rafts ◽  
Gm1 Ganglioside ◽  
Gel Phase

Immunogold electron microscopy of surface antigens of oral bacteria

1984 ◽  
Vol 30 (8) ◽  
pp. 1008-1013 ◽  
Author(s):  
C. Mouton ◽  
L. Lamonde
Keyword(s):  
Electron Microscopy ◽  
Surface Antigens ◽  
Oral Bacteria ◽  
Immunogold Electron Microscopy ◽  
Bacterial Surface ◽  
Outer Aspect ◽  
Bacteroides Gingivalis ◽  
Cell Envelopes ◽  
Inexpensive Procedure ◽  
Monospecific Antibodies

Colloidal gold particles 3–6 nm in diameter were prepared and stabilized with the IgG fraction of polyspecific rabbit antisera produced against four different oral bacteria. The immunogold markers were used in homologous reactions to label the bacteria in a preembedding procedure for electron microscopy. An indirect immunofluorescence procedure was concurrently used to optimize the labelling conditions before observation with the electron microscope. The immunogold markers labelled fibrillar structures extending outward 50–275 nm from the Gram-positive cell envelopes and a fuzzy 5–10 nm thick capsulelike layer on the outer aspect of Bacteroides gingivalis. The immunogold method appears to be a simple, rapid, and inexpensive procedure suitable for the study of bacterial surface antigens and can be upgraded with the use of monospecific antibodies.

scholarly journals Subcellular Distribution and Relative Expression of Fibrocyte Markers in the CD/1 Mouse Cochlea Assessed by Semiquantitative Immunogold Electron Microscopy

2011 ◽  
Vol 59 (11) ◽  
pp. 984-1000 ◽  
Author(s):  
Shanthini Mahendrasingam ◽  
Catherine Bebb ◽  
Ella Shepard ◽  
David N. Furness
Keyword(s):  
Electron Microscopy ◽  
Tissue Growth ◽  
Immunogold Electron Microscopy ◽  
Spiral Ligament ◽  
Type Ii ◽  
Relative Expression ◽  
Aquaporin 1 ◽  
Cellular Transplantation ◽  
S 100 ◽  
Type V

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Resinless section immunogold electron microscopy of karyo-cytoskeletal frameworks of eukaryotic cells cultured in vitro. Absence of a salt-stable nuclear matrix from mouse plasmacytoma MPC-11 cells

1991 ◽  
Vol 98 (1) ◽  
pp. 107-122
Author(s):  
X. Wang ◽  
P. Traub
Keyword(s):  
Electron Microscopy ◽  
Nuclear Matrix ◽  
Protein A ◽  
Mouse Skin ◽  
Nuclear Lamina ◽  
Immunogold Electron Microscopy ◽  
Cell Structures ◽  
Critical Point Drying ◽  
Cells Cultured

The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

Molecular Hybridization of Mouse Satellite DNA-Complementary RNA in Ultrathin Sections Prepared for Electron Microscopy

1974 ◽  
Vol 14 (2) ◽  
pp. 253-261
Author(s):  
J. JACOB ◽  
KATHERINE GILLIES ◽  
D. MACLEOD ◽  
K. W. JONES
Keyword(s):  
Electron Microscopy ◽  
Satellite Dna ◽  
Similar Distribution ◽  
Tissue Sections ◽  
Satellite Sequences ◽  
Interphase Cells ◽  
Nucleolar Chromatin ◽  
Ultrathin Sections ◽  
Mouse Satellite Dna

The feasibility of in situ hybridization in tissue sections prepared for electron microscopy has been examined using mouse satellite DNA-complementary RNA and mouse L cells. The results obtained are encouraging, although certain technical aspects require further clarification. In interphase cells, hybrid-forming sites occur in chromatin patches positioned along the nuclear envelope. It is also confirmed that satellite DNA occurs in nucleolus-associated chromatin. The results suggest that satellite sequences are present in intranucleolar and peri-nucleolar chromatin. A similar distribution is indicated for ribosomal cistrons.

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