scholarly journals Brain postsynaptic densities: the relationship to glial and neuronal filaments.

1980 ◽  
Vol 87 (2) ◽  
pp. 346-359 ◽  
Author(s):  
A Matus ◽  
G Pehling ◽  
M Ackermann ◽  
J Maeder
Keyword(s):  
Brain Tissue ◽  
Intermediate Filaments ◽  
Plasma Membranes ◽  
Subcellular Fractionation ◽  
Immunohistochemical Localization ◽  
Immunoperoxidase Staining ◽  
Protein Antigens ◽  
Synaptic Plasma Membranes ◽  
Intermediate Filament Proteins ◽  
Fractionation Schedule

Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of approximately 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F. Blomberg et al., 1977, J. Cell Biol., 74:214-225), we searched for evidence of neurofilament proteins among the PSD polypeptides. This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests. As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons. The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons. However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins. This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants. To test this possibility, the proteins of isolated brain intermediate filaments were labeled with 125I and added to brain tissue at the start of a subcellular fractionation schedule. The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization of PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence. Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.

scholarly journals Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization.

1982 ◽  
Vol 92 (3) ◽  
pp. 665-673 ◽  
Author(s):  
E B Lane
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Cell Line ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Antigenic Determinant ◽  
Protein Antigens ◽  
Molecular Weight Range ◽  
Intermediate Filament Proteins ◽  
Molecular Weights

The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation.

scholarly journals Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

1997 ◽  
Vol 138 (6) ◽  
pp. 1379-1394 ◽  
Author(s):  
Carlos Caulín ◽  
Guy S. Salvesen ◽  
Robert G. Oshima
Keyword(s):  
Intermediate Filaments ◽  
Cleavage Site ◽  
Uv Light ◽  
Tail Domain ◽  
Proteolytic Fragment ◽  
Intermediate Filament Proteins ◽  
Caspase Cleavage ◽  
Structural Cell ◽  
Caspase 6

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.

Sodium affinity of brain Na(+)-K(+)-ATPase is dependent on isozyme and environment of the pump

1990 ◽  
Vol 258 (5) ◽  
pp. C803-C811 ◽  
Author(s):  
J. L. Brodsky ◽  
G. Guidotti
Keyword(s):  
Plasma Membranes ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Synaptic Plasma Membranes ◽  
Apparent Dissociation Constant ◽  
Low Sodium ◽  
Physiological Relevance ◽  
Mouse Fibroblast Cell Line

The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.

The Role of Phosphoprotein B-50 in Phosphoinositide Metabolism in Brain Synaptic Plasma Membranes

1985 ◽  
pp. 399-414 ◽  
Author(s):  
W. H. Gispen ◽  
C. J. Van Dongen ◽  
P. N. E. De Graan ◽  
A. B. Oestreicher ◽  
H. Zwiers
Keyword(s):  
Plasma Membranes ◽  
Phosphoinositide Metabolism ◽  
Synaptic Plasma Membranes

scholarly journals Filaments and phenotypes: cellular roles and orphan effects associated with mutations in cytoplasmic intermediate filament proteins

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1703 ◽  
Author(s):  
Michael W. Klymkowsky
Keyword(s):  
Intermediate Filaments ◽  
Molecular Chaperones ◽  
High Probability ◽  
Essential Role ◽  
Loss Of Function ◽  
Intermediate Filament Proteins ◽  
Over Expression ◽  
Per Se ◽  
Metabolic Stresses ◽  
Encoding Genes

Cytoplasmic intermediate filaments (IFs) surround the nucleus and are often anchored at membrane sites to form effectively transcellular networks. Mutations in IF proteins (IFps) have revealed mechanical roles in epidermis, muscle, liver, and neurons. At the same time, there have been phenotypic surprises, illustrated by the ability to generate viable and fertile mice null for a number of IFp-encoding genes, including vimentin. Yet in humans, the vimentin (VIM) gene displays a high probability of intolerance to loss-of-function mutations, indicating an essential role. A number of subtle and not so subtle IF-associated phenotypes have been identified, often linked to mechanical or metabolic stresses, some of which have been found to be ameliorated by the over-expression of molecular chaperones, suggesting that such phenotypes arise from what might be termed “orphan” effects as opposed to the absence of the IF network per se, an idea originally suggested by Toivola et al. and Pekny and Lane.

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