scholarly journals Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies.

1994 ◽  
Vol 42 (11) ◽  
pp. 1499-1502 ◽  
Author(s):  
B P Hierck ◽  
L V Iperen ◽  
A C Gittenberger-De Groot ◽  
R E Poelmann
Keyword(s):  
Monoclonal Antibodies ◽  
Striated Muscle ◽  
Indirect Detection ◽  
Mouse Serum ◽  
Intermediate Filament Protein ◽  
Background Staining ◽  
Secondary Antiserum ◽  
Immunohistochemical Studies ◽  
Filament Protein

The indirect immunodetection method is powerful in detecting antigens in situ, but to date mouse monoclonal antibodies (MAbs) could not be used in immunohistochemical studies of murine tissues without severe background staining. We report here a modification of this method in which mouse MAbs are used to detect murine antigens in cryosections. Before application to the section, mouse MAbs and conjugated anti-mouse antiserum were allowed to complex in vitro. After blocking of the unbound secondary antiserum with normal mouse serum, standard immunohistochemistry was performed. Fifty percent of a randomly chosen panel of over 40 mouse MAbs recognized their antigens in our model system. Adaptation of, for example, the fixation protocol can probably even increase this number. An MAb to the intermediate filament protein desmin, staining both smooth and striated muscle, was used to demonstrate this technique in cryosections of 15-day-old mouse embryos. In contrast to standard immunohistochemistry with the same antibodies under the same conditions, background staining was completely absent with this technique. With this modification to the well-established indirect detection method, the usefulness of mouse MAbs is significantly increased.

scholarly journals Purification of desmin from adult mammalian skeletal muscle

1981 ◽  
Vol 195 (2) ◽  
pp. 345-356 ◽  
Author(s):  
J M O'Shea ◽  
R M Robson ◽  
M K Hartzer ◽  
T W Huiatt ◽  
W E Rathbun ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Average Diameter ◽  
Intermediate Filament Protein ◽  
Mammalian Skeletal Muscle ◽  
Cytoskeletal Function ◽  
10 Nm Filaments ◽  
And Function ◽  
Filament Protein

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.

scholarly journals Desmin-related cardiomyopathy: an unfolding story

2011 ◽  
Vol 301 (4) ◽  
pp. H1220-H1228 ◽  
Author(s):  
Patrick M. McLendon ◽  
Jeffrey Robbins
Keyword(s):  
Protein Aggregation ◽  
Striated Muscle ◽  
Intermediate Filament Protein ◽  
Cytoskeletal Organization ◽  
Sarcoplasmic Protein ◽  
Amyloid Oligomers ◽  
Abnormal Metabolism ◽  
Associated Proteins ◽  
Cellular Phenotypes ◽  
Filament Protein

The intermediate filament protein desmin is an integral component of the cardiomyocyte and serves to maintain the overall structure and cytoskeletal organization within striated muscle cells. Desmin-related myopathy can be caused by mutations in desmin or associated proteins, which leads to intracellular accumulation of misfolded protein and production of soluble pre-amyloid oligomers, which leads to weakened skeletal and cardiac muscle. In this review, we examine the cellular phenotypes in relevant animal models of desmin-related cardiomyopathy. These models display characteristic sarcoplasmic protein aggregates. Aberrant protein aggregation leads to mitochondrial dysfunction, abnormal metabolism, and altered cardiomyocyte structure. These deficits to cardiomyocyte function may stem from impaired cellular proteolytic mechanisms. The data obtained from these models allow a more complete picture of the pathology in desmin-related cardiomyopathy to be described. Moreover, these studies highlight the importance of desmin in maintaining cardiomyocyte structure and illustrate how disrupting this network can be deleterious to the heart. We emphasize the similarities observed between desmin-related cardiomyopathy and other protein conformational disorders and speculate that therapies to treat this disease may be broadly applicable to diverse protein aggregation-based disorders.

scholarly journals A Small Vimentin-Binding Molecule Blocks Cancer Exosome Release and Reduces Cancer Cell Mobility

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianping Wu ◽  
Qian Xie ◽  
Yanjun Liu ◽  
Yanan Gao ◽  
Zhipeng Qu ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Cell ◽  
Migration And Invasion ◽  
Intermediate Filament Protein ◽  
Cancer Cell Growth ◽  
Potential Applications ◽  
Health And Disease ◽  
Quantitative Analyses ◽  
Filament Protein

Vimentin is an intermediate filament protein with diverse roles in health and disease far beyond its structural functions. Exosomes or small extracellular vesicles (sEVs) are key mediators for intercellular communication, contributing to tissue homeostasis and the progression of various diseases, especially the metastasis of cancers. In this study, we evaluated a novel vimentin-binding compound (R491) for its anti-cancer activities and its roles in cancer exosome release. The compound R491 induced a rapid and reversible intracellular vacuolization in various types of cancer cells. This phenotype did not result in an inhibition of cancer cell growth, which was consistent with our finding from a protein array that R491 did not reduce levels of major oncoproteins in cancer cells. Morphological and quantitative analyses on the intracellular vacuoles and extracellular exosomes revealed that in response to R491 treatment, the exosomes released from the cells were significantly reduced, while the exosomes retained as intra-luminal vesicles inside the cells were subsequently degraded. Vim+/− cells had lower amounts of vimentin and accordingly, lower amounts of both the retained and the released exosomes than Vim+/+ cells had, while the vimentin-binding compound R491 inhibited only the release of exosomes. Further functional tests showed that R491 significantly reduced the migration and invasion of cancer cells in vitro and decreased the amount of exosome in the blood in mice. Our study suggests that vimentin promotes exosome release, and small-molecule compounds that target vimentin are able to both block cancer exosome release and reduce cancer cell motility, and therefore could have potential applications for inhibiting cancer invasive growth.

scholarly journals The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues

2021 ◽  
Author(s):  
Marina Spoerrer ◽  
Delf Kah ◽  
Richard C Gerum ◽  
Barbara Reischl ◽  
Danyil Huraskin ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Contractile Function ◽  
Three Dimensional ◽  
Mechanical Damage ◽  
Underlying Mechanism ◽  
Intermediate Filament Protein ◽  
Wild Type ◽  
Age Related ◽  
Contractile Forces

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

scholarly journals Vimentin binds to SARS-CoV-2 spike protein and antibodies targeting extracellular vimentin block in vitro uptake of SARS-CoV-2 virus-like particles

2021 ◽  
Author(s):  
Lukasz Suprewicz ◽  
Maxx Swoger ◽  
Sarthak Gupta ◽  
Ewelina Piktel ◽  
Fitzroy F Byfield ◽  
...  
Keyword(s):  
Cell Types ◽  
Cell Entry ◽  
Spike Protein ◽  
Intermediate Filament Protein ◽  
Surface Binding ◽  
Angiotensin Converting Enzyme 2 ◽  
Virus Like Particles ◽  
Direct Binding ◽  
Filament Protein

Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. In the case of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) has been identified as a necessary receptor, but not all ACE2-expressing cells are equally infected, suggesting that other extracellular factors are involved in host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens' cellular uptake. Here, we present evidence that extracellular vimentin might act as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry. We demonstrate direct binding between vimentin and SARS-CoV-2 virus-like particles coated with the SARS-CoV-2 spike protein and show that antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cell lines. Our results suggest new therapeutic strategies for preventing and slowing SARS-CoV-2 infection, focusing on targeting cell host surface vimentin.

scholarly journals Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons.

1991 ◽  
Vol 112 (6) ◽  
pp. 1205-1213 ◽  
Author(s):  
D E Weinstein ◽  
M L Shelanski ◽  
R K Liem
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Cell Lines ◽  
Intermediate Filament ◽  
Neuronal Cell ◽  
Acidic Protein ◽  
Intermediate Filament Protein ◽  
Astrocytoma Cell ◽  
Filament Protein ◽  
U251 Cells

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.

scholarly journals Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells.

1983 ◽  
Vol 96 (6) ◽  
pp. 1523-1531 ◽  
Author(s):  
G S Bloom ◽  
R B Vallee
Keyword(s):  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Immune Serum ◽  
Brain Cells ◽  
Intermediate Filament Protein ◽  
Antibody Staining ◽  
Filament Protein

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.

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