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.

Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits

1999 ◽  
Vol 77 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Jean-Martin Beaulieu ◽  
Janice Robertson ◽  
Jean-Pierre Julien
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Intermediate Filament Protein ◽  
Physiological Conditions ◽  
Filament Protein ◽  
Filament Network ◽  
Filament Type

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.Key words: peripherin, neurofilament, SW13 cells, intermediate filament.

scholarly journals Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

1993 ◽  
Vol 122 (6) ◽  
pp. 1323-1335 ◽  
Author(s):  
GY Ching ◽  
RK Liem
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Neuronal Cell ◽  
Intermediate Filament Protein ◽  
Amino Acid Residues ◽  
Intermediate Filament Proteins ◽  
Transfected Cells ◽  
Type Iv

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.

scholarly journals Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 423-434 ◽  
Author(s):  
G Manandhar ◽  
D Feng ◽  
Y-J Yi ◽  
L Lai ◽  
J Letko ◽  
...  
Keyword(s):  
Western Blotting ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Perinuclear Area

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes afterin vitrofertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result ofde novosynthesis at the onset of differentiation of the pluripotent blastomeres.

Differential in vitro effects of chemotherapeutic agents on primary cultures of human ovarian carcinoma

2004 ◽  
Vol 14 (4) ◽  
pp. 607-615 ◽  
Author(s):  
P. Kornblith ◽  
R. L. Ochs ◽  
A. Wells ◽  
M. J. Gabrin ◽  
J. Piwowar ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Human Tumor ◽  
Clinical Results ◽  
Chemotherapeutic Agents ◽  
Primary Ovarian Cancer ◽  
In Vitro Effects ◽  
Therapeutic Decision Making

The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process.

scholarly journals In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid

1996 ◽  
Vol 317 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Pere PUIGSERVER ◽  
Francisca VÁZQUEZ ◽  
María L. BONET ◽  
Catalina PICÓ ◽  
Andreu PALOU
Keyword(s):  
Retinoic Acid ◽  
Cultured Cells ◽  
Uncoupling Protein ◽  
Primary Cultures ◽  
Brown Adipocyte ◽  
Brown Adipose ◽  
Adipocyte Cell ◽  
Differentiation Stage

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium–late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.

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.

Immobilization and neutralization of Treponema pallidum attached to cultured mammalian cells

1985 ◽  
Vol 31 (12) ◽  
pp. 1152-1156
Author(s):  
Thomas Fitzgerald
Keyword(s):  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Cultured Cells ◽  
Treponema Pallidum ◽  
Immune Serum ◽  
Immune Reactivity ◽  
Complex Environment ◽  
In Vitro Effects

The in vitro effects of antibodies, complement, and (or) macrophages on Treponema pallidum have been previously characterized using relatively simple systems of organisms incubated with the immune components. In vivo, the more complex environment may alter immune reactivity. Experiments were performed to determine whether immobilizing and neutralizing antibodies retained their effectiveness in a more complex environment involving cultured mammalian cells. Two different protocols were used. In protocol A treponemes and normal or immune serum were mixed and added immediately to the cultured cells. In protocol B treponemes were preincubated for 18 h with cultured cells to maximize treponemal attachment; then normal or immune serum was added. With both protocols, attachment of organisms resulted in less effecient immobilization and neutralization. In further experiments, cultured cells were disrupted with Triton X, leaving cytoskeletal remnants on the vessel surface. Identical immobilization and neutralization experiments were performed in the presence of these remnants. In contrast to the findings with viable cultured cells, treponemal attachment to these nonviable remnants did not effect either antibody reaction. Attached organisms were immobilized or neutralized just as efficiently as unattached organisms. Results are discussed in terms of the altered immune reactivity in more complex in vitro environments.

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 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.

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