Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies

1989 ◽  
Vol 92 (3) ◽  
pp. 519-528 ◽  
Author(s):  
P. Draber ◽  
E. Draberova ◽  
I. Linhartova ◽  
V. Viklicky
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Antigenic Determinants ◽  
Alpha Tubulin ◽  
Domain Specific ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
C And N ◽  
Terminal Domains

A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6–11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.

scholarly journals Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.

1984 ◽  
Vol 98 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
W C Thompson ◽  
D J Asai ◽  
D H Carney
Keyword(s):  
Differential Staining ◽  
Hypotonic Medium ◽  
Cytoplasmic Microtubule ◽  
Alpha Tubulin ◽  
Apparent Correlation ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
Fibroblastic Cells ◽  
In Cells

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.

scholarly journals Human plasma and cerebrospinal fibronectins differ in the accessibility of the epitopes on the N-terminal domains.

2010 ◽  
Vol 57 (3) ◽  
Author(s):  
Małgorzata Pupek ◽  
Anna Lemańska-Perek ◽  
Jolanta Jasonek ◽  
Iwona Kątnik-Prastowska
Keyword(s):  
Cerebrospinal Fluid ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Antigenic Epitope ◽  
Cell Binding ◽  
Cerebrospinal Fluid Analysis ◽  
Fluid Analysis ◽  
C And N ◽  
The Mean ◽  
Terminal Domains

Three monoclonal antibodies specific to the central cell-binding and the C- and N-terminal domains of fibronectin (FN) were used to test antigenic epitope accessibility on human plasma and cerebrospinal fibronectins. In the plasma group, the mean N-terminal FN domain immunoreactivity was about one fourth that of the cell-binding and C-terminal domains, whereas in cerebrospinal fluid they were nearly equal. In the presence of 0.5-6 M urea N-terminal domain immunoreactivity in the plasma increased 3-6-fold, but it decreased 0.7-3-fold in the cerebrospinal fluid. Analysis of fibronectin domain immunoreactivities of the cell-binding and N-terminal domains by a panel of specific monoclonal antibodies may reveal N-terminal fibronectin domain accessibility for reaction with biological partner ligand(s) and/or processes in which FN could be implicated. Such determinations may have important clinical implications.

Evidence for heterogeneity in the 160/165 × 10(3) Mr glycoprotein components of desmosomes

1987 ◽  
Vol 88 (4) ◽  
pp. 513-520
Author(s):  
J.C. Jones ◽  
K.L. Vikstrom ◽  
R.D. Goldman
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Mouse Skin ◽  
Basal Cells ◽  
Antibody Preparation ◽  
Plaque Component ◽  
Mouse Tissues ◽  
Double Label ◽  
Cryostat Sections

We have prepared both monoclonal and polyclonal antibody preparations directed against the 160/165 × 10(3) Mr glycoproteins (desmogleins) of bovine tongue epithelial desmosomes. The polyclonal antibody preparation recognizes desmosomes in a number of mouse tissues, e.g. mouse skin, heart, bladder and trachea, as determined by immunofluorescence microscopy. Furthermore, the polyclonal antibodies recognize polypeptide(s), present in the high salt, Triton-insoluble residues (‘cytoskeleton preparations’) of mouse skin, heart, bladder and trachea, which comigrate with the 160/165 × 10(3) Mr glycoproteins of bovine tongue epithelial desmosomes as determined by ‘Western’ immunoblotting. Conversely, the monoclonal 160/165 × 10(3) Mr antibody preparation recognizes desmosomes of stratified squamous epithelial tissues but not desmosomes in other tissue types. Moreover, whereas the monoclonal antibodies recognize 160/165 × 10(3) Mr polypeptides in mouse skin cell cytoskeletons they show no immunoreactivity with the cytoskeleton preparations of mouse bladder, trachea and heart following immunoblotting. These results suggest therefore that although there are conserved epitopes of the 160/165 × 10(3) Mr glycoproteins there are also epitopes of these molecules which vary from tissue to tissue. Double label immunofluorescence observations of cryostat sections of mouse skin using the monoclonal antibodies and antibodies directed against desmoplakin, a plaque component of desmosomes, reveal that the monoclonal antibodies do not recognize certain desmosomes in basal cells which are recognized by desmoplakin antibodies. Indeed, double label observations of cryostat sections of mouse skin using the monoclonal antibodies and human autoantibodies which react with hemidesmosomal components suggest that the monoclonal antibodies stain desmosomes located along the apical surfaces of basal cells but fail to recognize desmosomes along the lateral surfaces of these same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of monoclonal antibodies to probe human apolipoprotein B structure and function

1985 ◽  
Vol 63 (8) ◽  
pp. 906-912 ◽  
Author(s):  
Ross W. Milne ◽  
Yves L. Marcel
Keyword(s):  
Monoclonal Antibodies ◽  
Limited Proteolysis ◽  
Ldl Receptor ◽  
Plasma Lipid ◽  
Density Lipoprotein ◽  
Lipid Vesicles ◽  
Cholesterol Homeostasis ◽  
Antigenic Determinants ◽  
Lipoprotein Subfractions ◽  
Apo B

Apolipoprotein (apo) B plays an important role in plasma lipid transport and in the maintenance of cholesterol homeostasis. Attempts to determine the structure of apo B have been hampered by technical obstacles resulting from its chemical and physical properties. Recently monoclonal antibodies (Mabs) against human apo B have been used as probes to study apo B structure and heterogeneity. Certain Mabs are capable of blocking binding of low density lipoprotein (LDL) apo B to the cell surface LDL receptor, which presumably reflects the proximity of their antigenic determinants to the receptor recognition domain. The distribution of antigenic determinants recognized by Mabs has been studied on the hepatic (apo B-100) and intestinal (apo B-48) forms of apo B and on fragments generated by limited proteolysis of apo B. Some Mabs are specific for apo B-100, whereas others cross-react with apo B-48. Apo B-100 specific Mabs coupled to Sepharose have been used to isolate separately apo-B-containing lipoproteins of intestinal and hepatic origin and their respective lipid and apolipoprotein compositions have been determined. Using the separated fractions it has been shown that apo B-100, but not apo B-48, can react with the LDL receptor. Most Mabs failed to react with apo B which had been delipidated and resolubilized, but in some cases immunoreactivity could be recovered if the solubilized apo B were reincorporated into lipid vesicles. These experiments showed that different determinants had different lipid requirements for their expression. Within an individual there is immunochemical heterogeneity in apo-B-containing lipoproteins in the expression of apo B antigenic determinants which can be detected by Mabs. Intersubject differences in reactivity of lipoprotein subfractions with Mabs have also been observed and in some cases appear to represent genetic polymorphism of apo B.

scholarly journals Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties.

1986 ◽  
Vol 103 (1) ◽  
pp. 13-22 ◽  
Author(s):  
M LeDizet ◽  
G Piperno
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Spatial Arrangement ◽  
Basal Bodies ◽  
Drug Resistant ◽  
Drug Induced ◽  
Alpha Tubulin ◽  
Tubulin Isoforms ◽  
Interphase Cells ◽  
Double Label ◽  
Cytoplasmic Microtubules

A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha-tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B-1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules.

scholarly journals Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits.

1987 ◽  
Vol 104 (6) ◽  
pp. 1563-1568 ◽  
Author(s):  
X J Chang ◽  
G Piperno
Keyword(s):  
Monoclonal Antibodies ◽  
Intermediate Filament ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antibodies ◽  
Nuclear Lamina ◽  
Helical Structure ◽  
Cross Reactivity ◽  
Cytoskeletal Proteins ◽  
Intermediate Filament Proteins ◽  
Double Label

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.

Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains

1996 ◽  
Vol 109 (9) ◽  
pp. 2253-2264 ◽  
Author(s):  
P.R. Odgren ◽  
G. Toukatly ◽  
P.L. Bangs ◽  
R. Gilmore ◽  
E.G. Fey
Keyword(s):  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Cell Types ◽  
Cdna Clones ◽  
Intermembrane Space ◽  
Western Blots ◽  
Double Label ◽  
Helical Region

We have identified and characterized a human protein of the mitochondria which we call mitofilin. Using monoclonal and polyclonal antibodies, we have isolated cDNA clones and characterized mitofilin biochemically. It appears as a 90 and 91 kDa doublet in western blots and is translated from a single 2.7 kb mRNA. Antibodies raised against cellular and bacterially-expressed protein given identical cytoplasmic immunofluorescence and immunoblot results. Mitofilin co-localizes with mitochondria in immunofluorescence experiments and co-purifies with mitochondria. Double label studies show co-localization only with mitochondria and not with Golgi or endoplasmic reticulum. Co-localization with mitochondria is retained when actin or tubulin are de-polymerized, and mitofilin is expressed in all human cell types tested. The cDNA encodes a polypeptide with a central alpha-helical region with predicted coiled coil domains flanked by globular amino and carboxy termini. Unlike coiled coil motor proteins, mitofilin is resistant to detergent extraction. The presence of mitochondrial targeting and stop-transfer sequences, along with the accessibility of mitofilin to limited proteolysis suggests that it resides predominantly in the intermembrane space, consistent with immuno-electron micrographs which show mitofilin mainly at the mitochondrial periphery. The cDNA sequence of mitofilin is identical to that recently reported by Icho et al. (1994; Gene 144, 301–306) for a mRNA preferentially expressed in heart muscle (HMP), consistent with the high levels of mitochondria in cardiac myocytes.

Immunocytochemical differentiation of microtubules in the cytoskeleton of Giardia lamblia using monoclonal antibodies to alpha-tubulin and polyclonal antibodies to associated low molecular weight proteins

1986 ◽  
Vol 80 (1) ◽  
pp. 233-252
Author(s):  
R. Crossley ◽  
J. Marshall ◽  
J.T. Clark ◽  
D.V. Holberton
Keyword(s):  
Monoclonal Antibodies ◽  
Giardia Lamblia ◽  
Polyclonal Antibodies ◽  
Rabbit Antiserum ◽  
Outer Edge ◽  
Basal Bodies ◽  
Alpha Tubulin ◽  
Electron Immunocytochemistry ◽  
Flagellar Proteins ◽  
Ventral Disc

In interphase trophozoites of Giardia lamblia, separate populations of microtubules constitute the four parts of the mastigont apparatus: flagella, ventral disc, funis and median body. Antigenic differences between the tubules have been investigated by light and electron immunocytochemistry after labelling with two monoclonal antibodies to alpha-tubulin (YL 1/2 and YOL 1/34 clones), and with polyclonal antibodies to Giardia tubule-associated proteins. Both anti-tubulins stained all tubules after isolated structures were fixed in formaldehyde, but different patterns of reactivity were shown by unfixed tubules. YL 1/2 antibodies labelled flagellar axonemes and basal bodies, funis and median body tubules. Disc microtubules were mostly unlabelled, but the antibody bound strongly to the outer edge of the disc where the ends of tubules are embedded. YOL 1/34 antibodies stained disc tubules uniformly, and cross-reacted with the median body but not with tubules of axonemes, basal bodies or funis. Antibodies to giardins 14A and 14B (approximately 30 000 Mr filament-forming proteins) localized these proteins in the microribbons attached to disc microtubules. The median body was also labelled by anti-giardins, indicating an ontogenetic relationship between this organelle and the ventral disc. A second set of approximately 30 000 Mr proteins with no immunoreactivity to anti-giardin was found in flagella purified without removing flagellar membranes. These polypeptides were Triton-soluble and therefore probably originated from an extra-axonemal site. A rabbit antiserum to the labile flagellar proteins specifically stained the two ventral flagella, but not the other six flagella on this cell.

An Immunoradiometric Assay for Factor VIII Related Antigen (VIIIRAg) Using Two Monoclonal Antibodies-Comparison with Polyclonal Rabbit Antibodies for Use in von Willebrand’s Disease Diagnosis

1984 ◽  
Vol 52 (03) ◽  
pp. 250-252 ◽  
Author(s):  
Y Sultan ◽  
Ph Avner ◽  
P Maisonneuve ◽  
D Arnaud ◽  
Ch Jeanneau
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Changes ◽  
Polyclonal Antibodies ◽  
Disease Diagnosis ◽  
Immunoradiometric Assay ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Structural Abnormalities ◽  
Antigenic Material ◽  
Von Willebrand

SummaryTwo monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand’s disease. The first antibody, an IgG1 was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megacaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient’s plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.

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