Monoclonal antibodies to plant nuclear matrix reveal intermediate filamentrelated components within the nucleus

1991 ◽  
Vol 98 (3) ◽  
pp. 293-302
Author(s):  
ALISON BEVEN ◽  
YUHONG GUAN ◽  
JAN PEART ◽  
CHRISTINE COOPER ◽  
PETER SHAW
Keyword(s):  
Monoclonal Antibodies ◽  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Protein S ◽  
Intermediate Filament Proteins ◽  
Daucus Carota L ◽  
Culture Cells ◽  
Matrix Fraction

We have prepared a nuclear matrix fraction from purified nuclei of carrot (Daucus carota L.) suspension culture cells, and used this fraction to produce a library of monoclonal antibodies. We report the preliminary characterisation of two antibodies-JIM 62 and JIM 63. The antibodies recognise a polypeptide doublet band at 92xl03Mr, which has been partially purified by differential urea extraction. Other intermediate filament antibodies-ME101, which recognises an epitope conserved among many intermediate filament proteins, and AFB, a monoclonal antibody to plant intermediate filament proteins, and an autoimmune serum directed against human lamina A and C (LSI), also label these bands, suggesting they are related to the intermediate filament/lamin family. IFA, another intermediate filament antibody, labels a band at approximately 60x103Mr, which is also enriched in the urea extracts of nuclear matrices. Immunofluorescence microscopy with JIM 63, ME 101, AFB and LSI shows network-like staining, often extending around the nucleolus. In many cases the staining reveals structures that appear to be bundles of fibres. JIM 63 also shows a weak staining of the nuclear rim in carrot nuclei, which can be greatly enhanced by treatment of the specimen with cold methanol after fixation. JIM 63 cross-reacts with all the other plant species we have tested. Vibratome sections of pea roots, extracted as for nuclear matrix preparation and stained with JIM 63 show a clear, strong nuclear rim labelling. Furthermore, JIM 63 strongly labels the nuclear lamina in rat liver nuclei. We suggest that the 92x103Mr protein(s) are related to intermediate filaments and/or lamins, and are distributed both within the nucleus and at the nuclear periphery.

Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells

1995 ◽  
Vol 108 (2) ◽  
pp. 635-644 ◽  
Author(s):  
P. Hozak ◽  
A.M. Sasseville ◽  
Y. Raymond ◽  
P.R. Cook
Keyword(s):  
Nuclear Membrane ◽  
Intermediate Filament ◽  
Mammalian Cells ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Human Cells ◽  
Lamin A ◽  
Intermediate Filament Proteins ◽  
Form Part ◽  
Dense Chromatin

The nuclear lamina forms a protein mesh that underlies the nuclear membrane. In most mammalian cells it contains the intermediate filament proteins, lamins A, B and C. As their name indicates, lamins are generally thought to be confined to the nuclear periphery. We now show that they also form part of a diffuse skeleton that ramifies throughout the interior of the nucleus. Unlike their peripheral counterparts, these internal lamins are buried in dense chromatin and so are inaccessible to antibodies, but accessibility can be increased by removing chromatin. Knobs and nodes on an internal skeleton can then be immunolabelled using fluorescein- or gold-conjugated anti-lamin A antibodies. These results suggest that the lamins are misnamed as they are also found internally.

Immunological characterization of lamins in the nuclear matrix of onion cells

1993 ◽  
Vol 106 (1) ◽  
pp. 431-439 ◽  
Author(s):  
A. Minguez ◽  
S. Moreno Diaz de la Espina
Keyword(s):  
Intermediate Filaments ◽  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Higher Plants ◽  
Nuclear Periphery ◽  
Immunogold Labelling ◽  
Root Cells ◽  
Intermediate Filament Proteins ◽  
Lamin B

We have used polyclonal and monoclonal antibodies against different lamins from vertebrates, and the IFA antibody recognizing all kinds of intermediate filament proteins, to investigate the lamins of the nuclear matrix of Allium cepa meristematic root cells. All the antibodies react in the onion nuclear matrix with bands in the range of 60–65 kDa, which are enriched in the nuclear matrix after urea extraction, and do not crossreact with other antibodies recognizing intermediate filaments in plants (AFB, anti-vimentin and MAC 322), ruling out crossreaction with contaminating intermediate filaments of cytoplasmic bundles. In 2-D blots the chicken anti-lamin serum reacts with one spot at 65 kDa and pI 6.8 and the anti B-type lamin antibodies with another one at 64 kDa and pI 5.75. Both crossreact with IFA. The lamin is localized at the nuclear periphery and the lamina by indirect immunofluorescence. Immunogold labelling of nuclear matrix sections reveals that the protein is not only associated with the lamina, but also with the internal matrix. Taken together these results reveal that higher plants, which do not possess an organized network of cytoplasmic intermediate filaments, nevertheless present a well-organized lamina containing lamins in which at least one of them is immunologically related to vertebrate lamin B. Our data confirm that lamins are very old members of the intermediate filament proteins that have been better conserved in plants during evolution than their cytoplasmic counterparts.

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.

Differential diagnosis of genitourinary tumors using monoclonal antibodies to intermediate filament proteins

Urology ◽  
1989 ◽  
Vol 33 (5) ◽  
pp. 433-439
Author(s):  
Steven A. Lofton ◽  
Allen M. Gown ◽  
Arthur M. Vogel ◽  
John N. Krieger
Keyword(s):  
Differential Diagnosis ◽  
Monoclonal Antibodies ◽  
Intermediate Filament ◽  
Intermediate Filament Proteins

A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix

1991 ◽  
Vol 98 (3) ◽  
pp. 281-291
Author(s):  
P. Belgrader ◽  
A.J. Siegel ◽  
R. Berezney
Keyword(s):  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Structural Integrity ◽  
Rnase A ◽  
Matrix Proteins ◽  
Intermediate Filament Proteins ◽  
Isolation And Characterization ◽  
Hela S3

Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.

scholarly journals Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells.

1981 ◽  
Vol 91 (1) ◽  
pp. 175-183 ◽  
Author(s):  
F G Falkner ◽  
H Saumweber ◽  
H Biessmann
Keyword(s):  
Drosophila Melanogaster ◽  
Monoclonal Antibodies ◽  
Heat Shock ◽  
Indirect Immunofluorescence ◽  
Intermediate Filament ◽  
Cross Reaction ◽  
Cytoplasmic Protein ◽  
Baby Hamster Kidney ◽  
Intermediate Filament Proteins ◽  
Bhk Cells

Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures.

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

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