A Monoclonal Antibody Raised Against Cytoplasmic Fibrillar Bundles from Carrot Cells, and its Cross-Reaction with Animal Intermediate Filaments

1989 ◽  
Vol 92 (3) ◽  
pp. 371-378 ◽  
Author(s):  
ALAN J. HARGREAVES ◽  
PETER J. DAWSON ◽  
GEOFFREY W. BUTCHER ◽  
AUDREY LARKINS ◽  
KIM C. GOODBODY ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Preprophase Band ◽  
Animal Cells ◽  
Suspension Cells ◽  
Band Formation ◽  
Type Iii ◽  
Carrot Cells ◽  
Spindle Poles ◽  
Cytoplasmic Microtubules

Carrot suspension cells contain cytoplasmic bundles of fibrils that are distinct from F-actin and microtubules and have some of the characteristics of intermediate filaments. In characterizing these fibrillar bundles further, we have raised a monoclonal antibody against them. This anti-fibrillar bundle antibody (AFB) immunoblots vimentin from a range of animal cells and tissues, as well as glial fibrillary acidic protein in brain and desmin in BHK fibroblasts, which are representatives of the type III intermediate filaments. Immunofluorescence staining of PtK2 cells indicates that AFB recognizes a network co-distributing with cytoplasmic microtubules. When this co-alignment is disturbed with the anti-microtubule agent, colcemid, the AFB staining segregates with the collapsed perinuclear whorls of vimentin. In carrot, AFB immunoblots the major bundle proteins but not plant tubulin. In plant as in animal cells, AFB immunofluorescently labels antigens that co-distribute with microtubules. In onion cells (which, unlike carrot, do not possess paracrystalline arrays of fibrils) AFB labels all four microtubule arrays throughout the cell cycle. The antigens do not, however, collapse around the spindle poles during mitosis. Double immunofluorescence, using anti-dog brain tubulin, indicates that the FB antigen is more diffusely distributed than tubulin; it is patchy and co-alignment is not exact, particularly during early preprophase band formation. Antigens in detergent-insoluble fibrils of carrot cells therefore exist both in animal type III intermediate filaments (IF), and in a more dispersed, microtubule-associated manner in onion meristematic cells. This constitutes an independent line of evidence for the existence of IF antigens in plants.

scholarly journals The mode of action of 2,4-D in counteracting the elongation of carrot cells grown in culture

1980 ◽  
Vol 45 (1) ◽  
pp. 257-268
Author(s):  
C.W. Lloyd ◽  
S.B. Lowe ◽  
G.W. Peace
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Dichlorophenoxyacetic Acid ◽  
Mitogenic Effect ◽  
Suspension Cells ◽  
Carrot Cells ◽  
Cytoplasmic Microtubules ◽  
Time Periods ◽  
Per Se ◽  
2,4 Dichlorophenoxyacetic Acid

The growth regulator 2,4-D (2,4-dichlorophenoxyacetic acid) has been used to investigate the inter-relationship between cell elongation and cell division in carrot suspension cells. Maintained in 1 mg/l. 2,4-D, dividing populations of cells remain spheroidal and in clusters. But when subcultured into lower levels or zero, 2,4-D they increasingly elongate at the expense of division. Over the range of 0 to 1.0 mg/l, 2,4-D, elongation and division are therefore inversely related. However, by suppressing the mitogenic effect with FUdR it can be shown that cells do elongate in 1 mg/l. 2,4-D—a concentration which otherwise produces dividing, spheroidal cells. This indicates that mitogenc levels of 2,4-D do not perturb structures which support cellular elongation. This conclusion is confirmed by immuno- and electron-microscopy which show that development of elaborate arrays of cytoplasmic microtubules is unaffected by 1 mg/l. 2,4-D when FUdR is present. It is concluded that over the time periods under study here, 2,4-D regulates cell size (and shape) by stimulating growing cells to enter the division cycle and not by inhibiting elongation per se.

A centrosomal antigen localized on intermediate filaments and mitotic spindle poles

1990 ◽  
Vol 97 (2) ◽  
pp. 259-271
Author(s):  
B. Buendia ◽  
C. Antony ◽  
F. Verde ◽  
M. Bornens ◽  
E. Karsenti
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mdck Cells ◽  
Large Fraction ◽  
Human Lymphocytes ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Xenopus Egg

A monoclonal antibody (CTR2611) raised against centrosomes isolated from human lymphocytes (KE37) stains the pericentriolar material and intermediate filaments in the same cells. In MDCK cells, where most of the microtubules do not originate from the pericentriolar region during interphase, the antigen is distributed along intermediate filaments. At the onset of mitosis, a large fraction of the CTR2611 antigen associates with the minus-end domain of the microtubules of the mitotic spindle but not with the pericentriolar region itself. Treatment of mitotic MDCK cells with taxol leads to the assembly of many microtubule asters in the cytoplasm at the expense of the mitotic spindle. The CTR2611 antigen is present in the center of each of these asters. Similar asters can also be produced in vitro by adding taxol to concentrated Xenopus egg mitotic cytoplasm. Again, the antigen is found close to the center of the asters. These results suggest that CTR2611 antigen is associated with a material involved in microtubule nucleation or microtubule minus-end stabilization. The monoclonal antibody recognizes a 74 × 10(3) Mr polypeptide and other polypeptides at 120 × 10(3) Mr and 170 × 10(3) Mr. The 74 × 10(3) Mr polypeptide is found in all species examined so far, suggesting that it contains a highly conserved epitope.

A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules

1993 ◽  
Vol 106 (4) ◽  
pp. 1263-1273 ◽  
Author(s):  
E. Draberova ◽  
P. Draber
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
3T3 Cells ◽  
Double Immunofluorescence ◽  
Interacting Protein ◽  
Cytoplasmic Microtubules ◽  
Microtubular Structures ◽  
Vimentin Filaments

A protein of M(r) 210,000 was identified in 3T3 cells by immunoblotting and by immunoprecipitation with a monoclonal antibody MA-01. The protein was thermolabile and was located on 3T3 microtubules prepared by taxol-driven polymerization in vitro. On fixed cells the MA-01 antigen was located on interphase and mitotic microtubular structures, vinblastine paracrystals, taxol bundles and colcemid-resistant microtubules. Microinjection experiments with purified MA-01 antibody followed by double immunofluorescence have shown that the injection of antibody led to disruption of vimentin filaments, whereas the distribution of cytoplasmic microtubules was unchanged. The collapse of vimentin filaments started 30 minutes after injecting the antibody at immunoglobulin concentrations 2 mg ml-1 or higher and reached its maximum 3–6 hours after the injection. Within 20 hours after the injection vimentin filaments became reconstituted. Microinjection of the antibody into cells pre-treated with vinblastine resulted in localization of the MA-01 antigen on vinblastine paracrystals as well as on coiled vimentin filaments. The data presented suggest that the MA-01 antigen is a new microtubule-interacting protein that mediates, directly or indirectly, an interaction between microtubules and vimentin intermediate filaments.

The role of F-actin in determining the division plane of carrot suspension cells. Drug studies

Development ◽  
1988 ◽  
Vol 102 (1) ◽  
pp. 211-221 ◽  
Author(s):  
C.W. Lloyd ◽  
J.A. Traas
Keyword(s):  
Preprophase Band ◽  
Cell Plate ◽  
Suspension Cells ◽  
Mitotic Apparatus ◽  
Division Plane ◽  
Division Site ◽  
Cytoplasmic Actin ◽  
Spindle Poles ◽  
Extraction Buffer ◽  
Actin Cables

Following the report that a network of F-actin is associated with the nucleus throughout the division cycle, we have examined the involvement of F-actin in determining the division plane of carrot suspension cells. This was achieved by treating cells with drugs and then staining the unfixed cells with rhodaminyl lysine phallotoxin in detergent extraction buffer. In interphase, actin cables radiate from the nucleus but at the cortex become more or less transversely arranged in the pattern already known for cortical microtubules. Concentration of the cortical F-actin into a band at preprophase draws most of the nucleus- associated actin into a transvacuolar disc, thereby forming the phragmosome within which mitosis and cytokinesis occur. In addition to this transversely aligned structure, F-actin is also associated with the spindle poles during mitosis but these filaments tend to align at right angles to the phragmosomal actin. F-actin therefore defines transverse and longitudinal vectors as division approaches. Depolymerization of F-actin with cytochalasin D can cause the spindle axis to reorientate such that the pole-pole axis comes to lie, abnormally, parallel with the phragmosome. The cytokinetic apparatus (the phragmoplast) develops centrifugally within the phragmosome. There has been considerable speculation on the nature of the elements that guide the phragmoplast to the cortical site previously occupied by the preprophase band of microtubules. This study demonstrates that F-actin bridges the leading margin of the outgrowing phragmoplast to the opposing cortex. Radial actin strands therefore provide a ‘memory’ of the predetermined division plane whose perimeter had been marked at preprophase by a band composed of microtubules and F-actin. This relationship was perturbed with the herbicide, chloroisopropylphenyl carbamate. Preprophase bands of actin appear to form normally in herbicide-treated cells. However, cytokinesis does not occur within this predicted plane since the drug perturbs the mitotic spindle, forming three nuclei which become separated by Y-shaped, actin-containing phragmoplasts. Cytoplasmic actin strands connect the edges of the phragmoplast to the cortex. It is suggested that the irregular distribution of F-actin, which radiates from the herbicide-altered mitotic apparatus, provides alternative paths for outgrowth of the abnormal phragmoplasts. Caffeine is known to cause failure of cell plate formation. But apart from inducing cytoplasmic ‘starbursts’ of F-actin in interphase cells it does not appear to have any effect on F-actin-containing division structures. It is concluded that the formation of a transvacuolar phragmosome, spindle alignment and the ‘correct’ outgrowth of a planar cytokinetic apparatus to the predetermined boundary of the division site all involve F-actin.

scholarly journals An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus.

1987 ◽  
Vol 105 (1) ◽  
pp. 387-395 ◽  
Author(s):  
J A Traas ◽  
J H Doonan ◽  
D J Rawlins ◽  
P J Shaw ◽  
J Watts ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Three Dimensional ◽  
Preprophase Band ◽  
Alternative Methods ◽  
Cell Periphery ◽  
Actin Network ◽  
Suspension Cells ◽  
Daucus Carota L ◽  
Culture Cells ◽  
Transvacuolar Strands

We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.

scholarly journals Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates.

1984 ◽  
Vol 98 (2) ◽  
pp. 449-456 ◽  
Author(s):  
C Walsh
Keyword(s):  
Monoclonal Antibody ◽  
Protein Synthesis ◽  
Indirect Immunofluorescence ◽  
Cell Protein ◽  
Basal Bodies ◽  
Immunofluorescence Method ◽  
Nonionic Detergent ◽  
Cytoplasmic Microtubules ◽  
Spherical Cells ◽  
Naegleria Gruberi

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.

PRIMARY BINDING SITE OF VON WILLEBRAND FACTOR IN THE SUBENDOTHELIUM WHICH MEDIATES PLATELET ADHESION IS NOT COLLAGEN

1987 ◽  
Author(s):  
Philip G de Groot ◽  
Jan A van Mourik ◽  
Jan J Sixma
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Collagen Type I ◽  
Extracellular Matrices ◽  
Type I ◽  
Type Iii ◽  
Von Willebrand ◽  
Willebrand Factor

We have studies the binding of von Willebrand factor (vWF) to extracellular matrices of endothelial cells and smooth muscle cells and to the vessel wall of human umbilical arteries in relation to its function in supporting platelet adhesion at high shear rates. CLB-RAg 38, a monoclonal antibody directed against vWF inhibits the binding of 125I-vWF extracellular matrices completely. The binding of 125I-vWF to subendothelium is not inhibited, because there are many different binding sites. CLB-RAg 38 inhibits platelet adhesion to extracellular matrices and subendothelium, in sofar as it is dependent on plasma vWF. CLB-RAg 38 has no effect on adhesion depending on vWF already bound to the matrix or subendothelium. CLB-RAg 38 does not inhibit binding of vWF to collagen type I and type III. Another monoclonal antibody against vWF, CLB-RAg 201, completely inhibits binding of vWF to collagen type I and type III. CLB-RAg 201 does not inhibit binding of 125I-vWF ot the extracellular matrices. CLB-RAg 201 partly inhibits platelet adhesion but this inhibition is also present when the adhesion depends on vWF already present in matrix or subendothelium, indicating that CLB-RAg 201 also inhibits the adhesion of platelets directly, this in contrast to CLB-RAg 38. The epitopes for CLB-RAg 201 and 38 were found on different tryptic fragments of vWF. These data indicate that vWF binds to subendothelium and to matrices of cultured cells by mechanism that is different from binding to collagen.

scholarly journals A monoclonal antibody distinguishes two types of phosphatidylinositol 4-kinase

1991 ◽  
Vol 273 (1) ◽  
pp. 63-66 ◽  
Author(s):  
G C Endemann ◽  
A Graziani ◽  
L C Cantley
Keyword(s):  
Monoclonal Antibody ◽  
Rat Brain ◽  
Rat Liver ◽  
Bovine Brain ◽  
Human Erythrocytes ◽  
Gene Products ◽  
Type Ii ◽  
Type Iii ◽  
Distinct Gene ◽  
Phosphatidylinositol 4

A monoclonal antibody has been developed against the type II PtdIns 4-kinase from bovine brain. This antibody, 4C5G, causes greater than 90% inhibition of the type II PtdIns 4-kinase from bovine brain, rat brain and human erythrocytes. However, it fails to inhibit type III PtdIns 4-kinase from bovine brain or PtdIns 3-kinase from rat liver. These results suggest that type II and type III PtdIns 4-kinases are distinct gene products, and that 4C5G will be useful in studying the function of the type II PtdIns 4-kinase.

scholarly journals The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

1999 ◽  
Vol 32 (10) ◽  
pp. 1177-1185 ◽  
Author(s):  
M. Garbuglia ◽  
M. Verzini ◽  
G. Sorci ◽  
R. Bianchi ◽  
I. Giambanco ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Type Iii
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