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.

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.

Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments

1992 ◽  
Vol 102 (1) ◽  
pp. 31-41 ◽  
Author(s):  
P.D. Kouklis ◽  
P. Traub ◽  
S.D. Georgatos
Keyword(s):  
Consensus Sequence ◽  
Sequence Motif ◽  
Sequence Motifs ◽  
3T3 Cells ◽  
Filament Assembly ◽  
In Vitro Assembly ◽  
Vimentin Filaments

Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain (‘coil 1a’ and ‘coil 2b’, respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.

Localization of a 210-kDa microtubule-interacting protein in the yeast Saccharomyces cerevisiae

1992 ◽  
Vol 38 (2) ◽  
pp. 149-152 ◽  
Author(s):  
J. Hašek ◽  
J. Jochová ◽  
P. Dráber ◽  
V. Viklický ◽  
E. Streiblová
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Key Words ◽  
Budding Yeast ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Labeling ◽  
Interacting Protein ◽  
Cytoplasmic Microtubules

Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae. In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution. This pattern was common for cells of all cell-cycle stages. The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole. Key words: immunoblotting, immunofluorescence, microtubule-interacting protein, Saccharomyces cerevisiae.

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 Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts.

1995 ◽  
Vol 131 (5) ◽  
pp. 1275-1290 ◽  
Author(s):  
G Gurland ◽  
G G Gundersen
Keyword(s):  
Intermediate Filaments ◽  
Perinuclear Region ◽  
3T3 Cells ◽  
Noticeable Effect ◽  
3T3 Fibroblasts ◽  
Rapid Regrowth ◽  
In Cells

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

scholarly journals Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

1997 ◽  
Vol 138 (6) ◽  
pp. 1379-1394 ◽  
Author(s):  
Carlos Caulín ◽  
Guy S. Salvesen ◽  
Robert G. Oshima
Keyword(s):  
Intermediate Filaments ◽  
Cleavage Site ◽  
Uv Light ◽  
Tail Domain ◽  
Proteolytic Fragment ◽  
Intermediate Filament Proteins ◽  
Caspase Cleavage ◽  
Structural Cell ◽  
Caspase 6

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.

A Call for a European Prohibition of Monoclonal Antibody Production by the Ascites Procedure in Laboratory Animals

1998 ◽  
Vol 26 (4) ◽  
pp. 523-536
Author(s):  
Coenraad F.M. Hendriksen
Keyword(s):  
Monoclonal Antibody ◽  
Laboratory Animals ◽  
Induction Method ◽  
Therapeutic Application ◽  
Monoclonal Antibody Production ◽  
In Vitro Methods ◽  
Appeals Process ◽  
Core Facilities ◽  
Mab Production

Monoclonal antibodies (mAbs) are particularly valuable in therapeutics and research. Unfortunately, one of the most familiar methods of producing mAbs, the ascites induction method, causes pain and distress to the animals used. In most cases, non-animal or in vitro alternatives can be employed to reduce or eliminate the use of animals for mAb production. Prohibition of the use of animals in the production of mAbs is recommended, except when the replacement in vitro methods prove to be insufficient, and in a limited number of other well-documented cases, such as an exceptional need for an emergency therapeutic application. A total ban on the use of animals for mAb production is impractical and it is imperative that an appeals process should accompany the prohibition. The need for the establishment of core facilities for in vitro mAb production is emphasised.

scholarly journals Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties

2021 ◽  
Vol 22 (3) ◽  
pp. 1124
Author(s):  
Mafalda Giovanna Reccia ◽  
Floriana Volpicelli ◽  
Eirkiur Benedikz ◽  
Åsa Fex Svenningsen ◽  
Luca Colucci-D’Amato
Keyword(s):  
Cell Line ◽  
Low Cost ◽  
Neural Cell ◽  
New Drugs ◽  
High Yield ◽  
Time Saving ◽  
Interacting Protein ◽  
Neuronal Marker ◽  
Differentiation Status

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.

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