Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein.

Abstract

T protein isolated from porcine brain microtubules was further purified by electrophoretic elution from polyacrylamide gels and used to prepare antisera in rabbits. The antiserum to T specifically stains mitotic spindles and a filamentous network within mouse fibroblasts when the indirect immunofluorescence technique is used. The staining of the filamentous network and mitotic spindles is identical to that observed when cells are treated with antiserum prepared against electrophoretically purified tubulin. The filamentous network observed with either serum is sensitive to Colcemid. Absorption of anti-T serum with electrophoretically purified tubulin does not remove the immunoflourescent staining of the mitotic spindle, whereas absorption with electrophoretically purified T protein does. Conversely, absorption of antitubulin serum with tubulin eliminates its ability to stain the mitotic spindle, whereas absorption with T has no effect. We conclude that r protein and tubulin are antigenically distinct proteins and that T is an integral part of microtubules in vivo. These results also provide evidence that T protein, or an antigenically related protein, is associated with microtubules not only in brain but also in other cell types.The development of procedures for the in vitro assembly of microtubules (1-3) has made possible the investigation of specific components in the polymerization process. Microtubules purified by successive cycles of assembly and disassembly have been found to contain, in addition to tubulin, a variety of accessory proteins whose presence is required under standard buffer conditions for assembly of tubulin (4-8). One of these accessory proteins, named r, has been isolated from tubulin by phosphocellulose chromatography (4) and has recently been purified on the basis of its ability to stimulate tubulin polymerization in vitro. It comprises most of the microtubuleinducing activity recoverable following the removal of tubulin on phosphocellulose (9,*). In the absence of , tubulin (under the standard ionic conditions) remains as a 6S subunit which is unable to polymerize into microtubules. In experiments testing competence of neural tubulin for growth onto flagellar nucleation sites, r has been shown to be required for elongation as well as initiation of microtubule assembly (10). Because both the initial rate of microtubule growth and the final level of assembly are functions of T concentrations, a stoichiometric rather than catalytic mode of action for T is indicated.Though T has been found to copurify with microtubules through multiple cycles of assembly at constant stoichiometry with tubulint, the in vivo association of T with microtubules has not been demonstrated. Indeed, other investigators have indicated that different accessory proteini (in particular highmolecular-weight proteins) may also be responsible for microtubule assembly in their preparations (5-8). Moreover, reports of the induction of microtubules or of microtubule-related structures from purified tubulin under nonphy...

Mapping the mammalian intercellular bridge

“…A similar function may also be carried out by proteins that are distributed throughout the bridge. Proteins localized in zone 1 may play a role in stabilizing the microtubules at the minus end, mediating Welsh et al, 1979Rattner et al, 1990Rattner et al, 1992aRattner et al, 1992bKingwell et al, 1987Ohata et al, 1990Andreassen et al, 1991Connolly et al, 1977Sellitto and Kuriyama, 1988Paddy and Chelsky, 1991Todorov et al, 1992Cooke et al, 1987Pankov et al, 1990Margolis et al, 1990Chevrier et al, 1992Izant et al, 1982Bulinski and Borisy, 1980Pepper et al, 1984Nickerson et al, 1992Newmeyer and Ohlsson-Wilhelm, 1985Bonifacino et al, 1985Yen et al, 1991 the severing process and integrating this portion of the bridge which extends into the cell cytoplasm.…”

Endothelial cell monolayer integrity. I. Characterization of dense peripheral band of microfilaments.

“…The preparation and characterization of rabbit-derived antisera to electrophoretically purified pig brain tubulin, 29 chicken breast muscle tropomyosin, 30 and pig uterine myosin 12 have been previously described. The antibody to chicken gizzard alpha-actinin and vinculin was kindly provided by S.Jon Singer, University of California, San Diego.…”

Monoclonal antibody against microtubule associated protein-1 produces immunofluorescent spots in the nucleus and centrosome of cultured mammalian cells.

“…Our results show a punctate nuclear pattern similar to that observed with anti-ankyrin antibody. This immunofluorescence image differs distinctly from that obtained by staining microtubules with antibodies against MAP-2 and tau proteins (5,6). Also shown is the localization of the immunofluorescence at the centrosome (12).…”

“…All the antibodies showed transient disappearance of the spotted nuclear pattern during prophase and its reappearance in the cytoplasm during telophase, then in the nucleus by early interphase. Localization of the immunofluorescence in the nucleus and the centrosome found with anti-MAP-1 antibody differs distinctly from the selective staining of microtubules with antibodies against tubulin, MAP-2 and tau proteins (5,6). Staining of the centrosome, but not the nucleus, by antiserum raised against MAP-1 has been reported (12).…”

“…MAP-2 projects from the surface of the microtubule in a periodic manner (2), interacts with actin filament (3), and is phosphorylated by cyclic AMP-dependent protein kinase (4). Antibodies against MAP-2 and tau proteins have been shown to be associated with microtubules in intact cells (5,6). …”