A probe for flagellar dynein in the mammalian mitotic apparatus

1981 ◽  
Vol 48 (1) ◽  
pp. 241-257
Author(s):  
G.W. Zieve ◽  
J.R. NcIntosh
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Indirect Immunofluorescence ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Structural Studies ◽  
Mitotic Apparatus ◽  
Specific Staining ◽  
Bovine Sperm ◽  
Sperm Proteins

An anti-serum has been prepared in rabbits that precipitates high-molecular-weight bovine sperm proteins, including the dyneins. The activity of the serum against the dyneins is demonstrated by the recognition of dynein polypeptides in stained electrophoretic profiles of sperm proteins and in immunoprecipitates of radiolabelled sperm proteins. In addition, the serum stains the sperm flagella when used in indirect immunofluorescence and quantitatively inhibits the motility of demembranated sperm reactivated with ATP. However, the serum has additional anti-sperm activities besides those directed against the dyneins as demonstrated by the staining of the acrosome in indirect immunofluorescence. When used to immunoprecipitate proteins from extracts of cultured cells, the serum precipitates 2 polypeptides; one has a molecular weight higher than the flagellar dyneins, one lower. No specific staining of cultured cells is observed when an affinity-purified anti-dynein fraction IgG is used to stain a variety of cultured cells including bovine fibroblasts. We interpret these data to suggest that flagellar dynein is not a component of the mammalian mitotic spindle and discuss how this conclusion is consistent with recent genetic and structural studies on the mitotic spindle.

scholarly journals Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence.

1978 ◽  
Vol 76 (3) ◽  
pp. 781-786 ◽  
Author(s):  
J A Connolly ◽  
V I Kalnins ◽  
D W Cleveland ◽  
M W Kirschner
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Indirect Immunofluorescence ◽  
Tau Protein ◽  
Intracellular Localization ◽  
Double Diffusion ◽  
Specific Staining ◽  
Intact Cells ◽  
Dividing Cells ◽  
Brain Microtubules

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.

scholarly journals High molecular weight MAPs are part of the mitotic spindle

1978 ◽  
Vol 77 (1) ◽  
pp. R9 ◽  
Author(s):  
P Sherline ◽  
K Schiavone
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Indirect Immunofluorescence ◽  
Mitotic Spindle ◽  
Microtubule Associated Proteins ◽  
Immunofluorescence Studies ◽  
Associated Proteins

We have found that the microtubule-associated proteins of high molecular weight are located in the mitotic spindle. Indirect immunofluorescence studies reveal that the pattern of distribution of these proteins is similar to that described for tubulin and corresponds to the known phases of mitosis.

Structural Studies on Mitotic Spindle Fibres

1978 ◽  
Vol 36 (3) ◽  
pp. 615-626
Author(s):  
J.R. Mcintosh
Keyword(s):  
Chemical Composition ◽  
Mitotic Spindle ◽  
Structural Studies ◽  
Mitotic Apparatus ◽  
Chemical Studies ◽  
Medical Interest ◽  
Spindle Fibres

The mitotic apparatus is a structure of obvious biological and medical interest, but it has proved to be a difficult cellular machine to understand. The chemical composition of the spindle is only slightly elucidated, largely because of the difficulties in preparing useful isolates of the structure. Chemical studies of the mitotic spindle have been reviewed elsewhere (Mcintosh, 1977), and will not be discussed further here. One would think that structural studies on the mitotic apparatus (MA) in situ would be straightforward, but even with this approach there is some disagreement in the results obtained with various methods and by different investigators. In this paper I will review briefly the approaches which have been used in structural studies of the MA, pointing out the strengths and problems of each approach. I will summarize the principal findings of the different methods, and identify what seem to be fruitful avenues for further work.

Structural studies of the capsular polysaccharide from Haemophilus pleuropneumoniae serotype 2

1987 ◽  
Vol 65 (5) ◽  
pp. 414-422 ◽  
Author(s):  
Eleonora Altman ◽  
Jean-Robert Brisson ◽  
Malcolm B. Perry
Keyword(s):  
Molecular Weight ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
High Molecular Weight ◽  
Capsular Polysaccharide ◽  
Structural Studies ◽  
High Molecular Weight Polymer ◽  
Molecular Weight Polymer ◽  
Structure Formula ◽  
Nuclear Magnetic

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 2 (ATCC 27089) is composed of D-glucose (two parts), D-galactose (one part), glycerol (one part), and phosphate (one part). Hydrolysis, dephosphorylation, methylation, enzymic studies, and 1H and 13C nuclear magnetic resonance experiments showed that the polysaccharide is a high molecular weight polymer of a tetrasaccharide repeating units, linked by monophosphate diester and having the following structure:[Formula: see text]

scholarly journals Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

1997 ◽  
Vol 138 (5) ◽  
pp. 1055-1066 ◽  
Author(s):  
Tirso Gaglio ◽  
Mary A. Dionne ◽  
Duane A. Compton
Keyword(s):  
Mitotic Spindle ◽  
Motor Proteins ◽  
Cultured Cells ◽  
Recent Observation ◽  
Somatic Cells ◽  
Cytoplasmic Dynein ◽  
Common Mechanism ◽  
Mitotic Apparatus ◽  
Free System ◽  
Spindle Poles

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.

Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells

1992 ◽  
Vol 101 (1) ◽  
pp. 125-137 ◽  
Author(s):  
S.X. Lin ◽  
C.A. Collins
Keyword(s):  
Cultured Cells ◽  
Fibroblast Cell ◽  
Cytoplasmic Dynein ◽  
Rat Tissues ◽  
Mitotic Apparatus ◽  
Specific Staining ◽  
Directed Movement ◽  
Double Labeling ◽  
Fluorescent Markers ◽  
High Degree

Polyclonal antisera have been raised against cytoplasmic dynein purified from calf brain and rat testis. These antibodies reacted most strongly with the 74 kDa dynein intermediate chain, but also recognized the 410 kDa heavy chain, and the 150 and 45 kDa polypeptides previously observed to copurify with cytoplasmic dynein from rat tissues. Localization studies were performed by indirect immunofluorescence microscopy using a fibroblast cell line. Dynein-specific staining appeared vesicular, distributed throughout the cell, but more concentrated near the nucleus. Double-labeling studies using fluorescent markers for membranous organelles indicated a co-localization of dynein with lysosomes. The distribution of the dynein-positive lysosomes was disrupted by treatment of the cells with microtubule-active drugs, and by acidification of the cytoplasm. Comparison of the distribution of lysosomes with peripheral microtubules indicated a high degree of coincidence. These results are consistent with the hypothesis that cytoplasmic dynein is involved in retrograde-directed movement of membranous organelles. In mitotic cells, dynein staining was also apparent along the microtubules of the mitotic apparatus, though vesicular staining was still conspicuous. The presence of dynein on vesicles as well as on spindle microtubules indicates that dynein distribution between these compartments may be regulated by distinct binding proteins.

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