scholarly journals Isolation of rat liver microtubule-associated proteins. Evidence for a family of microtubule-associated proteins with molecular mass of around 200,000 which distribute widely among mammalian cells.

1988 ◽  
Vol 263 (11) ◽  
pp. 5385-5389 ◽  
Author(s):  
S Kotani ◽  
H Murofushi ◽  
S Maekawa ◽  
H Aizawa ◽  
H Sakai
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Immunoreactive forms of erythrocyte spectrin and ankyrin in brain

1982 ◽  
Vol 299 (1095) ◽  
pp. 301-312 ◽  
Keyword(s):  
Molecular Mass ◽  
Erythrocyte Membranes ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Molecular Masses ◽  
Low Ionic Strength ◽  
Polypeptide Chains ◽  
The Brain ◽  
Total Membrane ◽  
Rotary Shadowing

Polypeptides immunologically related to erythrocyte spectrin and ankyrin have been detected in brain. The cross-reacting proteins include soluble as well as membrane-associated forms. A class of soluble cross-reacting polypeptides have been identified as high molecular mass microtubule-associated proteins (MAPS). MAP1, a group of polypeptides of molecular mass ca . 370 kDa contains a component that cross-reacts with anti-ankyrin IgG. MAP2, a polypeptide of molecular mass 300 kDa cross-reacts with anti-spectrin IgG, with the shared antigenic sites localized to the α chain of spectrin. The functional basis for structural homology between MAP1 and ankyrin may involve association with tubulin, since erythrocyte ankyrin binds to microtubules polymerized from pure brain tubulin. Spectrin did not associate with microtubules, but does have in common with MAP2 the ability to bind to actin (Brenner & Korn 1979; Sattilaro et al . 1981) and the shape of a flexible rod as visualized by rotary shadowing (Shotton et al . 1979; Voter & Erickson 1981). Immunoreactive forms of spectrin and ankyrin are also present in membrane fractions. A homologue of spectrin which constitutes 3% of the total membrane protein has been purified from low ionic strength extracts of membranes. This protein contains two non-identical polypeptide chains of molecular masses of 260 and 265 kDa, binds to F-actin, and displaces binding of erythrocyte spectrin to erythrocyte membranes. The brain protein pas been visualized by rotary shadowing as an extended rod-like molecule 195 nm in length. These studies indicate that the organization of proteins in the membrane-cytoskeleton complex of erythrocytes has direct relevance to other types of cells, and suggest the existence of families of proteins related to spectrin and ankyrin.

A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome

1994 ◽  
Vol 107 (2) ◽  
pp. 601-611 ◽  
Author(s):  
J.E. Dominguez ◽  
B. Buendia ◽  
C. Lopez-Otin ◽  
C. Antony ◽  
E. Karsenti ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Pericentriolar Material ◽  
Peptide Maps

The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

Identification of a 40 × 10(3) Mr centromere-associated protein in cultured mammalian cells

1990 ◽  
Vol 97 (4) ◽  
pp. 705-713
Author(s):  
R. Balczon ◽  
M.A. Accavitti ◽  
B.R. Brinkley
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Structure And Function ◽  
Interphase Nuclei ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules ◽  
And Function

Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant's reagent. One of the clones, hybridoma 32–9, produced antibodies that reacted with a 40 × 10(3) Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40 × 10(3) Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32–9 demonstrated that the 40 × 10(3) Mr antigen was associated with both spindle and cytoplasmic microtubules. In addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated proteins and microtubule-dependent ATPases, the 40 × 10(3) Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40 × 10(3) Mr protein remained associated with the insoluble cellular material. The 40 × 10(3) Mr antigen could be released from the insoluble pelleted material by extraction with 1 M NaCl. Once solubilized, the 40 × 10(3) Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.

A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton

1992 ◽  
Vol 103 (3) ◽  
pp. 665-675 ◽  
Author(s):  
A. Woods ◽  
A.J. Baines ◽  
K. Gull
Keyword(s):  
Molecular Mass ◽  
Trypanosoma Brucei ◽  
Minor Component ◽  
Relative Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Cytoplasmic Face ◽  
Associated Proteins ◽  
Cross Bridges ◽  
Main Component

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.

scholarly journals Mapmodulin, Cytoplasmic Dynein, and Microtubules Enhance the Transport of Mannose 6-Phosphate Receptors from Endosomes to theTrans-Golgi Network

1999 ◽  
Vol 10 (7) ◽  
pp. 2191-2197 ◽  
Author(s):  
Christian Itin ◽  
Nirit Ulitzur ◽  
Bettina Mühlbauer ◽  
Suzanne R. Pfeffer
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Cytoplasmic Dynein ◽  
Vesicle Transport ◽  
Microtubule Associated Proteins ◽  
Late Endosomes ◽  
Associated Proteins ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to thetrans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

scholarly journals CCDC74A/B are K-fiber crosslinkers required for chromosomal alignment

BMC Biology ◽  
2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Haining Zhou ◽  
Tao Zheng ◽  
Tianning Wang ◽  
Qi Li ◽  
Fulin Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Material ◽  
Fiber Formation ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Fiber Organization

Abstract Background Spindle microtubule organization, regulated by microtubule-associated proteins, is critical for cell division. Proper organization of kinetochore fiber (K-fiber), connecting spindle poles and kinetochores, is a prerequisite for precise chromosomal alignment and faithful genetic material transmission. However, the mechanisms of K-fiber organization and dynamic maintenance are still not fully understood. Results We reveal that two previously uncharacterized coiled-coil domain proteins CCDC74A and CCDC74B (CCDC74A/B) are spindle-localized proteins in mammalian cells. They bind directly to microtubules through two separate domains and bundle microtubules both in vivo and in vitro. These functions are required for K-fiber organization, bipolar spindle formation, and chromosomal alignment. Moreover, CCDC74A/B form homodimers in vivo, and their self-association activity is necessary for microtubule bundling and K-fiber formation. Conclusions We characterize CCDC74A and CCDC74B as microtubule-associated proteins that localize to spindles and are important K-fiber crosslinkers required for bipolar spindle formation and chromosome alignment.

Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules

1989 ◽  
Vol 67 (11-12) ◽  
pp. 791-800 ◽  
Author(s):  
E. Strömberg ◽  
L. Serrano ◽  
J. Avila ◽  
M. Wallin
Keyword(s):  
Molecular Mass ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Labile Fraction ◽  
Associated Proteins ◽  
Brain Microtubules

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly–disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (> 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.Key words: microtubules, microtubule-associated proteins, calcium, cod.

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