scholarly journals Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization.

1978 ◽  
Vol 76 (2) ◽  
pp. 547-555 ◽  
Author(s):  
N W Seeds ◽  
R B Maccioni
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Neuroblastoma Cells ◽  
Morphological Differentiation ◽  
Tubulin Polymerization ◽  
Associated Proteins ◽  
Neurite Formation ◽  
Brain Microtubules ◽  
Brain Tubulin

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.

scholarly journals The periodic association of MAP2 with brain microtubules in vitro.

1979 ◽  
Vol 80 (2) ◽  
pp. 266-276 ◽  
Author(s):  
H Kim ◽  
L I Binder ◽  
J L Rosenbaum
Keyword(s):  
Critical Concentration ◽  
Microtubule Assembly ◽  
Molar Ratio ◽  
Thin Sections ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins ◽  
Brain Microtubules ◽  
Brain Tubulin

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

1977 ◽  
Vol 35 ◽  
pp. 460-461
Author(s):  
Tai-Te Chao ◽  
John Sullivan ◽  
Awtar Krishan
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Tubulin Polymerization ◽  
Vinca Alkaloids ◽  
Antimitotic Activity ◽  
Transmission Electron ◽  
Flow Microfluorometry ◽  
Brain Tubulin

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.

scholarly journals Influence of the centrosome on the structure of nucleated microtubules.

1985 ◽  
Vol 100 (4) ◽  
pp. 1185-1191 ◽  
Author(s):  
L Evans ◽  
T Mitchison ◽  
M Kirschner
Keyword(s):  
Lattice Structure ◽  
Neuroblastoma Cells ◽  
Nucleation Sites ◽  
Self Assembled ◽  
Brain Microtubules

The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13-protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.

scholarly journals Tubulin phosphorylation by casein kinase II is similar to that found in vivo.

1987 ◽  
Vol 105 (4) ◽  
pp. 1731-1739 ◽  
Author(s):  
L Serrano ◽  
J Díaz-Nido ◽  
F Wandosell ◽  
J Avila
Keyword(s):  
Neuroblastoma Cells ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Carboxy Terminal Domain ◽  
Phosphatase Treatment ◽  
Microtubule Protein ◽  
Carboxy Terminal ◽  
Brain Tubulin

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.

scholarly journals Brain tubulin polymerization in the absence of "microtubule-associated proteins".

1976 ◽  
Vol 73 (12) ◽  
pp. 4397-4399 ◽  
Author(s):  
R. H. Himes ◽  
P. R. Burton ◽  
R. N. Kersey ◽  
G. B. Pierson
Keyword(s):  
Tubulin Polymerization ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Brain Tubulin

scholarly journals Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

1991 ◽  
Vol 2 (12) ◽  
pp. 1021-1033 ◽  
Author(s):  
P Rossino ◽  
P Defilippi ◽  
L Silengo ◽  
G Tarone
Keyword(s):  
Retinoic Acid ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Morphological Differentiation ◽  
Laminin Receptor ◽  
Human Neuroblastoma Cell ◽  
Type I ◽  
Human Neuroblastoma ◽  
Beta 1 Integrin

Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.

Microtubules in immature oocytes of Xenopus laevis

1985 ◽  
Vol 77 (1) ◽  
pp. 129-141
Author(s):  
S.R. Heidemann ◽  
M.A. Hamborg ◽  
J.E. Balasz ◽  
S. Lindley
Keyword(s):  
Light Microscopy ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Microtubule Assembly ◽  
Electron Microscopic ◽  
Immature Oocytes ◽  
Large Numbers ◽  
Brain Microtubules ◽  
Brain Tubulin

Previous work indicated that immature oocytes of Xenopus were incapable of assembling microtubules but that competence was achieved during maturation. We report here that small numbers of microtubules do exist in immature oocytes. Consistent with this finding, ultrastructural observations indicate that brain microtubules injected into immature oocytes persist in large numbers for at least 30 min. We report that the tubulin dimers of mature and immature oocytes are equally capable of assembling with brain tubulin in vitro. We confirmed previous results that injection of taxol into immature oocytes has no effect when assayed by light microscopy. However, ultrastructural observations suggest that some microtubule assembly is stimulated by taxol. We tested for the ability of immature oocytes to elongate microtubules from ‘seeds’ by injecting deciliated pellicles of Tetrahymena. No elongation was observed either by light or electron microscopic observation. We conclude that the immature oocyte is capable of very limited microtubule assembly and that a marked increase in assembly competence occurs during maturation. Our data suggest that the change in assembly competence during maturation is due to the release, activation or synthesis of a stimulatory co-factor.

A significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility

1993 ◽  
Vol 105 (2) ◽  
pp. 433-444 ◽  
Author(s):  
C.F. Chou ◽  
C.L. Riopel ◽  
L.S. Rott ◽  
M.B. Omary
Keyword(s):  
Cell Cycle ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cytosolic Fraction ◽  
Epithelial Cell Line ◽  
Sucrose Density Gradient Centrifugation ◽  
Alternate Mechanism ◽  
10 Nm Filaments

We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.

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