Characterization and Intracellular Distribution of Microtubule‐Associated Protein 2 in Differentiating Human Neuroblastoma Cells

Kirsch, Joachim; Zutra, Aliza; Littauer, Uriel Z.

doi:10.1111/j.1471-4159.1990.tb04593.x

Cited by 17 publications

(7 citation statements)

References 40 publications

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“…Microtubule Preparation Using Taxol. In some experiments, microtubules (MTs) were prepared from mouse or rat brain, and cultured cells based on the taxol polymerization method described by Kirsch et al (1990). Forebrain from 8-day or adult animals was homogenized at 4°C in 1.5 volumes of PEMF buffer (0.1 MPIPES, pH 6.6, 1 mM EGTA, 1 mM MgSO, and 25 mM NaF) containing 1 mM PMSF, 10 pg/ml leupeptin, 1 pg/ml pepstatin and 10 unit/ ml aprotinin using a glass reservoir/ teflon pestle homogenizer.…”

Section: Methodsmentioning

confidence: 99%

Distinct mode of microtubule‐associated protein 2 expression in the neuroblastoma/glioma cell line 108CC15/NG108‐15

Beaman-Hall

Vallano

1993

J. Neurobiol.

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The properties of microtubule-associated protein-2 (MAP-2) expression were examined in a transformed cell line, and compared to neurons from rodent brain where evidence supports both transcriptional and nontranscriptional regulation of MAP-2 synthesis. A monoclonal antibody that recognizes a common epitope in the adult (HMW MAP-2) and juvenile (MAP-2c) forms was used in an immunoblotting assay to assess the protein levels in actively dividing and differentiated neuroblastoma/glioma (108CC15, also designated NG108-15) cells. Multiply-phosphorylated MAP-2c was the predominant form in actively dividing cells, whereas HMW MAP-2 predominated in differentiated cells, which exhibited several other neuronal-like properties. A progressive increase in the levels of immunoreactive HMW MAP-2 was observed with increasing days of cell differentiation using dBcAMP as the inducing agent. However, the absolute levels of both HMW MAP-2 and MAP-2c in NG108-15 cells were significantly lower (at least 10-fold) than levels measured in rodent brain. To assess whether there are correspondingly lower levels of HMW MAP-2 and MAP-2c mRNAs in NG108-15 cells, relative to rodent brain, a highly sensitive RNA amplification assay (reverse transcription-polymerase chain reaction; RT-PCR) was developed. Oligonucleotide primers were designed to specify either HMW MAP-2 mRNA or MAP-2c mRNA, and whole tissue RNA extracted from adult and neonatal rodent brain was used to verify the reliability of the RT-PCR assay. Accordingly, PCR products of the predicted size, specificity, and abundance were obtained, with similar levels of HMW MAP-2 mRNA and proportionately higher levels of MAP-2c mRNA in neonatal brain, relative to adult brain. MAP-2c mRNA was the predominant transcript in actively dividing NG108-15 cells, and the amount of HMW MAP-2 mRNA gradually increased and became the predominant transcript in cells exposed to dBcAMP for 6-9 days. Thus, the observed changes in MAP-2-specific mRNAs during differentiation paralleled changes in expressed protein, suggesting that MAP-2 synthesis in NG108-15 cells is transcriptionally controlled. However, the levels of both MAP-2 mRNAs in NG108-15 cells were comparable to levels in rodent brain, despite the fact that MAP-2 protein levels are at least 10-fold lower in NG108-15 cells. These data suggest that the low levels of HMW MAP-2 and MAP-2c protein expression in NG108-15 cells are not due to correspondingly lower levels of MAP-2 mRNAs, and that transformed neuronal cell lines demonstrate a unique mode of MAP-2 regulation.

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Section: Methodsmentioning

confidence: 99%

Distinct mode of microtubule‐associated protein 2 expression in the neuroblastoma/glioma cell line 108CC15/NG108‐15

Beaman-Hall

Vallano

1993

J. Neurobiol.

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“…Microtubules were purified essentially as described by Kitsch et al [8]. Briefly, cells were scraped from the culture flasks using a rubber policeman, sedimented at 2000 rpm for 2 rain and washed three times in phosphate buffered saline (PBS) and twice in PEMF buffer (0.1 M PIPES-NaOH, 1 mM EGTA, I mM MgSO~, 25 mM NaF, pH 6.6).…”

Section: Virus and Microtubule Purificationmentioning

confidence: 99%

“…The role of microtubules in the intracellular transport of particles and organelles has been amply documented (reviewed in [15]). Anterograde and retrograde movement of organelles and proteins along microtubules has been shown to be driven by microtubule based motors [6,8,15,16]. Depolymerization of microtubules results in scattering of the Golgi apparatus.…”

Section: Introductionmentioning

confidence: 99%

African swine fever virus interaction with microtubules

Matos

Carvalho

1993

Biology of the Cell

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The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…About that time, Joachim Kirsch identified a new 250-kDa MAP2 isoform (designated MAP2d) in cultured human neuroblastoma cells. MAP2d was also observed in fetal human brain but was not found in adult human cerebellum (68). Analysis of human nerve cell-derived tumors showed that MAP2d is present in specimens from solid tumors diagnosed as neuroblastoma but hardly in ganglioneuroma tumors.…”

mentioning

confidence: 93%

“…By contrast, MAP1B is distributed along the entire length of the neurite. The differences in spatial distribution between MAP1B and MAP2 along the neurites illustrate the heterogeneity in microtubule composition in various parts of the cell process and may suggest a different function for MAP1B (68).…”

mentioning

confidence: 95%