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Local accumulations of B-50/GAP-43 evoke excessive bleb formation in PC12 cells

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Abstract

B-50 (GAP-43) is an axonal, plasma membrane-associated protein involved in growth cone morphology and function. We have conducted immunocytochemical, electron microscopic, and time-lapse experiments to visualize morphological consequences of local accumulations of B-50 at the plasma membrane of B-50-transfected PC-B2 cells, a clonal PC12 cell line with very low expression of endogenous B-50. The distribution of the transfected B-50 within these cells was inhomogeneous. At sites where the B-50 concentration was locally increased up to twofold, numerous filopodia were present in growth cone-like, substrate-attached regions. When local B-50 concentrations were even higher (up to 6.2-fold), blebs were formed, often containing vesicular structures, heavily decorated with B-50 immunoreactivity. Double labeling with f-actin binding phalloidin revealed that local B-50 accumulations were accompanied by increased actin filament concentrations. Colocalization of B-50 with actin filaments was prominent in filopodia, but was virtually absent in blebs, suggesting a disconnection of the bleb plasma membrane from the actin cytoskeleton. We conclude that B-50 evokes distinct effects on cell-surface activity in PC12 cells depending on its local concentration.

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Abbreviations

EM:

electron microscopy

EGFP:

enhanced green fluorescent protein

f-actin:

filamentous actin

FITC:

fluorescein isothiocyanate

GAP:

growth-associated protein

NGF:

nerve growth factor

PBS:

phosphate-buffered saline

PC12:

pheochromocytoma 12

PKC:

protein kinase C

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Authors and Affiliations

  1. Department of Physiological Chemistry, Rudolf Magnus Institute for Neurosciences, Utrecht University, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands

    L. H. J. Aarts & P. Schotman

  2. Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, Utrecht University, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands

    L. H. Schrama, A. B. Oestreicher & W. H. Gispen

  3. Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, Meyerhoferstrasse 1, D-69012, Heidelberg, Germany

    P. Verkade

  4. Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany

    P. Verkade

Authors
  1. L. H. J. Aarts
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  2. P. Verkade
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  4. A. B. Oestreicher
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  5. W. H. Gispen
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Additional information

During the preparation of this manuscript, we lost our colleague A. Beate Oestreicher after a courageous struggle against a fatal disease. With her we lost a very engaging and warm coworker and friend who is deeply missed.

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Aarts, L.H.J., Verkade, P., Schrama, L.H. et al. Local accumulations of B-50/GAP-43 evoke excessive bleb formation in PC12 cells. Mol Neurobiol 20, 17–28 (1999). https://doi.org/10.1007/BF02741362

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