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First published online April 8, 2004
Journal of Experimental Biology 207, 1625-1632 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00914

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Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium

Jolanta Wiejak, Liliana Surmacz and Elzbieta Wyroba^*

Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland

View larger version (74K): [in a new window]   Fig. 1. Redistribution of ß-adrenergic receptor immunoanalogue in isoproterenol-treated Paramecium cells, viewed by confocal microscopy. (A) Control cells, (B) isoproterenol-treated cells (10 µmol l–1) immunolabeled with antibodies against human ß2-adrenoreceptor and processed for confocal microscopy as described in Materials and methods. ß-adrenergic sites undergo translocation from the cell membrane (A) to the cytoplasmic compartment upon isoproterenol treatment (B). Bar, 15 µm.

View larger version (92K): [in a new window]   Fig. 2. Localization of dynamin and ß-adrenergic receptor (ßAR) immunoanalogue in isoproterenol-treated and untreated Paramecium cells using confocal laser scanning microscopy. Dual fluorochrome immunolabeling was performed as described in Materials and methods. Colocalization (yielding a yellow orange image) of ßAR (green) and dynamin (red) inside the cell in small punctate accumulations (arrows) was observed in the overlay of the series of confocal sections performed at 0.6 µm intervals (A–D). Such a pattern of colocalization was not observed in the untreated cells (E). Bar, 16 µm.

View larger version (86K): [in a new window]   Fig. 3. (A–D) Dynamin-dependent sequestration of ß-adrenergic receptor in isoproterenol-treated Paramecium cells, visualized by post-embedding immunogold electron microscopy. Immunolocalization of ßAR (10 nm gold particles) and dynamin (5 nm gold particles), was performed as described in Materials and methods. Colocalization of ßAR and dynamin in the small endocytic vesicles is observed (A, arrowheads; B–D). No or only scarce ßAR on the surface membrane is observed (A, arrow). (E) In untreated cells ßAR and dynamin colocalized on the plasma membrane. (F–H) Immunolocalization of ßAR alone (F) and dynamin alone (G,H), detected by anti-ßAR and anti-dynamin antibodies, respectively. A significant presence of ßAR on the cell surface (F) occurs, whereas dynamin was localized both on the membrane (H) and in the coated pits (G). Bars, 100 nm.

View larger version (34K): [in a new window]   Fig. 4. Identification of dynamin in Paramecium cells using antibodies against the C-terminal region of human dynamin 2. (A) SDS-PAGE (Ponceau Red stained) and (B) western blot analysis of protein fraction S2 (lane 1). The recombinant rat dynamin 2 (lane 2) was used as a positive control for immunoblot analysis. One immunoreactive band of 105 kDa was detected. Positions of the molecular marker are shown on the left. The western blots shown are representative of three independent experiments.

View larger version (68K): [in a new window]   Fig. 5. PCR amplification and cloning of the putative ßARK homologue in Paramecium. (A) PCR product of 400 bp (lane 2, arrowhead) was electrophoretically transferred onto DEAE-cellulose. DNA was eluted and used as a template for PCR reamplification resulting in the product of predicted molecular size of 400 bp (B; lane 2, arrowhead). Following subcloning into pGEM-T vector and transfection, the plasmid DNA was isolated from the positive colonies (as described in Materials and methods) and subsequently digested with EcoRI. The presence of an insert of the correct size was detected (C; lane 2, arrowhead). Lanes 1 in A–C are molecular mass standards.

View larger version (42K): [in a new window]   Fig. 6. Multiple alignment of ßARK genes from different species. The amino acid sequence of bovine ßARK1 (SEQ 1, Accession no. P21146), human ßARK2 (SEQ 2, Accession no. P35626) and deduced amino acid sequence of Paramecium gene fragment (SEQ 3, Accession no. AF346410) were aligned using the CLUSTAL W program. Identical residues are marked in blue and homologous residues in yellow. Conservative regions, i.e. the catalytic domain and autophosphorylation region, are indicated.