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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01091

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Expression of the endocytic proteins dynamin and amphiphysin in rat gastric enterochromaffin-like cells

Robert Zanner^1,*, Manfred Gratzl² and Christian Prinz^1,

¹ II Medizinische Klinik und Poliklinik, Technische Universität München, 81675 München, Germany
² Anatomisches Institut, Ludwig-Maximilians-Universität München, 80802 München, Germany

View larger version (189K): [in a new window]   Fig. 1. Typical electron micrograph of an isolated ECL cell. Numerous secretory vesicles are present, some of them containing an electron-dense core (arrows). Bar, 1 µm.

View larger version (32K): [in a new window]   Fig. 2. RT-PCR for dynamin isoforms. All three isoforms were present in ECL cells. Positive (PC12 cells/brain) and negative controls are also shown. MW, molecular weight markers.

View larger version (32K): [in a new window]   Fig. 3. Western blot analysis of dynamin protein expression in ECL cells. Dyn1 and dyn2 produced strong signals after short exposure times, whereas dyn3 was barely detectable. Exocrine chief cells served as a negative control for the dyn1 antibody. Ig, immunoglobulins.

View larger version (47K): [in a new window]   Fig. 4. Immunolocalization of dynamin isoforms in isolated ECL cells using subtype-specific antibodies. Dyn1 immunoreactivity was found in the cytoplasm and at the plasma membrane (A). Dyn2 immunoreactivity was also found in the cytoplasm, the perinuclear region and more strongly at the plasma membrane (B). Dyn3 was mainly detected in the perinuclear area (C). Negative controls (secondary antibodies only) did not produce unspecific staining (D-E'). Bars, 2 µm.

View larger version (48K): [in a new window]   Fig. 5. Immunohistochemical detection of amph1, amph2, and histidine decarboxylase (HDC), the marker enzyme for ECL cells, in the rat stomach. All three antigens were detected in cells of the basal mucosa (A-C). Immunostaining of serial thin sections identified ECL cells as the amph1 and amph2 positive mucosa cells (D,E).

View larger version (78K): [in a new window]   Fig. 6. Immunolocalization of amphiphysin isoforms in isolated ECL cells using subtype-specific antibodies. Both isoforms are found in the cytoplasm with bright spots at the plasma membrane. Bars, 2 µm.

View larger version (75K): [in a new window]   Fig. 7. Double immunolabeling of isolated ECL cells with antibodies against amph2 (A) and dyn1 (B). When images are superimposed, a partial colocalization at the plasma membrane can be observed (C). No staining is detectable in negative controls (secondary antibody only) (D-D"). Bars, 2 µm.

View larger version (51K): [in a new window]   Fig. 8. Detection of amph1 (A) and amph2 (B) in ECL cells by RT-PCR. Positive (brain) and negative controls are also shown. Owing to the inclusion of an alternative spliced site, two bands were detected for amph2 (B). Amplification of full-length amph2 from ECL cell cDNA produced several bands, implying the presence of multiple splice variants (C). MW, molecular weight markers. Overview of amph2 splice forms found in ECL cells (modified from Wigge et al., 1997) (D). Domain structure of the amph2 gene product. Binding sites for AP-2, clathrin and dynamin are marked. PRD, proline rich domain; SH3, src homology domain (E).

View larger version (56K): [in a new window]   Fig. 9. Co-immunoprecipitation of amphiphysin and dynamin from total protein extracts from brain and highly enriched isolated ECL cells. Immunoprecipitation was performed with the amph2-antibody. The control-ip did not show any bands.

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