Xenbase Image ID: 144596

Figure 3. A GFP-based BiFC assay detects and localizes P/rds self-assembly in transgenic X. laevis photoreceptor cells. We predicted that P/rds tetramerization, as a model for homotypic protein–protein interaction, could drive robust BiFC in situ. A, Confocal micrographs of ocular cryosections from 14 dpf tadpoles showing GFP (green), WGA-labeled photoreceptor OSs (red), and Hoechst stained nuclei (blue). GFP fluorescence in positive control animals expressing a xP/rds-GFP fusion protein was associated solely with OSs. Scale bars, 100 μm. Insets, Transgenic tadpoles were identified in vivo via widefield epifluorescence screening; GFP was clearly visible through the lens and cornea (arrowheads). B, Western blot analysis of whole eye lysates from 14 dpf tadpoles. Lane 1, Nontransgenic control; lane 2, xP/rds-GFP (predicted 66 kDa); lane 3, bP/rds-GFPa (predicted 58 kDa); lane 4, bP/rds-GFPa (predicted 58 kDa) + bP/rds-GFPb (predicted 49 kDa). The anti-GFP antisera reacted with both N- and C-terminal fragments of GFP (and therefore both fusion protein partners). Partners were expressed at predicted MWs as doublets, likely due to heterologous glycosylation. Proteolytic degradation of bP/rds-GFPa was alleviated by coexpression of bP/rds-GFPb. β-Tubulin reactivity was assayed as a loading control. C, Confocal micrographs of ocular cryosections showing BiFC (green), WGA labeling of OSs (white), anti-GFP labeling of transgenic proteins (red), and Hoechst stained nuclei (blue); Scale bars, 20 μm. Coexpression of complementary partners encoding N- and C-terminal GFP fragments (GFPa, GFPb) individually fused to bP/rds sequences produced strong BiFC in both ISs and OSs, consistent with an initial assembly process within the IS. Expression of a single partner (bP/rds-GFPa) did not generate BiFC. D, Distributions of transgenically expressed GFP fusion proteins in a modestly expressing OS, documented at the ultrastructural level by immunogold transmission electron microscopy (TEM) analysis; Scale bar, 2 μm. The bP/rds fusion proteins (detected with anti-GFP Pab290) were primarily localized at disk rims and incisures; occasional lamellar reactivity was observed. Similar results were seen for xP/rds-GFP transgenic expression, as reported previously (Loewen et al., 2003). These patterns are also similar to that previously reported for endogenous X. laevis P/rds (Kedzierski et al., 1996), detected here with Mab 1G9. E, Wider-view fields showing transgenically expressed bP/rds-GFPa + bP/rds-GFPb fusion proteins in modestly (left) and highly (right) expressing cells. Immunogold TEM analysis used anti-GFP Pab290 for labeling the transgenic proteins. COS, Cone outer segment. Scale bar, 2 μm. Image published in: Ritter LM et al. (2011) Copyright © 2011. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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