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Figure 4. Self-assembly and trafficking of OS and synaptic photoreceptor proteins fused to complementary Venus fragments produce distinctive patterns of BiFC. Confocal micrographs of tadpole ocular cryosections showing BiFC (green), WGA labeling of OSs (white), anti-GFP labeling of transgenic proteins (red), and Hoechst-stained nuclei (blue). Scale bars, 10 μm. P/rds oligomerization (resulting from Va-HA-P/rds + Vb-MYC-P/rds coexpression) generated robust Venus-based BiFC, essentially similar to that observed when GFP fragments were used (Fig. 2C). The representative cells shown illustrate substantial diffuse complex formation in ISs, with more robust intensities present in OSs (a). CNGB1 homotetramerization (driven by Va-HA-CNGB1 + Vb-MYC-CNGB1 coexpression) also produced BiFC, although the signals generated were far less intense (c). Typical distributions included punctate accumulations within ISs (c, d, arrowheads) and more diffuse localization within OSs that highlighted incisures. Weak synaptic signals were also frequently observed in positive cells. Expression of complementary Venus-rhodopsin partners (RHO-HA-Va + RHO-MYC-Vb) generated the most highly robust BiFC signals observed (e). The most intense BIFC was OS localized; however, substantial signals were present in IS plasma membranes, IS vesicles, and synaptic regions. RIB(B) homotypic interactions (generated by Va-HA-RIB(B) + Vb-MYC-RIB(B) coexpression) were observed to drive robust BiFC (g), seen as diffuse and punctate Venus fluorescence within ISs (g, arrow) and intense accretions within synaptic terminals (g, arrowhead). The BiFC generated by self-assembly of each protein [P/rds, CNGB1, RHO, and RIB(B)] was distributed as a subset of the total transgenic protein present, as visualized by IHC labeling using Pab290 (b, d, f, h) in a given cell/retina. 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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