AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

Christian Wahl-Schott,Elisabeth S. Butz,Ong N. P. Nguyen,Sybille Böhm,Stylianos Michalakis,Verena Hammelmann,Lisa M. Riedmayr,Martin Biel,Christian Schön,Elvir Becirovic

doi:10.3389/fnins.2016.00356

Abstract

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types.

Highlights

The outer segments (OS) of rod and cone photoreceptors are highly specialized cilia harboring all proteins involved in light-induced phototransduction (Arshavsky and Burns, 2012)
associated virus (AAV) for Fluorescence resonance energy transfer (FRET) in Photoreceptors proteins may randomly interact even though they are spatially separated in vivo, (iii) they do not allow for dynamic measurements, and (iv) exact quantification of interactions is rather difficult as many different technical parameters which might influence the interaction must be controlled rigorously
We propose the application of this method for FRET-based protein-protein interaction studies in other primary cilia of many cell types to study the pathogenesis of other ciliopathies

Summary

INTRODUCTION

The outer segments (OS) of rod and cone photoreceptors are highly specialized cilia harboring all proteins involved in light-induced phototransduction (Arshavsky and Burns, 2012). AAVs for FRET in Photoreceptors proteins may randomly interact even though they are spatially separated in vivo, (iii) they do not allow for dynamic measurements, and (iv) exact quantification of interactions is rather difficult as many different technical parameters which might influence the interaction must be controlled rigorously With regard to these obstacles, FRET is superior to the biochemical approaches. The complete procedure can be performed within 3 weeks on wild type animals and requires only limited expertise and special equipment (Figure 1) We show that this method is suitable for the detection and quantification of protein-protein interactions of proteins involved in the pathogenesis of hereditary degenerative retinal diseases. We propose the application of this method for FRET-based protein-protein interaction studies in other primary cilia of many cell types to study the pathogenesis of other ciliopathies

Experimental Procedure

STEPWISE PROCEDURES

ANTICIPATED RESULTS AND DISCUSSION