Let There Be Light!

Abstract

The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Förster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use.

An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

“…To investigate VAMP7-rhodopsin interactions in situ, we employed the established proximity ligation assay (PLA), modified for the analysis of vertebrate retinas, where it detects transient rhodopsin interaction with the ciliary targeting complex exclusively in the RIS (Wang and Deretic, 2015;Wang et al, 2017Wang et al, , 2012. Through the fluorescent signalwhich is only present when the PLA probes are <40 nm apart, a distance within the limits of protein-protein interactions (Raykova et al, 2016;Söderberg et al, 2006) we detected potential VAMP7-rhodopsin interaction sites along the entire Golgi/TGN-to-cilia pathway (Fig. 1E, red dots, arrows).…”

“…The PLA was performed using Duolink in situ Detection Reagents Red, containing specific secondary antibodies attached to oligonucleotides. Circular DNA is formed when oligonucleotides are less than <40 nm apart; it is amplified and hybridized to fluorescently labeled oligonucleotides that generate an output in a form of red fluorescent dots of ∼0.5 µm in size, which are visualized by microscopy (Raykova et al, 2016;Söderberg et al, 2006). Only previously validated antibodies were used for this assay.…”

The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis

“…The method can be considered as an in situ mimic of an "immunoprecipitation assay". Proximity ligation assays can identify a protein-protein interaction/association with a distance of 10-30 nm that is in the upper range of that observed using FRET (5-20 nm) and this methodology can detect authentic endogenous proteins in situ that does not rely on transfected or artificially GFPtagged protein vectors [45]. We evaluated whether IFITM1/3 and HLA-B co-associate using this methodology using antibodies to HLA-B and Mab-MHK (that can bind to both IFITM1 and IFITM3 proteins; Supplementary Fig.…”

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking

“…1E. To pinpoint the localization of GBF1 within the Golgi, we performed in situ proximity ligation assay (PLA), a molecular technique suitable for proteomic analysis, because a positive signal is possible only when the fluorescent PLA probes are <40 nm apart (Raykova et al, 2016;Söderberg et al, 2006). We employed PLA modified for studies of brain and retinal tissue (Blasic et al, 2012;Trifilieff et al, 2011;Wang and Deretic, 2015;Wang et al, 2012;Zulliger et al, 2015).…”