Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) for Nanoscale Imaging of Protein-Protein Interactions in Cells

Abstract

Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

“…A full proof of this active configuration would require quantitative PALM imaging in multiple colors or atomic scale structural models of Ras/Raf dimers; neither is yet available. Nevertheless, using a combined BiFC and PALM approach (BiFC-PALM), we have recently shown that the complex between KRas G12D and the Ras binding domain of CRaf (Ras-GTP/CRaf-RBD) can further aggregate to form dimers and occasional higher order structures in cells (27). This observation suggests that tetrameric Ras/Raf complexes do exist under physiological conditions.…”

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

“…In order to generate high-resolution maps of specific protein-protein interactions, specific pairs of fragments have been identified for photoactivatable or photoswitchable proteins and used for imaging protein-protein interactions in superresolution [86–89]. For example, a BiFC-PALM approach was recently developed using PAmCherry1 [88]. Nickerson et al .…”

Plasma membrane regulates Ras signaling networks

“…79 Recent dramatic advances in single molecule imaging techniques are giving us a deeper insight into interactions of Raf with Ras clusters. 81,82 Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) allowing detection of protein-protein interactions with 18 nm precision was used to identify K-Ras/Raf clusters of 30 nm in diameter and to observe the nanoscale clustering and diffusion of these complexes from the cell membrane. 81 Similarly, quantitative PALM imaging has allowed visualization of Raf multimers.…”

“…81,82 Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) allowing detection of protein-protein interactions with 18 nm precision was used to identify K-Ras/Raf clusters of 30 nm in diameter and to observe the nanoscale clustering and diffusion of these complexes from the cell membrane. 81 Similarly, quantitative PALM imaging has allowed visualization of Raf multimers. 82 This work suggests that the CAAX motif of Ras drives Raf molecules in clusters.…”