A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Abstract

Proximity between proteins plays an essential and ubiquitous role in many biological processes. Molecular tools enabling to control and observe the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced REcognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible, and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allowed furthermore the design of innovative biosensors for the study of various processes, such as signal transduction and apoptosis.

A tripartite chemogenetic fluorescent reporter for imaging ternary protein interactions

“…capable of complementation in presence of fluorogen even in absence of interaction. This system allowed us to develop a fluorogenic chemically induced dimerization tool to induce and visualize the proximity of two proteins 26 . This CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced Recognition) approach was made through bisection of pFAST at position 114-115, generating two fragments pFAST 1-114 (a.k.a.…”

“…The plasmid pAG1223 allowing the mammalian expression of Lyn11-mCherry- C FRB-pFAST 1-98 was constructed by replacing the PYP sequence by the sequence encoding for C FRB-pFAST 1-98 , amplified from pAG1184 and introducing the Lyn11 sequence, by using primers containing the sequence, in the vector pAG097 12 encoding for mCherry-PYP. The plasmid pAG1224 allowing the mammalian expression of MYC-pFAST 99-114 - N FRB-ECFP-Cb5 was generated by replacing the sequence coding for P2A- C FRB-pFAST 1-98 -IRES-HA-mTurquoise2 by the ECFP-Cb5 sequence, amplified from pAG1169 26 encoding FIRE mate-ECFP-Cb5, in the vector pAG1184. The plasmid pAG1239 allowing the mammalian expression MYC-pFAST 99-114 - N FRB-ECFP-Caax was generated by replacing the sequence encoding Cb5 by the Caax sequence, by using primers containing the sequence, in the vector pAG1224.…”

Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform

“…Recent estimates for the number of resident bacteria residing on and within a typical human host average about 3.8 x 10 13 cells, a roughly one-to-one ratio with the total number of host cells 1 . The vast majority of this population would be classified as anaerobic in nature, either requiring strictly anoxic (<0.5% oxygen) or microaerophilic (~2-8% oxygen) conditions.…”

“…That work included the origination of the FAST ligand, a hydroxybenzylidine-rhodanine conjugate whose fluorescence requires complexation with the FAST protein. Subsequent versions of FAST proteins have since been constructed, including: biochemically-improved 8 , red-shifted 9 , split 10 , promiscuous 11 , nano-sized 12 , and chemically-assisted tethering 13 versions. Further exciting advances in companion fluorogen chemistries beyond the original 4-hydroxybenzylidene-rhodanine (HBR) or 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) scaffolds have also been achieved 7 , including the introduction of a far-red emitting FAST fluorogen 9 , GFP-chromophore mimetics 14,15 , and stimuliresponsive derivatives 16 .…”

“…Overall, we observed very weak PL Blaze-lactose fluorescence from S. mutans and very bright fluorescence from a small subpopulation of E. coli cells, as shown by rodshaped fluorescence. To demonstrate that this effect was not due to impairments in FAST fluorescence, an outer membrane protein A 13 chimera with frFAST was constructed, which specifically localizes frFAST to the E. coli outer membrane 24 . When PL Blaze-lactose was added to E. coli cells expressing this construct, membrane and polar-localized fluorescence was readily discernable (Fig.…”