Illuminating pathogen–host intimacy through optogenetics
(A) Scheme of selected opto-tools to modulate and/or monitor second messengers, ions, and pH. Indicated proteins can be targeted to the prokaryotic/eukaryotic pathogens or even to the organelle of choice in the host cell by means of corresponding sorting signal sequence. For simplicity, only few selected organelles of a typical eukaryotic cell are shown to illustrate the concept. The actual proteins for individual applications may differ in their domain structure, mode of action, and light absorption. (B) Light-regulated methods to control gene expression, protein stability, and phosphoinositide signaling, as well as biosensors of lipids and lipid-derived metabolites. Upon illumination, an RNA-guided dCas9 binds to a CRY2-VP64 transactivation domain, which in turn allows otherwise repressed transcription of a gene. LoxP-mediated recombination at a target locus is achieved by a photo-dimerizable CRE recombinase. Light-activated degron: The protein of interest is fused to a photosensitive LOV2 and a proteasome targeting cODC1 domain. Optically induced degradation is facilitated by a conformational shift in the latter 2 domains. CRY2/CIBN fusion to inositol phosphatase enables a concurrent modulation and evaluation of phosphoinositide metabolism. Lipid-binding domains Lact-C2 and PKCδ-C1 fused to GFP allow fluorescent detection of subcellular PtdSer and DAG, respectively. Equally, a fusion of CFP and Venus with IP3-binding motif permits a FRET-based monitoring of IP3. Further details on indicated proteins can be found in S1 Appendix and references therein. CFP, cyan-fluorescent protein; CIBN, N-terminus of CIB1; CRE, cyclization recombinase; DAG, diacylglycerol; FRET, fluorescence-resonance energy transfer; GFP, green fluorescent protein; LACE, light-activated CRISPR-Cas9 effector.