Temperature-responsive optogenetic probes of cell signaling

Abstract

We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and PI3K signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

Oncogenic protein condensates suppress growth factor perception and modulate drug tolerance

“…To pinpoint the node in the EGFR/Erk pathway that was suppressed by EML4-ALK, we measured ppErk responses to optogenetic stimulation at successive nodes of the pathway in the presence or absence of drug ( Figure 3C,D ). We stimulated STE-1 cell lines that stably expressed either optoFGFR or optoSOS, which allows activation of Ras/Erk signals through light-induced membrane recruitment of the SOS catalytic domain(Benman et al, 2022; Toettcher et al, 2013) ( Figure 3D, S2 ). As before, light stimulation of optoFGFR yielded little increase in ppErk, but ALKi pretreatment enhanced ppErk induction ( Figure 3E, left ), consistent with relief of optoFGFR suppression by ALKi.…”

“…To locate the node in the EGFR/Erk pathway that was suppressed by EML4-ALK, we stimulated the pathway at successive nodes using optogenetics in the presence and absence of drug ( Figure 3C,D ). In addition to optoFGFR, we also generated STE-1 cells that expressed optoSOS, which allows activation of Ras/Erk signals through light-induced membrane recruitment of the SOS catalytic domain(Toettcher, Weiner, and Lim 2013; Benman et al 2022) ( Figure 3D, S2 ). As before, light stimulation of optoFGFR yielded little increase in ppErk, but ppErk induction was enhanced with pre-treatment with ALKi ( Figure 3E, left ), consistent with relief of optoFGFR suppression by ALKi.…”

“…For optogenetic control of FGFR signaling (Kim et al, 2014), the FGFR intracellular domain with a N-terminal myristoylation site was inserted upstream of mCh-Cry2 (Bugaj et al, 2013) in a CLPIT plasmid to create CLPIT Myr-mCh-FGFR(ICD)-Cry2. For optogenetic control of SOS signaling, we generated pHR sspB-SOScat-mCh-2A-iLid-CAAX by inserting an mCherry coding sequence to replace BFP, which we described previously (Benman et al, 2022). For live-cell tracking of ppErk activity, we generated pHR ErkKTR-mRuby2 by inserting an mRuby2 coding sequence in place of BFP in a construct we described previously (Benman et al, 2022).…”

“…For optogenetic control of SOS signaling, we generated pHR sspB-SOScat-mCh-2A-iLid-CAAX by inserting an mCherry coding sequence to replace BFP, which we described previously (Benman et al, 2022). For live-cell tracking of ppErk activity, we generated pHR ErkKTR-mRuby2 by inserting an mRuby2 coding sequence in place of BFP in a construct we described previously (Benman et al, 2022). Visualization of nuclei was achieved using pLenti PGK DEST-H2B-iRFP670 (Addgene plasmid #90237).…”

Oncogenic protein condensates suppress growth factor perception and modulate drug tolerance

“…To pinpoint the node in the EGFR/Erk pathway that was suppressed by EML4-ALK, we measured ppErk responses to optogenetic stimulation at successive nodes of the pathway in the presence or absence of drug (Figure 3C,D). We stimulated STE-1 cell lines that stably expressed either optoFGFR or optoSOS, which allows activation of Ras/Erk signals through light-induced membrane recruitment of the SOS catalytic domain 29,30 (Figure 3D, S2). As before, light stimulation of optoFGFR yielded little increase in ppErk, but ALKi pretreatment enhanced ppErk induction (Figure 3E, left), consistent with relief of optoFGFR suppression by ALKi.…”