Gene Constructs Design and Cloning

Two versions of photoactivatable ASAP1 (St-Pierre et al., 2014) were prepared. ASAP1 with three photoactivatable mutations (ASAP1-PATM; Berlin et al., 2015) was generated by two separate gene cloning steps. L163F and T164S mutations (correspond to L64F and T65S mutations in the original PA-GFP) were first introduced by polymerase chain reaction (PCR) using SM067A and SM067B primers. Then the T59H mutation that corresponds to the T203H mutation in PA-GFP construct was substituted in by PCR using SM068A and SM068B primers. A PCR with SM070 and SM071Xho1 primers was conducted to cut and paste the whole ASAP1-PATM insert into pcDNA3.1(+) backbone vector. A second type of photoactivatable ASAP1 was generated by switching the OPT (optimum) variant of circularly permuted superfolder GFP (cpsfGFP-OPT) in ASAP1 into a photoactivatable circularly permuted GFP from the short superfolder photoactivatable GCaMP6f (ssPA-GCaMP6f; Berlin et al., 2015). The synthesized gene fragment had Nhe1 and Xho1 restriction sites to facilitate cloning (Integrated DNA technologies, Coralville, IA, USA; Supplementary Data S1). Restriction digest was carried out with the two enzymes to cut and paste the synthesized gene fragment and it was then ligated into the pcDNA3.1(+) backbone vector to acquire ASAP1-ssPA version.

A FRET version PA-GEVI was prepared by mutating Nabi 2.242 developed by Sung et al. (2015). The original Nabi 2.242 has Clover and mRuby2 as a FRET pair. To replace Clover with PA-GFP, a 1,279 base pair long gene fragment that includes a Nhe1 site, PA-GFP, and an Apa1 site was synthesized (Integrated DNA Technologies, Coralville, IA, USA; Supplementary Data S1) and cloned into the Nabi 2.242 vector (Sung et al., 2015). Another set of PCR was conducted to produce PA-Nabi2.242 where SM100A and SM100B primers were used to introduce PA-Nabi2.242 into the pcDNA3.1(+) backbone vector.

Photoactivatable Bongwoori-R3 (PA-Bongwoori-R3) was prepared by replacing Bongwoori-R3’s (Lee et al., 2017) fluorophore with a mutated ecliptic pHluorin (Supplementary Data S1, Miesenböck et al., 1998). T203H and A227D mutations were introduced in the synthesized gene fragment to confer photoactivatability and voltage-sensitivity to the original ecliptic pHluorin. BamH1 and Xho1 restriction sites were used for cloning into the Bongwoori-R3 gene construct. A voltage-sensitive but non-photoactivatable version (ecliptic-Bongwoori-R3_A227D) and a photoactivatable but non-voltage-sensitive version (PA-Bongwoori-R3_T203H_D227A) were generated by PCR with SM105A and SM105B, and SM106A and SM106B primers, respectively. A cytoplasmic version of photoactivatable GFP (pPA-GFP-N1) was purchased from Addgene, USA (#11909).

A cytoplasmic version of photoactivatable ecliptic pHluorin was generated by a simple one-step PCR with SM103 and SM013 to acquire the ecliptic pHluorin part from PA-Bongwoori-R3_T203H_D227A. The polymerized insert was cut and ligated back into pcDNA3.1(+) backbone vector resulting in photoactivatable ecliptic pHluorin D227A (PA-ecliptic pHluorin).

All primers are listed in Supplementary Table S1. The sequences of newly generated gene constructs were verified commercially (Cosmogenetech, South Korea).