Aequorin

The strains expressing the codon-optimized aequorin gene were grown on minimal media for 2.5 days to achieve maximal conidiation. 10⁶ spores with liquid media were distributed to each well of a 96-well microtiter plate (Thermo Fischer, United Kingdom). Six wells were used in parallel for each treatment. The plates were incubated at 37°C for 18 h. The medium was then removed and the cells in each well were washed twice with PGM (20 mM PIPES pH 6.7, 50 mM glucose, 1 mM MgCl₂). Aequorin was reconstituted by incubating mycelia in 100 μL PGM containing 2.5 μM coelenterazine f (Sigma-Aldrich) for 4 h, at 4°C in the dark. After aequorin consititution, mycelia were washed twice with 1 mL PGM and allowed to recover to room temperature for 1 h [79 (link),80 (link)]. To chelate extracellular Ca²⁺, 1 mM EGTA or 8 mM BAPTA was added to each well 10 min prior to stimulus injection.
At the end of each experiment, the active aequorin was completely discharged by permeabilizing the cells with 20% (vol/vol) ethanol in the presence of an excess of calcium (3 M CaCl₂) to determine the total aequorin luminescence of each culture. Luminescence was measured with an LB 96P Microlumat Luminometer (Berthold Technologies, Germany), which was controlled by a dedicated computer running the Microsoft Windows-based Berthold WinGlow software. Conversion of luminescence (relative light units [RLU]) into [Ca²⁺]_c was done using Excel 2007 software (Microsoft). The relative light units (RLU) values were converted into [Ca²⁺]_c concentrations by using the following empirically derived calibration formula: pCa = 0.332588 (-log k) + 5.5593, where k is luminescence (in RLU) s^-1/total luminescence (in RLU) [77 (link)]. Error bars represent the standard error of the mean of six independent experiments, and percentages in the figures represent peak of [Ca²⁺]_c compared to that of the wild-type (100%).

Mammalian expression vectors were constructed using pcDNA3.1 and pcDNA5/FRT/TO (Invitrogen) and the open reading frames of chicken Opn4x, chicken Opn4m, mouse Opn4, amphioxus Opn4, human Opn4, human Rh1 and jellyfish opsin (see the electronic supplementary material).
To make an expression plasmid for a luminescent cAMP reporter, the region for the Glosensor cAMP biosensor was excised from pGlosensor 22F (Promega) and ligated into linearized pcDNA5/FRT/TO. All restriction enzymes were from New England Biolabs (NEB). A luminescent calcium reporter was synthesized using the photoprotein aequorin from Aequorea Victoria [34 (link)] (genbank accession no. AEVAQ440X; electronic supplementary material).

HeLa cells were transfected with mitochondrial Aequorin WT (mtAEQ WT). After 48 h, cells were incubated with 5 μM coelenterazine for 1.5 h in Krebs–Ringer modified buffer (KRB: 125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgSO4, 5.5 mM glucose, and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4, at 37°C) supplemented with 1 mM CaCl₂, and then transferred to the perfusion chamber.
Aequorin measurements were acquired in KRB supplemented with 1 mM CaCl₂, and the agonist was added to the same medium as indicated in figure legends. Cells were then lysed with Triton X-100 in a hypotonic Ca²⁺-rich solution (10 mM CaCl₂ in H2O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca²⁺] values using the algorithm reported in Rizzuto et al. (1992) (link).

HEK293T cells were transiently transfected with PERK full length-myc, PERK-K622A -myc, myc-empty vector, eGFP-empty vector, eGFP-tagged E-Syt1, eGFP-E-Syt1-ΔCDE, eGFP-E-Syt1-ΔSMP or eGFP-E-Syt1-ΔDE using Trans-IT X2 transfection reagent accordingly to the manufacturer’s instructions. HeLa cells were transiently transfected with human HRP-KDEL-myc, Mitochondrial Aequorin WT, mCherry-empty vector, mCherry-tagged E-Syt1, mCherry-E-Syt1-ΔCDE, eGFP-empty vector, eGFP-E-Syt1, eGFP-E-Syt1-ΔCDE, eGFP-Syt1-ΔSMP or eGFP-E-Syt1-ΔDE using Lipofectamin 2000 (Thermo Fisher Scientific) or electroporated with 4D-Nucleofector (Lonza Bioscience) using SE Cell Line kit (Lonza Bioscience). 24 h after transfections, cells were replated to (microscopy) culture dishes (Mattek corporation) or collected for lysate after 48 h. For Oxygen Consumption Rate (OCR) analysis, cells were plated after nucleofection, and OCR analysis was performed using a Seahorse XF24 (Agilent) Extracellular Flux Analyzer 24 h later.