supporting information (si appendix) - PNAS

SUPPORTING INFORMATION (SI APPENDIX) Phosphatases control PKA dependent functional microdomains at the outer mitochondrial membrane Alex Burdyga, Nicoletta C. Surdo, Stefania Monterisi, Giulietta Di Benedetto, Francesca Grisan, Elisa Penna, Luca Pellegrini, Mario Bortolozzi, PawelSwietach, Tullio Pozzan, and Konstantinos Lefkimmiatis

METHODS Cloning and adenovirus generation The open reading frames of H187, AKAR4 and PP2Aα, were released from pcDNA3 using double digestion with the restriction enzymes HindIII and EcoRI for the two sensors and BamHI and EcoRV for the phosphatase. After gel purification the full length H187, AKAR4 and PP2Aα fragments were introduced to the adenoviral precursor vector pDual CCM(+) (Vector Biolabs). Using single digestion with HindIII, the OMM targeting signal from yeast Tom70 was subsequently introduced to each sensor to create OMM-H187 and OMM-AKAR4 respectively. Vector Biolabs performed the amplification and purification of the viral particles and the final titers were Ad-H187 (2*1010); Ad-OMM-H187 (3.7×1010); Ad-AKAR4 (3.7×1010); Ad-OMM-AKAR4 (3.7×1010).

Immunofluorescence 24-hours after transfection cells were washed twice with cold PBS and then fixed with 4% Formaldehyde for 10 min at room temperature, followed by 3 washes with cold PBS complemented with 22 mg/ml glycine. Cells were permeabilized with PBS 0,1% Triton x-100 for 10 min at room temperature. After 3 washes with cold PBS complemented with 22 mg/ml glycine. Cells were blocked with BSA 5% www.pnas.org/cgi/doi/10.1073/pnas. 1806318115

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for 30 minutes. After over night incubation at 4oC with an anti-cMyc antibody (thermo scientific 9E10) (1:50) followed by washes and 1h incubation with the secondary Goat Anti-Mouse Alexa Fluor® 568 (Thermo-Fisher) (1:500). ProLong® Gold Antifade Mountant containing DAPI was used and imaging was performed by confocal microscopy. In vitro validation of the sensors HEK293 cells were grown in four 175cm2 culture flasks to approximately 60-80% confluence. Each flask was infected with a specific virus (5µl of cyto-AKAR4, 5µl of OMM-AKAR4, 2µl of cyto-H187, 2µl of OMM-H187). Twenty-four hours after infection the cells were washed with PBS and released from the flask's surface by trypsin (0.05%, Gibco). After two PBS washes the cells were mechanically lysed using a gauge-24 blunt-end needle. After elimination of cell debris (clarification) by centrifugation (1500rpm for 3 mins at 4oC) the supernatant was collected and kept at 4oC. For the H187-based sensors the supernatant was complemented with 1mM IBMX to inhibit non-specific PDE activity. Thereafter equal volumes of the supernatants containing H187 or OMM-H187 were treated with increasing doses of cAMP. Treatments were done for both versions of the sensor in parallel. After a 10 min incubation the FRET ratio was measured in 5-6 equal volume drops for each sample for each sensor. For the validation of the two AKAR4-based sensors equal volumes of the supernatants containing AKAR4 or OMM-AKAR4 were treated in parallel with increasing doses of a commercially available purified PKA catalytic subunit alpha (New England Biolabs). After 10 min incubation the FRET ratios for each point for each sensor were collected in parallel as described above.



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FRET ratio (R) data were fitted, as a function of substrate concentration S, to the Grynkiewicz equation, where Rmin, Rmax, n and KS are the minimal R, maximal R, Hill cooperativity number and binding constant, respectively. 𝑅 = 𝑅!"# + 𝑅!"# − 𝑅!"# ×

𝑆! 𝑆 ! + 𝐾! !

Confocal imaging For the co-culture images populations of NRVMs expressing soluble or targeted versions of our sensors were seeded onto glass coverslips coated with laminin. Twenty-four hours after infection the cells were loaded with Hoechst (10µg/ml) for 15min at 37oC and 1nM MitoTracker Red at room temperature for 3min. Subsequently cells were rinsed four times with Ringers modified buffer and imaged on a Zeiss LSM 700 laser scanning confocal microscope using a oil immersion 40x objective (NA 1.4). For the mitochondrial morphology NRVMs were treated with Iso; Fsk, H89 or DMSO. After 24h cells were loaded with 1nM MitoTracker Red at room temperature for 3min and images were collected using a 60x/1.35 Oil UPlanSApo objective for scanning images on an Olympus inverted IX81 Fluoview FV1000 confocal.

Real time PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen) as instructed by the manufaturer. Briefly, freshly isolated NRVMs were cultured into 6-well plates coated with laminin and treated for 24h. Total RNA was quantified and 0.8µg-1µg from each sample was used for the reverse transcription reaction using random hexamers (QuantiTect Reverse Transcription Kit form Qiagen). Multiplex real time reactions were performed on a StepOneTM Real-Time PCR System (ThermoFisher

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Scientific). The TaqMan Gene expression Assay Rn01448584-m1 for (Nr4A3) and Rn03928990-g1 for 18SrRNA (endogenous control) were used. Analysis was performed using the StepOneTM Software. Nr4A3 levels were calculated from at least 3 independent NRVM cultures. Immunoassay for cAMP Freshly isolated NRVMs were counted and equal numbers (3-4x105) plated in 12well plates and treated with the apposite drugs for 24h. Cyclic AMP levels were measured in duplicate for each condition using the Correlate-EIA Direct cAMP kit (Enzo Life Sciences) according to the manufacturer.



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Supplementary Figures & Legends

Supplementary Figure 1. Confocal photomicrographs of NRVMs in co-culture. A. NRVMs in co-culture, expressing the cytosolic cAMP sensor H187 or its OMM variant OMM-H187. B. NRVMs in co-culture, expressing the soluble PKA activity sensor AKAR4 or its OMM variant OMM-AKAR4. Each confocal experiment was repeated at least 3 independent times.



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Supplementary Figure 2. Comparison of minimal and maximal FRET ratios obtained in cells and in vitro. Calibration curves of cytosolic (dark traces) or OMM-targeted (red traces) variants of H187 (cAMP sensor) or AKAR4 (PKA activity sensor) were obtained in vitro (see methods). Shown to the left of the calibration data are FRET ratios measured for each sensor in intact NRVMs under basal conditions (R is expected to be close to Rmin) and in response to maximal stimulation of the cAMP-pathway with Fsk+IBMX (F/I; R is expected to be close to Rmax). Calibration curves were obtained from 4 independent experiments for H187-based sensors and 5 independent experiments for AKAR4-based sensors. The NRVM resting and saturation FRET ratios were calculated from 30-45 randomly selected cells for each sensor.



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Supplementary Figure 3. PDE inhibition does not affect the differences in PKA dependent phosphorylation between cytosol and OMM. A. Co-culture of cells expressing H187 or OMM-H187 subjected to increasing doses of the non-specific PDE inhibitor IBMX produced similar cAMP variations in the two compartments. Average ± SEM of 18 H187-expressing cells, 30 OMM-H187-expressing cells in 4 independent experiments. B. Co-culture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of IBMX. AKAR4 signals in response to IBMX were significantly higher at the OMM when compared to the cytosol. Average ± SEM of 11 AKAR4-expressing cells, 13 OMM-AKAR4-expressing cells in 4 independent experiments.



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C. Cytosolic fractions of NRVMs treated with Fsk combined with IBMX, the PKA inhibitor H89 or vehicle control (DMSO). The phosphorylation status was assessed by a phospho-PKA substrate antibody (RRX(S/T)P). GAPDH was used as cytosolic marker. Experiment repeated 3 times. D. HeLa cells were treated with Fsk for 10 min and thereafter fractionated into mitochondrial and soluble fractions. In order to determine the submitochondrial location of phosphorylated proteins mitochondrial and soluble

fractions

were

subjected

to

Proteinase

K

treatment.

Phosphorylation status of both fractions was evidenced by a phospho-PKA substrate antibody (RRX(S/T)P). The levels of the OMM marker protein Tom20 indicated correct action of Proteinase K. Purity and loading of the mitochondrial fractions was tested using an antibody cocktail against the human OXPHOS subunits, while GAPDH was used as a cytosolic marker. This experiment was repeated twice.



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Supplementary Figure 4. Dissecting the effects of PP2A/PP1 and PP2B. A. Cyclic AMP kinetics measured by OMM-H187 (red trace) or cytosolic H187 (black trace) in NRVMS. Challenge with Fsk 20 µM and IBMX 100 µM (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli the termination kinetics of the two sensors (depending on PDEs) was virtually

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overlapping. Representative traces of an experiment, average + SEM of 4 H187 and 3 OMM-H187 cells from a total of 12 H187-expressing cells, 15 OMM-H187-expressing cells in 4 independent experiments. B. Cyclic AMP kinetics measured by OMM-H187 (red trace) or cytosolic H187 (black trace) in NRVMS. Increasing doses of Iso (100pM and 1nM) induced submaximal production of cAMP. Upon rinsing the stimuli the termination kinetics of the two sensors was virtually overlapping. Representative traces of an experiment, average + SEM of 5 H187 and 3 OMM-H187 cells from a total of 18 H187-expressing cells, 16 OMM-H187-expressing cells in 4 independent experiments. C. In co-cultures of NRVMs expressing AKAR4 or OMM-AKAR4, phosphorylation in response to Fsk was significantly higher at the OMM when compared to the cytosol. However CalA induced a significant increase in cytosolic PKA activity as measured by AKAR4. Average ± SEM of 29 AKAR4-expressing cells, 19 OMM-AKAR4-expressing cells in 6 independent experiments. D. Co-culture of NRVMs expressing AKAR4 or OMM-AKAR4. Fsk induced PKAdependent phosphorylation at the OMM but not in the cytosol and this remained unchanged upon addition of the PP2B inhibitor CsA. E. Total cell lysates of NRVMs treated with increasing concentrations of CsA (50 nM, 200 nM, 1 µM) or vehicle control (DMSO). The phosphorylation status was assessed by a phospho-PKA substrate antibody (RRX(S/T)P) and a general phospho-Ser/Thr antibody. Alpha/beta actin was used as loading control and the experiment was repeated twice.

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Supplementary Figure 5: Effects of soluble and OMM-targeted PP2Aα on the starting ratio of OMM-AKAR4. Summary of the starting AKAR4 (32 cells) and OMM-AKAR4 (68 cells) ratios in the presence of soluble PP2Aα 1.2 µg/ml (22 cells) and 2.4 µg/ml (22 cells) as well as OMM-PP2Aα 1.2 µg/ml (56 cells) and 2.4 µg/ml (16 cells). (***