Results and discussion

Transcription

1 Supplementary Figure 1. Assessment of the cross-talk between FuraPE3 and D4ER fluorescent spectra. Cells were perifused with 15 mmol/l glucose (G15) in the presence of 250 µmol/l of the KATP channel opener diazoxide (Dz). Acetylcholine (ACh; 100 µmol/l) or KCl (K45; 45 mmol/l) was added as indicated. Cells were first infected with the D4ER adenovirus and then loaded for 13 min with 100 nmol/l FuraPE3/AM before imaging. The traces are representative of experiments performed on 33 cells.

2 Supplementary Figure 2. Assessment of the cross-talk between FuraPE3 and D4ER fluorescent spectra. Cells were perifused with 15 mmol/l glucose (G15) in the presence of 250 µmol/l of the KATP channel opener diazoxide (Dz). Acetylcholine (ACh; 100 µmol/l) or KCl (K45; 45 mmol/l) was added as indicated. Cells were infected with the D4ER adenovirus but not loaded with FuraPE3/AM before imaging. The traces are representative of experiments performed on 30 cells.

3 Supplementary Figure 3. Assessment of the cross-talk between FuraPE3 and D4ER fluorescent spectra. Cells were perifused with 15 mmol/l glucose (G15) in the presence of 250 µmol/l of the KATP channel opener diazoxide (Dz). Acetylcholine (ACh; 100 µmol/l) or KCl (K45; 45 mmol/l) was added as indicated. Cells were not infected with the D4ER adenovirus but were loaded for 13 min with 100 nmol/l FuraPE3/AM before imaging. Images above the panels show representative cells visualized at the different excitation and emission wavelengths used for FuraPE3 and D4ER imaging. The traces are representative of experiments performed on 15 cells.

4 Results and discussion In cells loaded with FuraPE3 and expressing D4ER, application of 45 mmol/l K and 100 µmol/l ACh induced antiparallel changes of the 340 and 380 nm excitation wavelengths of FuraPE3 and of the 475 and 540 nm emission wavelengths of D4ER (Suppl. Fig. 1). K45 increased both FuraPE3 (340/380 = [Ca 2+ ] c ) and D4ER (540/475 = [Ca 2+ ] ER ) ratios, whereas subsequent addition of ACh elicited the expected antiparallel changes in [Ca 2+ ] c and [Ca 2+ ] ER. To evaluate to which extent the D4ER signal might contaminate the FuraPE3 signal, experiments were performed on cells expressing D4ER but not loaded with FuraPE3 (Suppl. Fig. 2). This revealed that D4ER emits a substantial fluorescent signal when recorded at the wavelengths used for FuraPE3 measurement (keeping the same intensity of light and duration of acquisition of the excitation and emission wavelengths as used for Suppl. Fig. 1, 3). In average, the signal originating from D4ER (measured in 30 cells expressing D4ER but not loaded with FuraPE3) represents 48% and 60% of the global D4ER + FuraPE3 signal recorded, respectively, at 340 nm and 380 nm from 33 cells expressing D4ER and loaded with FuraPE3. This is expected from the overlap of the excitation spectrum of CFP in the D4ER indicator and the excitation spectra of FuraPE3. However, importantly, no change in the 340/380 ratio (FuraPE3 ratio) was observed in response to high K and ACh which, on the other hand, affected [Ca 2+ ] ER (Suppl. Fig. 2). This demonstrates that changes in the D4ER signal do not affect the FuraPE3 ratio. Nevertheless, the substantial contamination of the FuraPE3 signal by D4ER diminishes the amplitude of the true changes in the FuraPE3 ratio when [Ca 2+ ] c changes, which precludes any calibration of the [Ca 2+ ] c signal during simultaneous [Ca 2+ ] c and [Ca 2+ ] ER measurements. To evaluate to which extent the FuraPE3 signal contaminates the D4ER signal, cells were loaded with FuraPE3 but not infected with the D4ER adenovirus (Suppl. Fig.3). This showed that, in average, this signal originating from FuraPE3 (measured in 15 cells loaded with FuraPE3 but that did not express D4ER) represents only 4.1% and 7.3% of the global D4ER + FuraPE3 signal recorded, respectively, at 540 nm and 475 nm from 33 cells expressing D4ER and loaded with FuraPE3. Although changes in the FuraPE3 signal induced changes of the D4ER signal, the changes of the signal at both emission wavelengths of D4ER were of the same amplitude, so that, very importantly, changes in [Ca 2+ ] c did not induce apparent changes in the ratio of the emission wavelengths of D4ER (Suppl. Fig. 3). Overall these experiments demonstrate that, although there is some cross-talk between the two Ca 2+ probes, any change in one signal does not affect the ratio of the other.

5 Supplementary Figure 4. [Ca 2+ ] c measurements (FuraPE3) in β-cells from the experiments illustrated in Fig. 4I. SERCA3 +/+ (solid line) or SERCA3 -/- (dashed line) mice perifused with 15 mmol/l glucose (G15) and 250 µmol/l diazoxide (Dz). Cells were stimulated with 15 mmol (K15) and 45 mmol/l (K45) K when indicated. K45 elicited a large [Ca 2+ ] c hump immediately after the rise in [Ca 2+ ] c in SERCA3 +/+ β-cells only. Values are means ± SE of 37 SERCA3 +/+ and 54 SERCA3 -/- β-cells from 3 islet preparations.

6 Supplemenatry Figure 5. [Ca 2+ ] c and metabolism dependence of Ca 2+ uptake by the ER via pumps other than SERCAs. [Ca 2+ ] ER (D4ER) was measured in β-cells from SERCA3 +/+ mice pretreated for 30 min with 1 µmol/l thapsigargin (pre Thapsi) and perifused in a glucose-free medium (G0) before being submitted to 20 mmol/l glucose (G20), 250 µmol/l diazoxide (Dz), 45 mmol/l K (K45) or 100 µmol/l ACh as indicated on top of the panels. Values are means ± SE for cells from 3 experiments with 3 islet preparations.

7 Supplementary Figure 6. Quenching experiments with Mn 2+ support the existence Ca 2+ influx in hyperpolarized β-cells. After 1h loading with 2 µmol/l FuraPE3/AM, β-cells from SERCA3 +/+ mice were perifused with 15 mmol/l glucose (G15) and 250 µmol/l diazoxide (Dz) throughout. 45 mmol/l (K45) K and 500 µmol/l MnCl 2 (Mn) were added when indicated. The fluorescence of FuraPE3 was recorded at 510 nm upon excitation at 340, 360 or 380 nm. The dashed line in panel A illustrates that the ratio 340/380 nm does no longer accurately reflect [Ca 2+ ] c because of the quenching of the Fura fluorescence by Mn 2+. The traces are representative of experiments performed on 32 cells from 3 islet preparations.

8 Results and discussion The 340 and 380 nm excitation wavelengths of FuraPE3 are Ca 2+ sensitive and hence report the changes in [Ca 2+ ] c, whereas the 360 nm excitation wavelength is Ca 2+ insensitive (= isosbestic point). Mn 2+ can enter through Ca 2+ permeable channels and has the characteristic to quench the fluorescence of Fura. The quenching of the fluorescence of Fura trapped within the cell by Mn 2+ applied in the extracellular medium is often used as an indicator of Ca 2+ entry (1, 2). A first application of K45 elicited the expected antiparallel changes in fluorescence upon excitation at 340 and 380 nm, but did not affect the fluorescence upon excitation at 360 nm, which indicates that 360 nm was the true isosbestic point (does not report changes in Ca 2+ ). Application of Mn 2+ significantly decreased the fluorescence at 360 nm in cells hyperpolarized with diazoxide (see slope of trace 1), i.e. under conditions where voltage-dependent Ca 2+ are closed. This unequivocally shows that there is already a significant entry of Ca 2+ (reported by Mn 2+ ) when the cell is hyperpolarized and [Ca 2+ ] c stays at basal level. As expected, subsequent application of K45 in the presence of Mn 2+ strongly accelerated the speed of the quenching (see steeper slope of trace 2), because of Mn 2+ entry through opened voltage-dependent Ca 2+ channels.

9 Supplementary Figure 7. Characteristics of Ca 2+ uptake by the ER in response to activation of cell metabolism. [Ca 2+ ] ER (D4ER) was measured in -cells from SERCA3 +/+ (A) or SERCA3 -/- (B) mice perifused with a Ca 2+ free medium (Ca0-EGTA) throughout (A) or only at the beginning of the experiment (B). A: 10 mmol/l -ketoisocaproate (KIC) was added when indicated. B: The medium contained 250 µmol/l diazoxide (Dz) throughout, and the glucose concentration was increased from 0 (G0) to 20 mmol/l (G20) before the readmission of 2.5 mmol/l CaCl 2 (Ca2.5). A, B: 100 µmol/l acetylcholine (ACh) was added at the end of the experiments. Values are means ± SE for 17 (A) and 23 (B) cells from 3 islet preparations.

10 Supplementary Figure 8. RNA extraction from freshly isolated mouse control tissues for ryanodine receptor (Ryr) expression (gastrocnemius muscle, total heart and brain cortex) was done using TRIzol Reagent as described before (3). Total RNA from freshly isolated islets, FACS purified β-cells and MIN6 cells was extracted as described in ref. 3 using the Absolutely RNA microprep (Stratagene, CA). FACS purified β-cells were obtained from islets of RIPYY mice expressing EYFP under the rat insulin promoter, i.e. specifically in β-cells (4), and contained > 99% pure β-cells. The abundance of mrna signals corresponding to Ryr1, Ryr2, Ryr3 and the 3 SERCA pumps Atp2a1 (SERCA1), Atp2a2 (SERCA2) and Atp2a3 (SERCA3) were quantified with previously published (3) methods using two Affymetrix microarray platforms: Gene array 1.0 ST (exon oriented) and arrays (3 UTR oriented). Gene array 1.0 ST image files were generated using the Affymetrix GeneChip command console (AGCC). Raw data were analyzed with RMA sketch using the standard settings for Gene 1.0 ST arrays of Expression Console in the AGCC software (affymetrix). The arrays were analyzed using GCOS with the MAS5 algorithm. The fluorescence intensity of each individual chip was scaled to a target intensity of 150 using the global scaling method. Data were log2 transformed for normalization. Two reference genes, beta actin (Actb) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh), were used to assess expression levels in all tested conditions. The identifiers of the informative probe sets for both arrays are shown in the table below. In order to estimate background level of signals for genes which are considered as non expressed, we analyzed the mean signal intensity of a set of 41 olfactory receptor genes for which probe sets exist both on the Gene 1.0 ST arrays and arrays. Mean background signal intensity for this set of negative control genes set was 24±3 for the Gene 1.0 ST arrays and 17±7 for the arrays. probeset probeset GENE array array Ryr _at Ryr _at Ryr _at Atp2a _at Atp2a _at Atp2a _a_at Actb _a_at Gapdh _s_at

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12 Results and discussion Analysis of gene expression by two different methods (Gene array 1.0 ST (exon oriented) and arrays (3 UTR oriented) give very similar results in all tissues. As expected, skeletal muscles (gastrocnemius) and the heart strongly express mrna levels of Ryr1 and Ryr2, respectively. The brain expresses moderate levels of Ryr2 and Ryr3 mrna. However, FACS purified mouse pancreatic β-cells, mouse islets and the mouse insulin-secreting cell line, MIN6 cells, do not express significant amounts of Ryr mrna since the signals for the 3 Ryr isoforms are close to that of the background signal of a set of 41 olfactory receptor genes which are considered to be non expressed in any of the tissue studied. To validate further the method of gene expression analysis, we also evaluated the gene expression of the three SERCA isoforms (Atp2a1 to 3). ATP2a2 (SERCA2) gene (irrespective of both SERCA2a or SERCA2b isoforms) is expressed in all tested tissue, whereas ATP2a1 (SERCA1) and ATP2a3 (SERCA3) genes are specifically expressed in skeletal muscle cells and pancreatic β-cells, respectively. On the other hand, the 2 references genes Actb and Gapdh are, as expected for references genes, expressed at fairly constant levels across all tested tissues. References 1. Liu YJ, Gylfe E. Store-operated Ca 2+ entry in insulin-releasing pancreatic beta-cells. Cell Calcium 22: , Vasudevan SR, Lewis AM, Chan JW, Machin CL, Sinha D, Galione A, Churchill GC: The calciummobilizing messenger nicotinic acid adenine dinucleotide phosphate participates in sperm activation by mediating the acrosome reaction. J Biol Chem 285: Thorrez L, Laudadio I, Van Deun K, Quintens R, Hendrickx N, Granvik M, Lemaire K, Schraenen A, Van Lommel L, Lehnert S, Aguayo-Mazzucato C, Cheng-Xue R, Gilon P, Van Mechelen I, Bonner- Weir S, Lemaigre F, Schuit F. Tissue-specific disallowance of housekeeping genes: the other face of cell differentiation. Genome Res 21: Quoix N, Cheng-Xue R, Guiot Y, Herrera PL, Henquin JC, Gilon P. The GluCre-ROSA26EYFP mouse: a new model for easy identification of living pancreatic alpha-cells. FEBS Lett 581: