nature | methods

Simultaneous intracellular chloride and pH measurements using a GFP-

based sensor

Daniele Arosio, Fernanda Ricci, Laura Marchetti, Roberta Gualdani, Lorenzo Albertazzi & Fabio Beltram

Supplementary figures and text:

Supplementary Figure 1 Thermodynamics of H+ and Cl– binding to ClopHensor

Supplementary Figure 2 ClopHensor thermodynamics in vivo

Supplementary Figure 3 Cl– association and dissociation kinetics

Supplementary Figure 4 Time-lapse signal stability and corrections

Supplementary Figure 5 Visualization of Cl– fluxes and pH changes in real-time

Supplementary Figure 6 Useful concentration ranges

Supplementary Table 1 Cl– binding kinetic constants

Supplementary Table 2 Primer list

Supplementary Data Sequences of ClopHensor, NPY-ClopHensor and PalmPalm-ClopHensor

Nature Methods: doi:10.1038/nmeth.1471

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Supplementary Figure 1. Thermodynamics of H+ and Cl- binding to ClopHensor (a) Chloride dissociation constant (Cl

dK ) dependence on pH for purified ClopHensor.

Reported values were derived from Langmuir fitting of chloride-binding curves (e.g. Fig. 1f) collected at various pH, 37 °C and 1 M ionic strength, with fluorescence spectrophotometer (■) or confocal microscope (○ and □). The continuous line was drawn after fitting with Eq. S4 and best-fit parameters ± s.d., pKa = 6.81 ± 0.05, Cl

dK1 = 13.1 ±

0.5 mM. The inset depicts the linkage binding scheme for the infinite cooperativity between H+ and Cl- binding to the sensor molecule (M). (b) Net number of H+ exchanged upon Cl- binding (see Eq. 7 in Ref.#12); i.e. binding of one chloride ion to the anionic form of the sensor is linked to the binding of one proton. As a result two intrinsic molecular properties, pKa and

CldK1 , fully characterize

ClopHensor in terms of H+ and Cl- binding.

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Supplementary Figure 2 ClopHensor thermodynamics in vivo Calibration curves of the biosensor signals in vivo in WSS-1 cells and validation of pKa and Cl

dK1 estimation.

(a) Green-to-red ratio for 3 sets of cell cultures ([Cl-] i = 0, 50 and 90 mM) red line was drawn using Eq. S6 and the best-fit (± s.d.) parameter pKa = 6.84 ± 0.05, in agreement to the value (6.78) derived from green-to-cyan ratio (Fig. 2d). (b) Cl- calibration at pH = 6.1 (~fully protonated sensor). Red line was drawn using Eq. S7 and the best-fit (± s.d.) parameter )pH(Cl

dK = 14.4 ± 2.0 mM.

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Supplementary Figure 3 Cl- association and dissociation kinetics Stopped flow analysis of the purified ClopHensor (see Methods online). This figure represents a typical kinetic-fluorescence trace (pH=7.0, [Cl-] = 69 mM). The exponential time constant (τ = 175 ± 4 ms) was obtained from fitting with single time-component function. Under the condition of pseudo-first order kinetic, we extended our kinetic Cl- binding analysis at different pH and chloride concentration values. The recorder kinetic traces were always monoexponential; so a simple relationship between decay rate and [Cl-] (1/τ=kon [Cl-] + koff) allowed us to calculate kon and koff (Supplementary Table 1 online).

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a

b

Supplementary Figure 4 Time-lapse signal stability and corrections Laser intensities in laser scanning confocal microscope exhibit considerable fluctuations in the minute timescale Ref.#18,19. Real-time excitation ratio imaging acquisitions are particularly hampered by this problem. Nevertheless, it could be remedied, in agreement with previous reports Ref.#18,19, by using the transmission detector to update laser-intensity fluctuations dynamically. (a) By inserting a home-made beam sampler into the optical excitation path we measured a significant correlation (r = 0.91) between laser power fluctuations and average intensity (of images) measured with the transmission detector during a 10-minute interval. (b) Stability of cyan-to-red ratio: RCl vs time for 4 cellular ROI before (left panel) and after (right panel) correction for laser-intensity fluctuations α2 = I543 / I458 (inset).

Nature Methods: doi:10.1038/nmeth.1471

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Supplementary Figure 5 Visualization of Cl- fluxes and pH changes in real-time The ability of the ClopHensor to track rapid [Cl-] i changes was tested in WSS-1 cells Ref.#21, which exogenously express GABAA receptors. Cl- fluxes were triggered by puff (500 ms) of 10 mM GABA. (a) [Cl-] i maps at three representative time points during GABAA activation. Calibration bar from 0 to 90 mM, scale bar 10 µm. (b) Temporal evolution of [Cl-] i and (c) pHi within labeled cellular ROI. 3-channel images were collected every 1.666 s, at 400 Hz line frequency, 512×512 pixel format and 1 Airy pinhole aperture. Note that the observed dynamics, in agreement to what reported in hippocampal neurons [Slemmer, J.E., Matsushita, S., De Zeeuw, C.I., Weber, J.T. & Knopfel, T. Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice. Eur J Neurosci 19, 2915-2922 (2004)], was in the minute timescale and so favorably compares with the kinetic of sensor's Cl- response (Supplementary Fig. 3).

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Supplementary Figure 6 Useful concentration ranges Among the parameters most relevant for practical work [Schulte, A., Lorenzen, I., Bottcher, M. & Plieth, C. A novel fluorescent pH probe for expression in plants. Plant Methods 2, 7 (2006)], the useful concentration range may be identified with the range in which a linear relationship holds between the logarithm of ratio and the logarithm of analyte concentration. Panels (b) and (c) show the identification of these ranges for pH and Cl-, respectively. Cl- calibration data (c) were collected at pH 6.3. The useful Cl- concentration range does depend on pH. Panel (a) depicts the ClopHensor useful ranges for pHi and [Cl-] i quantifications (red-contour area) compared to typical intracellular values (grey hatched area) and LDCV values (blue circle).

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Supplementary Table 1 Cl- binding kinetic constants. Data reported are best-fit ± s.d. parameters.

pH kon (s-1 mM-1) koff (s-1) koff/ kon (mM)

5.0 (3.9 ± 0.1) · 10-2 0.6 ± 0.1 15 ± 3

7.1 (4.0 ± 0.1) · 10-2 1.2 ± 0.1 30 ± 5

8.2 (3.5 ± 0.2) · 10-2 2.4 ± 0.2 69 ± 8

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Supplementary Table 2 Primer list

BsaI-E2-fw 5’-ATGGTAGGTCTCAGCGCCGTGAGCAAGGGCGAGGAGCTG-3’

HindIII-E 2-fw 5’-CCCAAGCTTGGGATGGCTAGCTGGAGCCACCCG-3’ E2-BamHI-rv 5’-CCGGAATTCCGGTTACGCGGATCCGCGCTTGTACA

GCTCGTCCAT-3’ BamHI-linker-fw 5’-CTAGTTCCACGTGGATCTGCCTCAGGAGCAGTGAG

CAAGGGCGAGGAG-3’ DsRed-fw 5’-CGCGGATCCGCGTCTGGTGGTGGTGGTGGTCTAGT

TCC ACGTGGATCT-3’ DsRed-BsaI-rv 5’-ATGGTAGGTCTCATATCACTGGGAGCCGGAGTGGC

GGGCCTC-3’ DsRed-XbaI-rv 5’-TGCTCTAGAGCACTACAGGAACAGGTGGTGGCGGC

CCTC-3’ NPY-fw 5’-CTAGCatgctaggtaacaagcgactggggctgtccggactgaccctcgccctg

tccctgctcgtgtgcctgggtgcgctggccgaggcgtacccctccaagccggacaacccgggcgaggacgcaccagcggaggacG-3’

NPY-rv 5’-CTAGCgtcctccgctggtgcgtcctcgcccgggttgtccggcttggaggggtacgcctcggccagcgcacccaggcacacgagcagggacagggcgagggtcagtccggacagccccagtcgcttgttacctagcatG-3’

Gap20-fw 5’-CTAGCatgctgtgctgtatgagaagaaccaaacaggttgaaaagaatgatgaggaccaaaagatcG-3’

Gap20-rv 5’-CTAGCatgctgtgctgtatgagaagaaccaaacaggttgaaaagaatgatgaggaccaaaagatcG-3’

Nature Methods: doi:10.1038/nmeth.1471

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Supplementary Data Sequences of ClopHensor, NPY-ClopHensor and PalmPalm-ClopHensor

Nature Methods: doi:10.1038/nmeth.1471

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Nature Methods: doi:10.1038/nmeth.1471

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Nature Methods: doi:10.1038/nmeth.1471