Optical estimation of absolute membrane potential using fluorescence lifetime imaging

In the present study, we use VF-FLIM to provide the first cell-resolved, direct visualization of voltage changes induced by growth factor signaling. For long term Vmem recordings during growth-related processes, an optical approach is more attractive than an electrode-based one. Electrophysiology becomes increasingly challenging as time scale lengthens, especially if cells migrate, and washout of the cytosol with pipette solution can change the very signals under study (Horn and Korn, 1992; Malinow and Tsien, 1990). Previous attempts to electrophysiologically record Vmem in EGF-stimulated A431 cells were unsuccessful due to these technical challenges (Pandiella et al., 1989). Because whole cell voltage-clamp electrophysiology was intractable, the Vmem response in EGF-stimulated A431 cells was addressed indirectly through model cell lines expressing EGFR exogenously (Pandiella et al., 1989), bulk measurements on trypsinized cells in suspension (Magni et al., 1991), or cell-attached single channel recordings (Peppelenbosch et al., 1991; Lückhoff and Clapham, 1994; Mozhayeva et al., 1989). By stably recording Vmem during EGF stimulation, VF-FLIM enables direct study of Vmem signaling in otherwise inaccessible pathways.

In conjunction with physiological manipulations and pharmacological perturbations, we explore the molecular mechanisms underlying EGF-induced hyperpolarization. We find that signaling along the EGF-EGFR axis results in a robust hyperpolarizing current carried by K+ ions, passed by the Ca2+-activated K+ channel KCa3.1, and mediated by intracellular Ca2+ (Figure 5C). We achieve a complete loss of the hyperpolarizing response to EGF by altering the K+ driving force (‘High K+’ Figure 5A, Figure 5—figure supplement 1B), blocking calcium-activated K+ currents directly (‘CTX’ and ‘TRAM-34’, Figure 5A, Figure 5—figure supplement 1D,E), or intercepting cytosolic Ca2+ (‘BAPTA-AM’, Figure 5A, Figure 5—figure supplement 1G). These results, combined with transcriptomic evidence that KCa3.1 is the major KCa channel in A431 cells (Thul et al., 2017), indicate that KCa3.1 mediates the observed hyperpolarization. Interestingly, under some conditions where K+-mediated hyperpolarization is blocked (‘CTX,’ ‘high K+', ‘BAPTA-AM’), VF-FLIM reveals a small, secondary depolarizing current not visible during normal EGF stimulation. This current likely arises from initial Ca2+ entry into the cell, as previously observed during EGF signaling (Pandiella et al., 1987; Marquèze-Pouey et al., 2014). Although we have obtained direct and conclusive evidence of EGF-induced hyperpolarization in A431 cells, the interactions between this voltage change and downstream targets of EGFR remain incompletely characterized. Enhancing EGF signaling by blockade of cellular tyrosine phosphatases with orthovanadate (Reddy et al., 2016) correspondingly increases EGF-mediated hyperpolarization (‘Na3VO4’ Figure 5A, Figure 5—figure supplement 1H), but inhibition of downstream kinase activity appears to have little effect on hyperpolarization (‘wortmannin’ Figure 5A, Figure 5—figure supplement 1I).

In the context of RTK signaling, Vmem may serve to modulate the driving force for external Ca2+ entry (Huang and Jan, 2014; Yang and Brackenbury, 2013) and thereby act as a regulator of this canonical signaling ion. Alternatively, Vmem may play a more subtle biophysical role, such as potentiating lipid reorganization in the plasma membrane (Zhou et al., 2015). Small changes in Vmem likely affect signaling pathways in ways that are currently completely unknown, but high throughput discovery of Vmem targets remains challenging. Combination of electrophysiology with single cell transcriptomics has begun to uncover relationships between Vmem and other cellular pathways in excitable cells (Cadwell et al., 2016); such approaches could be coupled to higher throughput VF-FLIM methods to explore pathways that interact with Vmem in non-excitable contexts.

VF-FLIM represents a novel and general approach for interrogating the roles of membrane potential in fundamental cellular physiology. Future improvements to the voltage resolution could be made by use of more sensitive indicators, which may exhibit larger changes in fluorescence lifetime (Woodford et al., 2015). VF-FLIM can be further expanded to include the entire color palette of PeT-based voltage indicators (Huang et al., 2015; Deal et al., 2016), allied with targeting methods to probe absolute membrane potential in heterogeneous cellular populations (Liu et al., 2017; Grenier et al., 2019), and coupled to high-speed imaging techniques for optical quantification of fast voltage events (Raspe et al., 2015; Gao et al., 2014).

VoltageFluor (VF) dyes VF2.1.Cl and VF2.0.Cl were synthesized in house according to previously described syntheses (Woodford et al., 2015). VFs were stored either as solids at room temperature or as 1000x DMSO stocks at −20°C. VF stock concentrations were normalized to the absorption of the dichlorofluorescein dye head via UV-Vis spectroscopy in Dulbecco’s phosphate buffered saline (dPBS, Thermo Fisher Scientific, Waltham, MA) pH 9 with 0.1% sodium dodecyl sulfate (w/v, SDS). Di-8-ANEPPS was purchased from Thermo Fisher Scientific. Di-8-ANEPPS was prepared as a 2 mM (2000x) stock solution in DMSO and stored at −20°C. Di-8-ANEPPS concentrations were determined via UV-Vis spectroscopy in methanol (ε at 498 nm: 41,000 cm−1 M−1 according to the manufacturer’s certificate of analysis).

All salts and buffers were purchased from either Sigma-Aldrich (St. Louis, MO) or Fisher Scientific. TRAM-34, 4-aminopyridine, and charybdotoxin were purchased from Sigma-Aldrich. Gefitinib, wortmannin, sodium orthovanadate, and BAPTA-AM were purchased from Fisher Scientific. Canertinib was a gift from the Kuriyan laboratory at UC Berkeley. Gefitinib, wortmannin, canertinib, and TRAM-34 were made up as 1000x-10000x stock solutions in DMSO and stored at −20°C. Charybdotoxin was made up as a 1000x solution in water and stored at −80°C. 4-aminopyridine was made up as a 20x stock in imaging buffer (HBSS) and stored at 4°C. Recombinantly expressed epidermal growth factor was purchased from PeproTech (Rocky Hill, NJ) and aliquoted as a 1 mg/mL solution in water at −80°C.

Solid sodium orthovanadate was dissolved in water and activated before use (Gordon, 1991). Briefly, orthovanadate solutions were repeatedly boiled and adjusted to pH 10 until the solution was clear and colorless. 200 mM activated orthovanadate stocks were aliquoted and stored at −20°C.

Unless otherwise noted, all imaging experiments were performed in Hank’s Balanced Salt Solution (HBSS; Gibco/Thermo Fisher Scientific). HBSS composition in mM: 137.9 NaCl, 5.3 KCl, 5.6 D-glucose, 4.2 NaHCO3, 1.3 CaCl2, 0.49 MgCl2, 0.44 KH2PO4, 0.41 MgSO4, 0.34 Na2HPO4. High K+ HBSS was made in-house to 285 mOsmol and pH 7.3, containing (in mM): 120 KCl, 23.3 NaCl, 5.6 D-glucose, 4.2 NaHCO3, 1.3 CaCl2, 0.49 MgCl2, 0.44 KH2PO4, 0.41 MgSO4, 0.34 Na2HPO4. Nominally Ca2+/Mg2+ free HBSS (Gibco) contained, in mM: 137.9 NaCl, 5.3 KCl, 5.6 D-glucose, 4.2 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4.

All cell lines were obtained from the UC Berkeley Cell Culture Facility and discarded after twenty-five passages. A431, HEK293T, MCF-7, and MDA-MB-231 cells were authenticated by short tandem repeat (STR) profiling. All cells were routinely tested for mycoplasma contamination. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L D-glucose supplemented with 10% FBS (Seradigm (VWR); Radnor, PA) and 2 mM GlutaMAX (Gibco) in a 5% CO2 incubator at 37°C. Media for MCF-7 cells was supplemented with 1 mM sodium pyruvate (Life Technologies/Thermo Fisher Scientific) and 1x non-essential amino acids (Thermo Fisher Scientific). Media for CHO.K1 (referred to as CHO throughout the text) cells was supplemented with 1x non-essential amino acids. HEK293T and MDA-MB-231 were dissociated with 0.05% Trypsin-EDTA with phenol red (Thermo Fisher Scientific) at 37°C, whereas A431, CHO, and MCF-7 cells were dissociated with 0.25% Trypsin-EDTA with phenol red at 37°C. To avoid potential toxicity of residual trypsin, all cells except for HEK293T were spun down at 250xg or 500xg for 5 min and re-suspended in fresh complete media during passaging.

For use in imaging experiments, cells were plated onto 25 mm diameter poly-D-lysine coated #1.5 glass coverslips (Electron Microscopy Sciences) in six well tissue culture plates (Corning; Corning, NY). To maximize cell attachment, coverslips were treated before use with 1–2 M HCl for 2–5 hr and washed overnight three times with 100% ethanol and three times with deionized water. Coverslips were sterilized by heating to 150°C for 2–3 hr. Before use, coverslips were incubated with poly-D-lysine (Sigma-Aldrich, made as a 0.1 mg/mL solution in phosphate-buffered saline with 10 mM Na3BO4) for 1–10 hr at 37°C and then washed twice with water and twice with Dulbecco’s phosphate buffered saline (dPBS, Gibco).

A431, CHO, HEK293T, and MCF-7 were seeded onto glass coverslips 16–24 hr before microscopy experiments. MDA-MB-231 cells were seeded 48 hr before use because it facilitated formation of gigaseals during whole-cell voltage clamp electrophysiology. Cell densities used for optical resting membrane potential recordings (in 103 cells per cm2) were: A431 42; CHO 42; HEK293T 42; MCF-7 63; MDA-MB-231 42. To ensure the presence of single cells for whole-cell voltage clamp electrophysiology, fast-growing cells were plated more sparsely (approximately 20% confluence) for electrophysiology experiments. Cell densities used for electrophysiology (in 103 cells per cm2) were: A431 36–52; CHO 21; HEK293T 21; MCF-7 63; MDA-MB-231 42. To reduce their rapid growth rate, HEK293T cells were seeded onto glass coverslips in reduced glucose (1 g/L) DMEM with 10% FBS, 2 mM GlutaMAX, and 1 mM sodium pyruvate for electrophysiology experiments.

Cells were loaded with 1x VoltageFluor in HBSS for 20 min in a 37°C incubator with 5% CO2. For most experiments, 100 nM VoltageFluor was used. Serum-starved A431 cells were loaded with 50 nM VoltageFluor. After VF loading, cells were washed once with HBSS and then placed in fresh HBSS for imaging. All imaging experiments were conducted at room temperature under ambient atmosphere. Cells were used immediately after loading the VF dye, and no cells were kept for longer than an hour at room temperature.

Pipettes were pulled from borosilicate glass with filament (Sutter Instruments, Novato, CA) with resistances ranging from 4 to 7 MΩ with a P97 pipette puller (Sutter Instruments). Internal solution composition, in mM (pH 7.25, 285 mOsmol/L): 125 potassium gluconate, 10 KCl, 5 NaCl, 1 EGTA, 10 HEPES, 2 ATP sodium salt, 0.3 GTP sodium salt. EGTA (tetraacid form) was prepared as a stock solution in either 1 M KOH or 10 M NaOH before addition to the internal solution. Pipettes were positioned with an MP-225 micromanipulator (Sutter Instruments). A liquid junction potential of −14 mV was determined by the Liquid Junction Potential Calculator in the pClamp software package (Barry, 1994) (Molecular Devices, San Jose, CA), and all voltage step protocols were corrected for this offset.

For VF-FLIM and CAESR, electrophysiology recordings for VF-FLIM and CAESR were made with an Axopatch 200B amplifier and digitized with a Digidata 1440A (Molecular Devices). The software package used was pClamp 10.3. Signals were filtered with a 5 kHz low-pass Bessel filter. Correction for pipette capacitance was performed in the cell attached configuration. Voltage-lifetime calibrations were performed in V-clamp mode, with the cell held at the potential of interest for 15 or 30 s while lifetime was recorded. Potentials were applied in random order, and membrane test was conducted between each step to verify the quality of the patch. For single cell patching, recordings were only included if they maintained a 30:1 ratio of membrane resistance (Rm) to access resistance (Ra) and an Ra value below 30 MΩ throughout the recording. Due to the reduced health of HEK293T cells transfected with CAESR, recordings were used as long as they maintained a 10:1 Rm:Ra ratio, although most recordings were better than 30:1 Rm:Ra. Only recordings stable for at least four voltage steps (roughly 2 min) were included in the dataset.

For di-8-ANEPPS, electrophysiology recordings were made in the same manner as the above, with the following minor differences. Signals were digitized with a Digidata 1550B; the pClamp 10.6 software package was used (Molecular Devices). Potentials were applied in the order 0 mV, −80 mV, +40 mV, −40 mV, +80 mV for ten seconds at each step. Patch parameters were tested at the beginning and end of the patch program, rather than between each step. Only patches that retained a 30:1 ratio of Rm to Ra and access resistance below 30 MΩ throughout the recording were included in the dataset.

For electrophysiology involving small groups of cells (Figure 2—figure supplement 3), complete voltage clamp across the entire cell group was not possible. Recordings were used as long as Ra remained below 30 MΩ for at least three voltage steps. Most recordings also retained Rm:Ra ratios greater than 20:1.

A431 cells were serum starved prior to epidermal growth factor studies. Two days before the experiment, cells were trypsizined and suspended in complete media with 10% FBS. Cells were then spun down for 5 min at 500xg and re-suspended in reduced serum DMEM (2% FBS, 2 mM GlutaMAX, 4.5 g/L glucose). Cells were seeded onto 25 mm coverslips in six well plates at a density of 84 × 103 cells per cm2. 4–5.5 hr before the experiment, the media was exchanged for serum-free DMEM (0% FBS, 2 mM GlutaMAX, 4.5 g/L glucose).

After 4–5.5 hr in serum-free media, cells were loaded with 50 nM VF dye as described above. In pharmacology experiments, the drug or vehicle was also added to the VF dye loading solution. All subsequent wash and imaging solutions also contained the drug or vehicle. For changes to buffer ionic composition, VoltageFluor dyes were loaded in unmodified HBSS to avoid toxicity from prolonged incubation with high K+ or without Ca2+. Immediately prior to use, cells were washed in the modified HBSS (120 mM K+ or 0 mM Ca2+) and recordings were made in the modified HBSS.

For analysis of short-term responses to EGF (3 min time series), VF lifetime was recorded in 6 sequential 30 s exposures. Immediately after the conclusion of the first frame (30–35 s into the recording), EGF or vehicle (imaging buffer only) was added to the indicated final concentration from a 2x solution in HBSS imaging buffer. For analysis of long-term responses to EGF (15 min time series), EGF addition occurred in the same way, but a gap of 150 s (without laser illumination) was allotted between each 30 s lifetime recording. Times given throughout the text correspond to the start of an exposure. Voltage changes at 2.5 min were calculated from the difference between an initial image (taken before imaging buffer vehicle or EGF addition) and a final image (a 30 s exposure starting 2.5 min into the time series).

The CAESR plasmid was obtained as an agar stab (FCK-Quasar2-Citrine, Addgene #59172), cultured overnight in LB with 100 μg/mL ampicillin, and isolated via a spin miniprep kit (Qiagen). HEK293T cells were plated at a density of 42,000 cells per cm2directly onto a six well tissue culture plate and incubated at 37°C in a humidified incubator for 24 hr prior to transfection. Transfections were performed with Lipofectamine 3000 according to the manufacturer’s protocol (Thermo Fisher Scientific). Cells were allowed to grow an additional 24 hr after transfection before they were plated onto glass coverslips for microscopy experiments (as described above for electrophysiology of untransfected HEK293T cells).

Average voltage changes 2.5 min after addition of EGF to serum deprived A431 cells were determined at different EGF concentrations, and these means were fit to a four parameter logistic function in MATLAB (MathWorks, Natick, MA).

If intracellular and extracellular concentrations, as well as relative permeabilities, of all ionic species are known, the Goldman-Hodgkin-Katz (GHK) equation (Equation 1) can be used to calculate the resting membrane potential of a cell (Hodgkin and Katz, 1949). In practice, the intracellular ion concentrations [X]in and relative permeabilities Px are difficult to determine, so the GHK equation is not a substitute for direct measurement of Vmem. To obtain a range of reasonable Vmem values in systems where these concentrations and relative permeabilities are not known, we calculated possible Vmem using the ‘standard’ parameters derived from Hodgkin and Katz (1949), as well as a value above and a value below each ‘standard’ point. The values evaluated were the following: PK 1; PNa 0.01, 0.05, 0.2; PCl 0.2, 0.45, 0.9; [K+]in 90, 150, 200 mM; [Na+]in 5, 15, 50 mM; [Cl-]in 2, 10, 35 mM. Extracellular ion concentrations [X]out were known (see Materials and methods). In Equation 1, R is the universal gas constant, T is the temperature (293 K for this experiment), and F is Faraday’s constant.(1)Vmem=RTFlnPK[K+]out+PNa[Na+]out+PCl[Cl-]inPK[K+]in+PNa[Na+]in+PCl[Cl-]out

Fluorescence lifetime imaging was conducted on a LSM 510 inverted scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with an SPC-150 or SPC-150N single photon counting card (Becker and Hickl GmbH, Berlin, Germany) (Scheme 1). 80 MHz pulsed excitation was supplied by a Ti:Sapphire laser (MaiTai HP; SpectraPhysics, Santa Clara, CA) tuned to 958 nm and frequency-doubled to 479 nm. The laser was cooled by a recirculating water chiller (Neslab KMC100). Excitation light was directed into the microscope with a series of silver mirrors (Thorlabs, Newton, NJ or Newport Corporation, Irvine, CA).

Excitation light power at the sample was controlled with a neutral density (ND) wheel and a polarizer (P) followed by a polarizing beamsplitter (BS). Light was titrated such that VoltageFluor lifetime did not drift during the experiment, no phototoxicity was visible, and photon pile-up was not visible on the detector. For recordings at high VoltageFluor concentrations (Figure 1—figure supplement 2, Figure 2—figure supplement 4), reduced power was used to avoid saturating the detector. For optical voltage determinations using 50 or 100 nM VoltageFluor, typical average power at the sample was 5 μW.

Fluorescence emission was collected through a 40x oil immersion objective (Zeiss) coated with immersion oil (Immersol 518F, Zeiss). Emitted photons were detected with a hybrid detector, HPM-100–40 (Becker and Hickl), based on a Hamamatsu R10467 GaAsP hybrid photomultiplier tube. Detector dark counts were kept below 1000 per second during acquisition. Emission light was collected through a 550/49 bandpass filter (Semrock, Rochester, NY) after passing through a 488 LP dichroic mirror (Zeiss). The reference photons for determination of photon arrival times were detected with a PHD-400-N high speed photodiode (Becker and Hickl). Data were acquired with 256 time bins in the analog-to-digital-converter and either 64 × 64 or 256 × 256 pixels of spatial resolution (see discussion of pixel size below).

Routine evaluation of the proper functioning of the lifetime recording setup was performed by measurement of three standards (Figure 1—source data 2): 2 μM fluorescein in 0.1 N NaOH, 1 mg/mL erythrosin B in water (pH 7), and the instrument response function (IRF). The IRF was determined from a solution of 500 μM fluorescein and 12.2 M sodium iodide in 0.1 N NaOH. Because of the high concentration of iodide quencher, the IRF solution has a lifetime shorter than the detector response time, allowing approximation of the instrument response function under identical excitation and emission conditions as data acquisition (Liu et al., 2014).

Signal from photons detected in a TCSPC apparatus are convolved with the instrument response (IRF). IRFs can be approximated by the SPCImage fitting software, but consistency of lifetime fits on VF-FLIM datasets was improved by using a measured IRF. Measured IRFs were incorporated by the iterative reconvolution method using SPCImage analysis software (Becker, 2012).

All VoltageFluor lifetime data were fit using SPCImage (Becker and Hickl), which solves the nonlinear least squares problem using the Levenberg-Marquadt algorithm. VF2.1.Cl lifetime data were fit to a sum of two exponential decay components (Equation 2). Attempts to fit the VF2.1.Cl data with a single exponential decay (Equation 3) were unsatisfactory.(2)Ft=a1e-tτ1+a2e-tτ2

The fluorescence lifetime of VF2.0.Cl was adequately described by a single exponential decay for almost all data (Equation 3). A second exponential component was necessary to fit data at VF2.0.Cl concentrations above 500 nM, likely attributable to the concentration-dependent decrease in lifetime that was observed high VF concentrations.(3)Ft=ae-tτ

For all data fit with the two component model, the weighted average of the two lifetimes, τm (Equation 4), was used in subsequent analysis.(4)τm=a1τ1+a2τ2a1+a2