Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca²⁺ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

<div><p>The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic <em>beta-</em>cells in response to D-glucose.</p> </div

Characterization of D1GO-Cam.

<p>(<b><i>A</i></b>) Schematic representation of D1GO-Cam. D1GO-Cam consists of the orange fluorescence protein, (mKO_κ) as the FRET acceptor and a circular permutated green fluorescent protein (cp173-mEGFP) as the FRET donor. In contrast to existing cameleons the FRET acceptor (mKO_κ) is on the N-terminus in front of the Ca²⁺ sensitive domain, while the FRET donor (cp173-mEGFP) is on the C-terminus. FRET between cp173-mEGFP and mKO_κ is increasing upon binding of Ca²⁺ to approved CaM/M13 sequences of the design 1 (D1) of D1GO-Cam. The flash symbols indicate optimal excitation and emission wavelength for imaging GO-Cams. (<b><i>B</i></b>) Representative recordings of cytosolic Ca²⁺ oscillations upon cell stimulation with 100 µM histamine in intact HeLa cells expressing D1GO-Cam. The black curves represent original traces of the FRET donor GFP (upper panel) and the respective FRET signal (lower panel). Original traces (black) were corrected for photobleaching using the red curves (representing the bleaching functions regarding a one phase exponential decay) yielding the corrected traces for the GFP (FRET donor, green trace, upper panel) and the FRET channel (orange trace, lower panel), respectively. (<b><i>C</i></b>) Ratios of FRET/GFP of raw and corrected values from panel B. (<b><i>D</i></b>) Representative tracings of cytosolic Ca²⁺ signals measured with either D1GO-Cam (orange curve) or fura-2 (green curve) in ionomycine (3 µM) treated HeLa cells at various Ca²⁺ concentrations. (<b><i>E</i></b>) Comparative statistics of delta maximal ratios in percentage of D1GO-Cam (white column, n = 8) and fura-2 (black column, n = 8) at different Ca²⁺ concentrations ([Ca²⁺]) in ionomycin (3 µM) treated HeLa cells. The average of delta maximal ratios at 2.0 µM Ca²⁺ were defined as 100%. (<b><i>F</i></b>) The <i>in situ</i> Ca²⁺ concentration response curve of D1GO-Cam was calculated from experiments using ionomycin (10 µM) treated HeLa cells. The actual Ca²⁺ concentrations plotted were determined using respective fura-2 or Magfura-2 signals, which were recorded simultaneously. The curve shown here represents an average of 7 independent experiments.</p

Mitochondrial targeting of 4mtD1GO-Cam.

<p>(<b><i>A</i></b>) Mitochondrial targeting of 4mtD1GO-Cam was visualized on an array confocal laser scanning microscope in Ea.hy926, HeLa, and INS-1 cells. (<b><i>B</i></b>) The efficiency of mitochondrial targeting of the cameleons 4mtD3cpv and 4mtD1GO-Cam was tested in HeLa cells. The proportion of mitochondrial fluorescence was calculated from both sensors in percentage to that of respective mis-targeted cytosolic fluorescence in individual HeLa cells expressing either 4mtD3cpv (n = 23) or 4mtD1GO-Cam (n = 41). *P<0.05 vs. 4mtD3cpv.</p

Comparison of cell type specific coupling between [Ca²⁺]_cyto and [Ca²⁺]_mito.

<p>(<b><i>A–E</i></b>) Fura-2/AM loaded cells expression 4mtD1GO-Cam were used to simultaneously record [Ca²⁺]_cyto (green traces) and [Ca²⁺]_mito (orange traces) in response to cell stimulation with IP₃-generating agonists. Respective zooms into the rising events are presented on right panels. (<b><i>A</i></b>) Representative traces of cytosolic and mitochondrial Ca²⁺ signals in Ea.hy926 in response to 100 µM histamine in the presence of 2 mM Ca²⁺. (<b><i>B</i></b>) Temporal correlation between [Ca²⁺]_cyto and [Ca²⁺]_mito in HeLa cells in response to 100 µM histamine in the presence of 2 mM Ca²⁺. (<b><i>C</i></b>) INS-1 cells were treated with a mixture of 100 µM CCh and 100 µM ATP in the absence of Ca²⁺. (<b><i>D</i></b>) [Ca²⁺]_cyto and [Ca²⁺]_mito in percentage of the respective maximal increases in response to the treatments shown in panels <i>A-C</i> are plotted against each other. Curves are representative for at least 4 independent experiments. (<b><i>E</i></b>) [Ca²⁺]_cyto and [Ca²⁺]_mito are plotted against each other for the Ca²⁺ extrusion phases during and after the removal of the IP₃-generating agonists as indicated in panels <i>A-C</i>. (<b><i>F</i></b>) Representative time course of changes of [Ca²⁺]_cyto in EA.hy926 cells in response to 100 µM histamine in the presence of 2 mM Ca²⁺ simultaneously measured with fura-2 and D1GO-Cam. The right panel shows the zoom into the rising event upon cell treatment with histamine.</p

Determination of spectral overlaps between fura-2 and cameleons.

<p>(<b><i>A</i></b>) Spectral overlaps between fura-2 and cameleons are demonstrated by plotting the Ca²⁺ induced changes of the fluorescence intensities at different excitation wavelength ranging from 315 nm –525 nm. Spectral scans were performed with Ea.hy926 cells on a digital wide field fluorescence microscope with an exposure time of 100 ms at each wavelength. Cells were either loaded with fura-2 or transiently transfected with D3cpv or D1GO-Cam. Emissions from the individual excitation wavelengths were taken at 480 nm (CFP of D3cpv), 510 nm (Fura-2 or GFP from D1GO-Cam), 535 nm (FRET of D3cpv), and 560 nm (FRET of D1GO-Cam), respectively. During the scans cells were stimulated with 10 µM ionomycin in the presence of 2 mM Ca²⁺ to induce strong changes of the fluorescence of the Ca²⁺ sensitive probes. (<b><i>B</i></b>) Representative glucose-induced oscillations of cytosolic Ca²⁺ within same individual INS-1 cells measured simultaneously with fura-2 (dotted blue line) and D1GO-Cam (continuous orange line). Curves are presented in percentage of the respective maximal delta ratio value of the fura-2 signal or the D1GO-Cam signal, respectively.</p

Correlation of repetitive cytosolic and mitochondrial Ca²⁺ transients and glucose-induced Ca²⁺ oscillations in INS-1 cells.

<p>(<i>A</i>) Fura-2/AM loaded INS-1 cells expressing 4mtD1GO-Cam were repetitively stimulated with short pulses of 130 mM K⁺. The time course of both, [Ca²⁺]_cyto (green trace) and [Ca²⁺]_mito (orange trace) is plotted as representative curves. (<b><i>B</i></b>) Representative curves demonstrating [Ca²⁺]_cyto and [Ca²⁺]_mito of single individual INS-1 cells that were 3 times treated with pulses of high K⁺, with a longer recovery time between the second and third addition of 130 mM K⁺. (<b><i>C</i></b>) Temporal correlation between [Ca²⁺]_cyto and [Ca²⁺]_mito of glucose (16 mM) induced Ca²⁺ oscillations in a single individual INS-1 cell. [Ca²⁺]_cyto and [Ca²⁺]_mito were measured simultaneously using fura-2/AM loaded cells expressing 4mtD1GO-Cam. Blue arrows between minute 3 and 7 indicate clear cytosolic Ca²⁺ signals that were not transferred into mitochondria. (D) Zoom into a typical set of glucose induced Ca²⁺ oscillations in INS-1 cells from the curves presented in panel C. (E) Temporal correlations between [Ca²⁺]_cyto (green trace) and [Ca²⁺]_mito (orange trace) of single isolated Ca²⁺ transients of INS-1 cells in response to 16 mM glucose from 3 independent experiments. (F) Statistical evaluation of the temporal correlation between [Ca²⁺]_cyto and [Ca²⁺]_mito of the single isolated Ca²⁺ transients shown in panel A by calculating the average ratio of [Ca²⁺]_cyto/[Ca²⁺]_mito (n = 8). (G) Representative traces of [Ca²⁺]_cyto and [Ca²⁺]_mito of fast subsequent cytosolic Ca²⁺ transients of INS-1 cells in response to 16 mM glucose. (H) Statistical evaluation of the temporal correlation between [Ca²⁺]_cyto and [Ca²⁺]_mito of fast subsequent Ca²⁺ transients by calculating the maximal ratios of [Ca²⁺]_cyto/[Ca²⁺]_mito of respective subsequent signals from 3 independent experiments (n = 3). (J) Representative traces of [Ca²⁺]_cyto and [Ca²⁺]_mito of complex glucose induced Ca²⁺ signal clusters in INS-1 cells.</p