mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo

Abstract

Genetically Encoded Ca²⁺ Indicators (GECIs) are extensively used to study organelle Ca²⁺ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca²⁺ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described.

Keywords: Biological Sciences Tools; Cell Biology; Optical Imaging.

Graphical abstract

Figure 1

Optimization of Mitochondrial and Cytosolic Cameleons (A–D) Cloning and localization of the mitochondrial probe. (A) Schematic representation of the cloning strategy used to modify the mitochondria targeting sequence (4mt) with the elongated version (4mt*) and to substitute ECFP with mCerulean3 in 4mtD3cpv Cameleon probes. (B) Confocal images of 4mtD3mC3. The mitochondrial localization of the probe was evaluated as the co-localization with a mitochondrial marker, TOM20. Yellow color indicates co-localization of the mitochondrial maker and 4mtD3mC3. Scale bar, 10 μm. (C) The bar chart represents the mean ± SEM of the number of cells (N) showing a proper mitochondrial localization, normalized to the number of transfected cells for each field. N ≥ 17 cells for each condition. (D) Mitochondria-targeting sequence cleavage. Twenty-four hours after transfection, total proteins were extracted and subjected to Western blot analysis with antibodies anti-GFP. (E–H) The newly generated mitochondrial mCerulean3-based Cameleon is a functional and brighter probe compared with the original 4mtD3cpv. (E) The bar chart represents the mean ± SEM of ECFP and mCerulean3 fluorescence normalized to cpV fluorescence, in HeLa cells expressing mitochondrial Cameleon probes. N ≥ 39 cells for each condition. (F) The bar chart shows the R_max/R_min of the mitochondrial probes as mean ± SEM (normalized to 4mtD3cpv) of N ≥ 24 cells for each condition. (G) Representative kinetics of mitochondrial Ca²⁺ uptake in HeLa cells expressing 4mtD3cpv (black), 4mtD3mC3 (gray) and 4mtD3mC3+16 (light gray). HeLa cells transiently transfected with 4mtD3cpv, 4mtD3mC3, or 4mtD3mC3+16 were treated with 100 μM histamine (Hist) and perfused with 600 μM EGTA where indicated. (H) Left. Image of HeLa cells expressing 4mtD3mC3+16 along with the analyzed ROIs. Right. Representative kinetics of single mitochondrial Ca²⁺ uptake analysis in HeLa cells expressing 4mtD3mC3+16, using the protocol described for panel G. Data are plotted as ΔR/R₀ as defined in the Transparent Methods section. (I–M) Cloning and localization of the mitochondrial probe. (I) Schematic representation of the cloning strategy used to substitute ECFP with mCerulean3 in D3cpv Cameleon probes. (J) Fluorescence microscope image of the donor (cyan) and the acceptor (yellow) cytosolic D3mC3. Scale bar, 10 μm. (K–M) The newly generated mCerulean3-based Cameleon is a functional and brighter probe compared with the original D3cpv. (K) Representative kinetics of cytosolic Ca²⁺ uptake in HeLa cells expressing D3cpv or D3mC3+16 employing the same protocol as in panel A. Data are presented ΔR/R₀. (L) The bar chart represents the mean ± SEM of ECFP and mCerulean3 fluorescence, normalized to cpV fluorescence, in HeLa cells expressing cytosolic Cameleon probes. N ≥ 27 cells for each condition. (M) The bar chart shows the R_max/R_min of the cytosolic probes as mean ± SEM (normalized to D3cpv) of N ≥ 18 cells for each condition. Statistical significance (*p < 0.05) was detected by Wilcoxon test for comparison between two groups and by one-way ANOVA and Bonferroni post hoc for comparison among three different groups. See also Figure S1 and Table S7.

Figure 2

Ca²⁺ Affinity and Mitochondrial Ca²⁺ Uptake upon Stimulation (A–C) Titration protocol. (A) Representative kinetics of R% in permeabilized HeLa cells transiently expressing the 4mtD3cpv. Where indicated, digitonin-permeabilized cells were perfused with an intracellular-like medium without energy sources and containing different [Ca²⁺] together with 5 μM of the mitochondrial uncoupler, FCCP. In the representative trace 10 μM CaCl₂ was perfused; 5 mM CaCl₂ was finally added to reach the maximal FRET. (B and C) In situ Ca²⁺ titration mCerulean3-based Cameleon (red trace) or original Cameleon (black trace). The graph represents the data as mean ± SEM of N ≥ 5 cells for each [Ca²⁺], at pH 8.0 for mitochondrial (B) and at pH 7 for cytosolic probes (C). (D–G) Stimulated mitochondrial Ca²⁺uptake. Average kinetics of mitochondrial Ca²⁺ uptake in HeLa (D) or MEFs (F) cells expressing 4mtD3mC3+16 (gray trace), 4mtD3cpv (black trace), or mt-Aequorin (mt-aeq, blue trace) employing the same protocol as in Figure 1G, with the only exception to MEFs, stimulated with ATP, 100 μM. Data are presented as [Ca²⁺], mean ± SEM. The bar chart shows the average of the mitochondrial Ca²⁺ peaks elicited by histamine application in HeLa cells (E) and ATP application in MEF cells (G) as mean ± SEM of N ≥ 18 and 7 cells, respectively, for each condition. Statistical significance (*p < 0.05) was detected by one-way ANOVA and Bonferroni post hoc.

Figure 3

FLIM Analysis of FRET Efficiency and Alterations in MCUC Components Protein Level Effect on Mitochondria [Ca²⁺] in Resting Conditions (A) Normalized fluorescence decay of 4mtD3cpv at minimum FRET (black squares) and maximum FRET (blue dots). The full lines are the fitting curves with a two-exponential model. For comparison, the decays of 4mt-CFP expressed in mitochondria are reported (red lines). The insets are the false-color FLIM images (amplitude-weighted lifetime) at minimum and maximum FRET, in the 1- to 4-ns range. Data are from N ≥ 15 cells for each condition. (B) Normalized fluorescence decay of 4mtD3mC3+16 at minimum FRET (black squares) and maximum FRET (blue dots). The full lines are the fitting curves with a two-exponential model. For comparison, the decay of mCerulean3 expressed in mitochondria is reported (red line, one-exponential fitting model). The insets are the false-color FLIM images (amplitude-weighted lifetime) at minimum and maximum FRET, in the 2- to 5-ns range. Data are from N ≥ 15 cells for each condition. (C) FLIM images of 4mtD3mC3+16 in intact HeLa cells in resting conditions. FLIM images report the amplitude-weighted lifetime in the 2- to 3-ns range. Scale bar, 10 μm. (D) The bar chart represents the <τ> mean ± SEM of N ≥ 27 cells expressing 4mtD3mC3+16 and overexpressing MCU, MICU1, or MCU and MICU1. (E) The bar chart represents the mean ± SEM of the R (cpV/mCerulean3), normalized to control, of N ≥ 25 cells expressing 4mtD3mC3+16 and overexpressing MCU, MICU1, or both. (F) The bar chart represents the mean ± SEM of R (% of max, where maximum is the R at pH 9.0) of N ≥ 13 cells transfected with mt-SypHer in the presence of void vector, MCU, MICU1, or MCU and MICU1. (G) The bar chart represents the <τ> mean ± SEM of N ≥ 15 cells expressing 4mtD3mC3+16 and overexpressing MCU, MICU1, or MCU and MICU1, including only data that display the negative rise term in the acceptor channel. See also Figure S2 and Tables S1–S6.

Figure 4

Monitoring [Ca²⁺] Changes in Cardiomyocytes Representative traces of spontaneous Ca²⁺ oscillations in neonatal rat cardiomyocytes expressing AAV9-CMV-D3mC3+16 (A) or AAV9-CMV-4mtD3mC3+16 (B). Representative traces of spontaneous Ca²⁺ oscillations in adult cardiomyocytes isolated from mice injected with AAV9-CMV-D3mC3+16 (C) or AAV9-CMV-4mtD3mC3+16 (D). Representative traces of [Ca²⁺] changes in response to caffeine in adult cardiomyocytes from mice injected with AAV9-CMV-D3mC3+16 (E) or AAV9-CMV-4mtD3mC3+16 (F). Representative traces of spontaneous Ca²⁺ oscillations in human induced pluripotent stem cell-derived cardiomyocytes expressing AAV9-CMV-D3mC3+16 (G) or AAV9-CMV-4mtD3mC3+16 (H). Data are plotted as ΔR/R₀. See also Figure S3.

Figure 5

Monitoring [Ca²⁺] Changes in the Brain: Hippocampus Slices from Mouse, Somatosensory Cortex Slices from Mouse, and In Vivo Mouse Cortex (A and B) Average traces of Ca²⁺ rise induced by DHPG (50 μM) in neurons of hippocampal slices from mice injected with AAV9-syn-D3mC3+16 (A) or AAV9-syn-4mtD3mC3+16 (B) of N ≥ 4 cells. (C and D) Representative traces of Ca²⁺ rise induced by DHPG in neurons of hippocampal slices from mice injected with AAV9-syn-D3mC3+16 (C) or AAV9-syn-4mtD3mC3+16 (D). (E) Average traces of Ca²⁺ rise induced by carbachol (CCH, 500 μM) in neurons of hippocampal slices from mice injected with AAV9-syn-D3mC3+16 (black) or AAV9-syn-4mtD3mC3+16 (gray). N ≥ 5. (F) Average traces of Ca²⁺ rise induced by perfusion of NMDA 50 μM in neurons of hippocampal slices from mice injected with AAV9-syn-D3mC3+16 (black) or AAV9-syn-4mtD3mC3+16 (gray). N ≥ 10. (G) Average traces of Ca²⁺ rise induced by neuronal depolarization induced by NMDA receptor activation with 1 mM NMDA (puff administration) in neurons of cortical slices from mice slices from mice injected with AAV9-syn-D3mC3+16 (black) or AAV9-syn-4mtD3mC3+16 (gray). N ≥ 22. (H) Average traces of Ca²⁺ rise induced by neuronal depolarization with 30 mM KCl in neurons of cortical slices from mice slices from mice infected with AAV9-syn-D3mC3+16 (black) or AAV9-syn-4mtD3mC3+16 (gray). N ≥ 5. (I and J) In vivo experiment showing representative traces of Ca²⁺ rise induced by NMDA receptor activation with 1 mM NMDA (puff administration) in neurons from mice injected with AAV9-syn-D3mC3+16 (I) or AAV9-syn-4mtD3mC3+16 (J). [Ca²⁺] changes have been measured in neuronal cell bodies. Data are plotted as ΔR/R₀. See also Figures S4–S7.