Figures and data
Properties of low affinity variants measured in purified protein at room temperature
Biophysical properties of purified LA-HaloCaMP in comparison to previous sensors.
ER-HaloCaMP585 is a bright and highly responsive red ER Ca2+ sensor.
(A) Chai Discovery-predicted structure of low affinity HaloCaMP showing the mutations in the CaM domain in turquoise, Ca2+ binding sites in yellow and JF585-HTL in red. The HaloTag is shown in grey. (B) Screening for low affinity HaloCaMP variants: in vitro Ca2+ titrations of different purified HaloCaMP variants conjugated with JF585-HTL. (C) Top: targeting scheme for expression in the ER by adding the N-terminal signal peptide of calreticulin (CALR sig peptide) and the C-terminal KDEL retention motif. Bottom: high resolution image of a HeLa cell expressing ER-localized LA-HaloCaMP reconstituted with JF585-HTL shows the ER structure. Pseudocolor scale shows low to high fluorescence intensity. Scale bar, 1 µm. (D) Comparative responsiveness ofER-HaloCaMP585, LAR-GECOl and R-CEPIAer. (E) Quantification ofthapsigargin response (1 µM) in both HeLa cells and cortical neuron somas. (F-H) Comparison of relative brightness of LAR-GECO, R-CEPIAer and ER-HaloCaMP585 using identical illumination and detection conditions in either HeLa cells or cortical neurons. (F) Quantification of sensor brightness in intact HeLa cells or cortical neuron somata. (G) Representative images of brightness level in neuronal somata. Scale bar, 10 µm (H) Quantificationof the ability to detect ER tubules in axons using red ER calcium sensors. Data are represented as mean ± SEM. See Supplementary Table STl for details on statistical tests and sample sizes.
ER-HaloCaMP585 enables ER Ca2+ measurements in subcellular locales with low ER content.
(A-D) Axonal ER Ca2+ responses to stimulation of 20 action potentials (AP) at 20 Hz measured using different indicators. (A) On the left: schematic representation ofER-HaloCaMP585 expressed within a single ER tubule of axon. Right panel shows a representative image of an axon expressing ER-HaloCaMP585 and changes in fluorescence induced by stimulation. Scale bar, 4 µm. Pseudocolor scale of intensity shown below. (B) Axonal ER Ca2+ responses to stimulation of 20AP at 20 Hz before and after 15 minute thapsigargin treatment using ER-HaloCaMP585. (C) Quantification of activity-driven ER Ca2+uptake peak response upon 20 AP (20 Hz) stimulation using ER-GCaMP6-210 (green), ER-HaloCaMP585 (red) and ER-HaloCaMP585 (dark red) in the presence of thapsigargin. (D) Quantification of relative brightness measured with ER-GCaMP-210 or ER-HaloCaMP585 in axons. (E-H) Dendritic ER Ca2+responses to glutamate uncaging in spines. (E) On the left: schematic representation of ER-HaloCaMP585 in dendrites and location of glutamate uncaging on a spine. Right panel shows the dendrites of a neuron expressing ER-HaloCaMP585, (pseudocolor scale relative to the image showing low to high intensity). Scale bar, 4 µm. (F) Dendritic ER Ca2+ responses adjacent to the site of glutamate uncaging measured with ER-HaloCaMP585 or ER-GCaMP-210. Blue ticks indicate the 6 uncaging pulses of 100 ms at 0.25 Hz, 7.2 mW 720 nm uncaging pulses. (G) Quantification of dendritic ER Ca2+peak release. (H) Corresponding quantification of fluorescence recovery 15 s after glutamate uncaging.
Far-red ER Ca2+ measurements using ER-HaloCaMP635.
(A) High resolution image of a HeLa. cell expressing ER-HaloCaMPla reconstituted with JF635-HTL shows the ER structme (pseudocolor scale below showing low to high intensity). Scale bar, 12 µm. (B) Average fluorescence intensity over time, upon thapsigargin treatment (I µM) in HeLa cells, measured with ER-HaloCaMP635. Light gray indicates cells in ‘fyrode’s solution, dark grey indicates when thapsigargin is adeed. (C) Quantification of the thapsigargin response (I µM) in HeLa. cells, measured with different sensors. (D-F) Axonal ER Ca2+ responses to 20 AP (20 Hz) stimulation, using different indicators. (D) On the left: schematic representation of ER-HaloCaMP635 expressed within a single ER tubule of axon. On the right representative image of a neuron expressing ER-HaloCaMP635, zoomed on an axon. Scale bar, 4 µm. Corresponding kymograph (pseudocolor scale below showing low to high intensity). (E) Corresponding fluorescence intensity over time, before and after thapsigargin treatment (I µM). (F) Quantification of activity-driven ER Ca2+ uptake peak response upon 20 AP (20 Hz) stimulation. Data are represented as mean ± SEM. See Supplementary Table STI for details on statistical tests and sample sizes.
Mito-HaloCaMP is a tunable and highly responsive mitochondrial Ca2+ sensor.
Top: targeting scheme for expression in the mitochondria by adding 4 times COX8 mitochondrial targeting sequence (MTS). Bottom: high resolution image of a HeLa cell expressing Mito-HaloCaMP reconstituted with JF585-HTL shows the mitochondrial structures (pseudocolor scale below showing low to high intensity). Scale bar, 2 µm. (B) Average fluorescence intensity over time, upon histamine treatment (10 µM), measured with red mitochondrial calcium indicators in HeLa cells. (C) Corresponding quantification of the peak response. (D) Comparison of relative brightness of mito-RCaMPlb and mito-HaloCaMP585 using identical illumination and detection conditions in intact HeLa cells. (E) On the left: schematic representation of mito-HaloCaMP585 expressed within a single axon. On the right representative image of a neuron expressing mito-HaloCaMP585, zoomed on an axon. Scale bar, 4 µm. Corresponding kymograph (pseudocolor scale below showing low to high intensity). (F) Corresponding average fluorescence intensity over time upon 20 AP (20 Hz) stimulation, measured with red mitochondrial calcium indicators in axons of cortical neurons. (G) Quantification of activity-driven mitochondrial Ca2+uptake peak response upon 20 AP (20 Hz) stimulation or 100 AP (100 Hz) with red mitochondrial calcium indicators.
Far-red mit(rHaloCaMP imaging enables multiplexing Ca2+ signaling.
(A) HeLa cell expressing cytosolic RCaMPlh (red), ER-GCaMP6-210 (green) and MitoHaloCAMP635. Zoomed image shows individual mitochondria. Intensity bar: low to high fluorescence. Scale bar, 2 µm. (B) Averaged responses to histamine (dark grey) in the cytosol (cyto, brown), mitochondria (mito, pwple), and endoplasmic reticulum (ER, green). Vertical colored bars indicate the time at which each organelle response is maximal (C) Time-to-peak Ca2+ responses in each compartment, calculated relative to the cytosolic peak set to t=O. (D) Correlation between ER, cytosolic and mitochondrial Ca2+ signals during histamine. Each dot represents a single cell. Dot size and color intensity are scaled to reflect mitochondrial ΔF/F. (E) Top panel: single cell examples showing low, medium and high mitochondrial Ca2+ responses for similar ER and cytosol Ca2+ dynamics. Lower panel: quantification of ER-mitochondrial Ca2+ transfer during histamine. Mitochondria and cytosol peak responses were used to calculate the coupling factor (see Methods). The line represents a fit to a Hill curve, and the gray shading indicates the 99% confidence interval of the fit. R2(coefficient of determination) = 0.93. (F-1) Axonal Mito-HaloCaMP635 Ca2+ responses to 20 AP (20 Hz) and 100 AP (100 Hz) stimulation (F) On the left: schematic representation of Mito-HaloCaMP635 expressed within a single axon. On the right representative image of a neuron expressing mito-HaloCaMP635, zoomed on an axon. Scale bar, 4 µm. Corresponding kymograph of response, scale bar indicates 5 sec. Pseudocolor scale shows low to high intensity. (G) Average fluorescence intensity over time upon 20 AP (20 Hz) stimulation. (H) Average fluorescence intensity over time upon 100 AP (100 Hz) stimulation. (I) Quantification of activity-driven mitochondrial Ca2+ uptake peak response upon 100 AP (100 Hz) measured with Mito-HaloCaMP635, Mito-GCaMP6f and Mito-HaloCaMP585. Data are represented as mean ± SEM. See Supplementary Table STl for details on statistical tests and sample sizes.
Visualization of ER Ca2+ dynamics in rat and fly brain tissue using ER-BaloCaMP
(A) Representative images of an organotypic hippocampal slice with individual CA3 neurons expressing ER-HaloCaMP585. A magnified view of neurons is shown on the right. Scale bar, 300 µm. (B) ER Ca2+ decreases after a pulse of 1 min application of 50 µM DHPG and overshoots later during washout. Line and shading are mean ± SEM. (C) Quantification of ER Ca2+ release by DHPG in individual neurons using ER-HaloCaMP585. (D) Scheme representing the fly brain and the pars intercerebralis region in which Myosupressin (Ms) neurons are located. ER-HaloCaMP is expressed in Ms neurons, denoted by Ms > ER-HaloCaMP585. (E) Representative 2-photon images of mTagBFP2 and ER-HaloCaMP reconstituted with JF585-HTL. (F) ER Ca2+ depletion by thapsigargin in Ms neurons using ER-HaloCaMP585. (G) Quantification of ER Ca2+ depletion by thapsigargin in individual fly brains using ER-HaloCaMP585. See Supplementary Table STl for details on statistical tests and sample sizes.
Biochemical properties of LA-HaloCaMPl.
(A) Predicted structural models of low-affinity HaloCaMP (LA-HaloCaMP) and HaloTagCPY generated using Chai Discovery. Left: LA-HaloCaMP is shown with HaloTag (gray), the CaM-binding peptide (purple), calmodulin (CaM, cyan) and bound calcium ions (yellow). JF585,HTL is represented in red Right: HaloTagCPY is modeled as a fusion ofHaloTag (PDB ID: 6Y7B) and the CaM--peptide complex (PDB ID: lCDL). (B) In vitro calcium titration of purified LA-HaloCaMP585 protein at room temperature (RT, blue) and 37°C (red), showing EC50 of 86 µM at room temperature (RT) and 89 µMat 37°C. (C) In-cell calibration at 37°C of LA-HaloCaMP5ss in permeabilized HeLa cells, yielding an EC50 of 115 µM. (D) Excitation and emission spectra of LA-HaloCaMP labeled with JF585-HTL in the presence of Ca2+ (red) or Ca2+-free buffered with NTA (gray). Excitation spectra (dashed lines) and emission spectra (solid lines) were normalized to their respective maxima. Data are represented as mean ± SEM. See Supplementary Table STl for details on sample sizes.
Photostability comparisons in axons and dendrites after light exposure
(A) Imaging of ER-GCaMP6-210 in either axons (top) or dendrites (bottom) shows a quick lossin fluorescence compatible with photoswitching of a fraction of the sensor population, resulting in a first decay in fluorescence that is quickly stabilized. (B) Comparative measurements of ER-HaloCaMPsis585 in axons (top) or dendrites (bottom) show that signals were stable right after the illumination of the sample starts, showing improved photostability. Data are represented as mean ± SEM. See Supplementary Table STl for details on sample sizes.
Correlation analysis of Cai+ retpolllM and non-uturatlon of die sensors during histamine.
(A) Single-cell Ca2+ peak responses to histamine in theERand cytosol (left), ER and mitochondria (middle), or mitochondria and cy10S01 (right). (B) Correlation matrix of the data shown in (A), calculated under two assumptions: linear relationships (Pearson’s) and mono10nic relationships (Spearman’s). Each cell shows the respective correlation coefficient and asterisks indicate statistically significant deviation from zero. (C) Paired single-cell Ca2+ peak responses in the ER (left), cytosol (middle), and mitochondria (right) after histamine and subsequent ionomycin treatment. Ionomycin saturates each sensor and provides a signal that is always higher than the histamine peak response, confirming that none of the sensors was saturated by histamine. See Supplementary Table STl for details on statistical tests and sample sizes.
Simultaneous measurements of ER-HaloCaMP585 and mTagBFP2 in Ms fly neurons expressing ER-HaloCaMP585
(A) Mean relative fluorescence changes normalized to baseline over time in response to ER calcium depletion induced by thapsigargin in LA-HaloCaMP585 and mTagBFP2. Shaded areas represent SEM. (B) Quantification of fluorescence changes for ER-HaloCaMP585 (left, red) and mTagBFP2 (right, blue) at baseline and after thapsigargin application in paired samples. ER-HaloCaMP585 fluorescence significantly decreased whereas mTagBFP2 fluorescence remained stable (n.s., not significant). See Supplementary Table STl for details on statistical tests and sample sizes
