An expanded palette of bright and photostable organellar Ca²⁺ sensors

Reviewer #1 (Public review):

Summary:

The manuscript by Moret et al. details the development and characterisation of novel ER- and mitochondria-targeted genetically encoded chemogenic Ca2+ sensors.

Strengths:

Compared to existing probes, these sensors exhibited superior responsiveness, brightness, and photostability within the red and far-red emission spectrum, enabling triple compartment Ca2+ measurements (ER, mitochondria, cytosol) and the detection of Ca2+ dynamics in axons and dendrites.

Weaknesses:

The data are robust and convincing, although the manuscript text lacks precision.

Reviewer #2 (Public review):

Summary:

Moret et al. present an engineered family of fluorescent calcium indicators based on HaloCamp, a HaloTag-based sensor system that utilizes Janelia Fluorophores (JF dyes) to report calcium dynamics. By introducing single or multiple amino acid substitutions, the authors reduce HaloCamp's calcium affinity, making these low-affinity variants well-suited for imaging calcium transients in high-calcium environments such as the endoplasmic reticulum (ER) and mitochondria. The study validates the sensors' dissociation constants (Kd), spectra, and multiplex capabilities. It demonstrates improved performance compared to existing tools when targeted to subcellular compartments in mammalian cells and cultured neurons. The sensors can be tuned across the red-to-far-red spectrum via JF585 and JF635 labeling, enabling flexible multiplexed imaging. For example, the authors show that HaloCamp can be targeted to mitochondria and used alongside other green and red sensors, allowing simultaneous imaging of calcium dynamics in the cytosol, ER, and mitochondria. Overall, they achieve their goals, and the data demonstrate that HaloCamp variants are effective for detecting ER and mitochondrial calcium changes under physiological conditions. The presented experiments support the conclusions. However, some key aspects, such as sensor kinetics and axonal validation, would benefit from further analysis.

This work is likely to have an important impact on the fields of calcium imaging and organelle physiology. The modular design of HaloCamp and its compatibility with a wide range of fluorophores offer a broad application range for cell biologists and neuroscientists.

Strengths:

(1) The authors introduce the first tunable, dye-based, low-affinity HaloTag calcium sensors for subcellular imaging, addressing a significant unmet need for ER and mitochondrial calcium detection.

(2) The ability to pair HaloCamp with JF585 and JF635 extends the spectral range, facilitating multiplexed imaging with existing calcium indicators.

(3) The sensors are validated in a range of subcellular compartments (ER, mitochondria, cytosol) in both mammalian cells and neurons.

(4) The authors successfully demonstrate simultaneous imaging of three compartments using orthogonal sensors, a technically impressive feat.

(5) Kd values are measured, and fluorescent responses are tested under physiologically relevant stimulation.

Weaknesses:

(1) The authors do not quantify the kinetics (e.g., decay tau or off-rate) of the fluorescent signals, particularly after stimulation. For example, in the ER imaging experiments in neurons, the decay of the HaloCamp fluorescence after field stimulation (20 APs @ 20 Hz) is not analyzed or compared to ER-GCaMP6-210 or R-CEPIer.

(2) It remains unclear whether the observed decay represents the sensor's off-kinetics or actual physiological calcium clearance from the ER. A comparison between sensors or an independent measurement of ER clearance rates in vitro would clarify this.

(3) The choice of 20 APs at 20 Hz is not justified. Specifically, single APs or low-frequency stimulations are not tested, leaving unclear what the detection threshold of the new sensors is.

(4) In neuron experiments, the authors report measuring ER calcium in axons based presumably on morphology, but no specific justification for selection, markers, or post hoc labeling is described.

(5) Figure 5 assumes that all three indicators (cytosolic, ER, and mitochondrial) are fast enough to report calcium dynamics in response to histamine. This assumption is not fully validated. Cross-controls (e.g., expressing GCaMP6-210 in mitochondria and HaloCamp in the ER) would strengthen confidence that the sensors are correctly reporting dynamic changes.

(6) It is not clear why Thapsigargin leads to depletion in HeLa cells and neurons in experiments shown in Figure 1E, but not in 2B upon field stimulation.