@article {69733, title = {An expanded palette of bright and photostable organellar Ca2+ sensors}, journal = {bioRxiv}, year = {2025}, month = {01/2025}, abstract = {

The use of fluorescent sensors for functional imaging has revolutionized the study of organellar Ca2+ signaling. However, understanding the dynamic interplay between intracellular Ca2+ sinks and sources requires bright, photostable and multiplexed measurements in each signaling compartment of interest to dissect the origins and destinations of Ca2+ fluxes. We introduce a new toolkit of chemigenetic indicators based on HaloCaMP, optimized to report Ca2+ dynamics in the endoplasmic reticulum (ER) and mitochondria of mammalian cells and neurons. Both ER-HaloCaMP and Mito-HaloCaMP present high brightness and responsiveness, and the use of different HaloTag ligands enables tunable red and far-red emission when quantifying organelle Ca2+ dynamics, expanding significantly multiplexing capacities of Ca2+ signaling. The improved brightness of ER-HaloCaMP using either red or far-red HaloTag ligands enabled measuring ER Ca2+ fluxes in axons of neurons, in which the ER is formed by a tiny tubule of 30-60 nanometers of diameter that impeded measurements with previous red ER Ca2+ sensors. When measuring ER Ca2+ fluxes in activated neuronal dendritic spines of cultured neurons, ER-HaloCaMP presented increased photostability compared to the gold-standard ER Ca2+ sensor in the field, ER-GCaMP6-210, while presenting the same responsiveness. On the other hand, Mito-HaloCaMP presented higher responsiveness than current red sensors, and enabled the first measurements of mitochondrial Ca2+ signaling in far-red in cell lines and primary neurons. As a proof-of-concept, we used 3-plex multiplexing to quantify interorganellar Ca2+ signaling. We show that effective transfer of Ca2+ from the ER to mitochondria depends on the ER releasing a critical amount of Ca2+. When this threshold is not met, the mobilized Ca2+ is diverted to the cytosol instead. Our new toolkit provides an expanded palette of bright, photostable and responsive organellar Ca2+ sensors, which will facilitate future studies of intracellular Ca2+ signaling.

}, doi = {10.1101/2025.01.10.632364}, url = {https://www.biorxiv.org/content/early/2025/01/12/2025.01.10.632364}, author = {Moret, Agathe and Farrants, Helen and Fan, Ruolin and Zingg, Kelsey and Gee, Christine E. and Oertner, Thomas G. and Rangaraju, Vidhya and Schreiter, Eric R. and de Juan-Sanz, Jaime} } @article {68915, title = {Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.}, journal = {ACS Sensors}, volume = {8}, year = {2023}, month = {2023 Aug 25}, pages = {3014-3022}, abstract = {

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.

}, issn = {2379-3694}, doi = {10.1021/acssensors.3c00484}, author = {Shen, Yi and Wen, Yurong and Sposini, Silvia and Vishwanath, Anjali Amrapali and Abdelfattah, Ahmed S and Schreiter, Eric R and Lemieux, M Joanne and de Juan-Sanz, Jaime and Perrais, David and Campbell, Robert E} } @article {48266, title = {Axonal Endoplasmic Reticulum Ca(2+) Content Controls Release Probability in CNS Nerve Terminals.}, journal = {Neuron}, volume = {93}, year = {2017}, month = {2017 Jan 30}, pages = {867-81}, abstract = {

Although the endoplasmic reticulum (ER) extends throughout axons and axonal ER dysfunction is implicated in numerous neurological diseases, its role at nerve terminals is poorly understood. We developed novel genetically encoded ER-targeted low-affinity Ca(2+) indicators optimized for examining axonal ER Ca(2+). Our experiments revealed that presynaptic function is tightly controlled by ER Ca(2+) content. We found that neuronal activity drives net Ca(2+) uptake into presynaptic ER although this activity does not contribute significantly to shaping cytosolic Ca(2+) except during prolonged repetitive firing. In contrast, we found that axonal ER acts as an actuator of plasma membrane (PM) function: [Ca(2+)]ER controls STIM1 activation in presynaptic terminals, which results in the local modulation of presynaptic function, impacting activity-driven Ca(2+) entry and release probability. These experiments reveal a critical role of presynaptic ER in the control of neurotransmitter release and will help frame future investigations into the molecular basis of ER-driven neuronal disease states.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2017.01.010}, author = {de Juan-Sanz, Jaime and Holt, Graham T and Schreiter, Eric R and de Juan, Fernando and Kim, Douglas S and Ryan, Timothy A} }