Abstract
Ca2+ channels are essential to cell birth, life, and death. They can be externally activated by optogenetic tools, but this requires robust introduction of exogenous optogenetic genes for expression of photosensitive proteins in biological systems. Here we present femtoSOC, a method for direct control of Ca2+ channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on the plasma membrane for multiphoton excitation, we directly induced Ca2+ influx in cultured cells. Mechanistic study reveals that photoexcited flavins covalently bind cysteine residues in Orai1 via thioether bonds, which facilitates Orai1 polymerization to form store-operated calcium channels (SOCs) independently of STIM1, a protein generally participating in SOC formation, enabling all-optical activation of Ca2+ influx and downstream signaling pathways. Moreover, we used femtoSOC to demonstrate direct neural activation both in brain slices in vitro and in intact brains of living mice in vivo in a spatiotemporal-specific manner, indicating potential utility of femtoSOC.
Highlights
Basic performance and validation of femtoSOC As schematically shown in Fig. 1a, the basic operation of femtoSOC is to scan a small area of the plasma membrane of a target cell with a femtosecond pulse laser whose power is typical for two-photon microscopy without the need for any special preparation for the cell
We found that no Ca2+ rise was present either after localizing the femtosecond-laser focus inside the cell nucleus in normal medium
Using localized fluorescent Ca2+-indicative proteins (Lck-GCaMP5G, GCaMP6s, and CEPIA3mt) transfected into cells (Supplementary information, Fig. S3), we visualized Ca2+ dynamics in the plasma membrane, cytoplasm, and mitochondria, respectively and found that the Ca2+ diffusion in both the plasma membrane and cytoplasm clearly initiated from the femtoSOC laser illumination area (Fig. 1f)
Summary
Ca2+ channels are essential to cell birth, life, and death.[1,2] Among Ca2+ channels, store-operated calcium channels (SOCs) are of prominent importance in virtually all cells including excitable cells such as neurons and skeletal muscle cells since they maintain cellular Ca2+ balance and regulate Ca2+ influx from extracellular milieu for various vital physiological functions such as cell development, growth, differentiation, and apoptosis.[3,4,5] They play an important role in the control of gene expression, secretion, and immune response.[6,7,8] The last two decades have seen an extensive study of the molecular mechanism of Ca2+ influx through SOCs, leading to a number of findings about the structure and function of SOCs.[5,7,9,10,11] According to a widely accepted theory, SOCs are plasma membrane ion channels mainly composed of the pore-forming subunit calcium release-activated calcium channel protein 1 (Orai1) and are activated to open in response to the depletion of Ca2+ in the lumen of the endoplasmic reticulum (ER) sensed by the ER-localized protein stromal interaction molecule 1 (STIM1), which polymerizes and relocates near the plasma membrane, where it covalently binds Orai[1] and triggers its formation to be hexamers for the formation of SOCs.[12,13,14]In the last several years, efforts have been made to develop techniques for external activation of SOCs. While these optogenetic techniques are effective and powerful, they require time-consuming, but robust introduction of exogenous optogenetic genes that express the photosensitive proteins into biological systems transiently or stably,[17,18] due to which the strength of the SOC activation varies, depending on their expression level
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