A Synthetic Photoactivated Protein to Generate Local or Global Ca2+ Signals

Abstract

Ca(2+) signals regulate diverse physiological processes through tightly regulated fluxes varying in location, time, frequency, and amplitude. Here, we developed LOVS1K, a genetically encoded and photoactivated synthetic protein to generate local or global Ca(2+) signals. With 300 ms blue light exposure, LOVS1K translocated to Orai1, a plasma membrane Ca(2+) channel, within seconds, generating a local Ca(2+) signal on the plasma membrane, and returning to the cytoplasm after tens of seconds. With repeated photoactivation, global Ca(2+) signals in the cytoplasm were generated to modulate engineered Ca(2+)-inducible proteins. Although Orai1 is typically associated with global store-operated Ca(2+) entry, we demonstrate that Orai1 can also generate local Ca(2+) influx on the plasma membrane. Our photoactivation system can be used to generate spatially and temporally precise Ca(2+) signals and to engineer synthetic proteins that respond to specific Ca(2+) signals.

From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression

“…22,32,33 The present study adds to a growing number of examples that demonstrate the potential of engineered photoreceptors for optogenetics. 16 However, it is noteworthy that despite their indisputable potential, no single engineered photoreceptor 16,[19][20][21][22][23][32][33][34][35][36] has found broad deployment in optogenetics to date. Arguably, the main factors that have impeded their wider adoption are high background activity, low dynamic range, and difficulty in implementation.…”

Algal photoreceptors: in vivo functions and potential applications

“…In the dark state, the LOV domain prevents the interaction of S1K with the plasma membrane Ca 2+ entry channel Orai1. Illumination with blue light changes the conformation of LOV-Jα and allows for translocation of S1K to the Orai1 channel, which opens and produces a local Ca 2+ entry at the plasma membrane (Pham et al 2011).…”

“…For this purpose, Pham et al fused the C-terminal fragment S1K of the calcium-sensor protein STIM1 to LOV-Jα (Fig. 9b) (Pham et al 2011). In the dark state, the LOV domain prevents the interaction of S1K with the plasma membrane Ca 2+ entry channel Orai1.…”

“…a Generation of light-driven membrane depolarization using ChR2 to control neural spiking (Boyden et al 2005). b Blue-light-activated LOV-Jα allows for translocation of the S1K domain to Orai1, a plasma membrane Ca 2+ channel, which generates a local Ca 2+ signal on the plasma membrane (Pham et al 2011). In the dark, LOV blocks the interaction of S1K with Orai1.…”

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes

“…This method does not depend on the relative intensities of the two channels and therefore alleviates the interpretation of true partial or complete colocalization to intermediate PC values (range from 21 to 1: negative values not used for colocalization). All images before automatic colocalization analysis from raw images were corrected post-acquisition using mean background subtraction (Comeau et al, 2006;Pham et al, 2011). …”