Tuning Protein Dynamics to Sense Rapid Endoplasmic‐Reticulum Calcium Dynamics

Abstract

Multi-scale calcium (Ca 2+ )d ynamics,e xhibiting wide-ranging temporal kinetics,constitutes aubiquitous mode of signal transduction. We report an ovel endoplasmicreticulum (ER)-targeted Ca 2+ indicator,R -CatchER,w hich showed superior kinetics in vitro (k off ! 2 10 3 s À1 ,k on ! 7 10 6 M À1 s À1 )a nd in multiple cell types.R -CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints,e nabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose ap rinciple to rationally design genetically encoded Ca 2+ indicators with as ingle Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore.T he design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range.Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery.

Arc ubiquitination regulates endoplasmic reticulum-mediated Ca2+release and CaMKII signaling

“…5F,G; two-way ANOVA, Genotype: F(1, 32) = 9.120, p = 0.0049, Time: F(1, 32) = 39.59, p < 0.0001, Interaction: F(1, 32) = 6.651, p = 0.0147; Sidak’s post-hoc test, WT vs KR at End p = 0.0008), PD (Figures 1H,I; two-way ANOVA, Genotype: F(1, 32) = 7.966, p = 0.0081, Time: F(1, 32) = 24.04, p < 0.0001, Interaction: F(1, 32) = 7.966, p = 0.0063; Sidak’s post-hoc test, WT vs KR at End p = 0.0006), and SD (Figures 5L,M; two-way ANOVA, Genotype: F(1, 32) = 32.51, p < 0.0001, Time: F(1, 32) = 36.77, p < 0.0001, Interaction: F(1, 32) = 48.36, p < 0.0001; Sidak’s post-hoc test, WT vs KR at End p < 0.0001) of KR neurons. However, KR SB showed no difference in release compared to WT neurons (Figures 1J,K; two-way ANOVA, Genotype: F(1, 32) = 0.3066, p = 0.5836, Time: F(1, 32) = 8.865, p = 0.0055, Interaction: F(1, 32) = 0.6407, p = 0.4294), which related to our previous findings that Ca 2+ release at secondary branchpoints is reduced (Deng et al, 2021; Reddish et al, 2021).…”

“…G-CatchER + is specifically targeted and expressed in the ER lumen; thus, a decrease in G-CatchER + fluorescence reflects Ca 2+ being released from the ER (Reddish et al, 2021). As the subcellular distribution of Ca 2+ signaling-related proteins, ER complexity, intracellular [Ca 2+ ], and ER Ca 2+ release can vary throughout the neuron (Sharp et al, 1993; Terasaki et al, 1994; Spacek and Harris, 1997; Blaustein and Golovina, 2001; Cui-Wang et al, 2012; Krijnse-Locker et al, 2017), we examined various neuronal subregions separately: soma, primary branchpoints (PBs), primary dendrites (PDs), secondary branchpoints (SBs), and secondary dendrites (SDs) (Deng et al, 2021; Reddish et al, 2021) (Figures 1A-1C). G-CatchER + fluorescence traces showed a striking depletion of ER Ca 2+ stores in KR neurons after DHPG application (Figure 1).…”

“…For highly illuminated laminated optical sheet (HILO) microscopy, a fiber-coupled 488 nm laser (Oxxius) was first collimated and then focused at the back focal plane of a 100X oil TIRF objective (N.A. 1.49, Nikon) using an achromatic lens with a focal length of 200 mm (Thorlabs) as previously described (Deng et al, 2021; Reddish et al, 2021). The position of the focused laser at the back focal plane of the objective was controlled by a 2D translational stage (Thorlabs).…”

“…Live cell images were analyzed as previously described (Deng et al, 2021; Reddish et al, 2021) in ImageJ (NIH) by manually drawing a region of interest around clearly visible sub-regions of neurons (soma, primary branchpoints (PB)s, primary dendrites (PD)s, secondary branchpoints (SB)s, and secondary dendrites (SD)s) and fluorescence traces extracted. Raw pixel values were normalized to the average baseline fluorescence to generate a profile plot.…”

“…The copyright holder for this preprint this version posted September 4, 2022. ; https://doi.org/10.1101/2022.09.02.506383 doi: bioRxiv preprint WT and ArcKR (KR) primary hippocampal neuron cultures were transfected with the ER-localized genetically-encoded calcium indicator G-CatchER + (Reddish et al, 2021) and imaged on a highly inclined laminated optical sheet (HILO) microscope during the addition of 100 µM S-3,5-DHPG. As the subcellular distribution of Ca 2+ signaling-related proteins, ER complexity, intracellular [Ca 2+ ], and ER Ca 2+ release can vary throughout the neuron (Sharp et al, 1993;Terasaki et al, 1994;Spacek and Harris, 1997;Blaustein and Golovina, 2001;Cui-Wang et al, 2012;Krijnse-Locker et al, 2017), we examined various neuronal subregions separately: primary branchpoints (PBs), primary dendrites (PDs), secondary branchpoints (SBs), and secondary dendrites (SDs) (Deng et al, 2021;Reddish et al, 2021) (Figures 1A-1C).…”