Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA

Abstract

The endoplasmic reticulum (ER) and mitochondria accumulate Ca2+ within their lumens to regulate numerous cell functions. However, determining the dynamics of intraorganellar Ca2+ has proven to be difficult. Here we describe a family of genetically encoded Ca2+ indicators, named calcium-measuring organelle-entrapped protein indicators (CEPIA), which can be utilized for intraorganellar Ca2+ imaging. CEPIA, which emit green, red or blue/green fluorescence, are engineered to bind Ca2+ at intraorganellar Ca2+ concentrations. They can be targeted to different organelles and may be used alongside other fluorescent molecular markers, expanding the range of cell functions that can be simultaneously analysed. The spatiotemporal resolution of CEPIA makes it possible to resolve Ca2+ import into individual mitochondria while simultaneously measuring ER and cytosolic Ca2+. We have used these imaging capabilities to reveal differential Ca2+ handling in individual mitochondria. CEPIA imaging is a useful new tool to further the understanding of organellar functions.

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca 2+ signaling

“…24 h after seeding (15 000 cells in a chamber), HeLa cells were transfected with pCMV24-CEPIA3mt vector (Addgene plasmid #58219), originally developed by Iino and co-workers [36]. The measurements were performed 48 h after transfection in modified Krebs solution, using Zeiss Axio Observer Z1 Inverted Microscope equipped with a × 20 air objective (filter set 62 HE, excitation at 474 ± 28 nm and emission 527 nm) and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany).…”

TRPM7 senses oxidative stress to release Zn 2+ from unique intracellular vesicles

“…The addition of 100 μM ATP to intact cells triggered Ca 2+ spikes in M7Vs that were synchronous with ER Ca 2+ release (simultaneously monitored with the red fluorescent, ER-targeted Ca 2+ sensor, RCEPIAer) ( Fig. S4C) (53) or cytosolic spikes (simultaneously monitored with cytosolic red fluorescent R-GECO1) (Fig. S4D) (54).…”

Imaging approaches to measuring lysosomal calcium

“…Once Ca 2þ -reporter proteins were cloned or engineered (e.g., luminescent aequorin or various fluorescent constructs), this offered more precise control over reporter location by judicious introduction of targeting sequences for a broader array of organelles involved in Ca 2þ signaling, including mitochondria (Rizzuto, Simpson, Brini, & Pozzan, 1992;Suzuki et al, 2014), ER (Suzuki et al 2014), Golgi (Lissandron, Podini, Pizzo, & Pozzan, 2010Pinton, Pozzan, & Rizzuto, 1998), peroxisomes (Drago, Giacomello, Pizzo, & Pozzan, 2008;Lasorsa et al, 2008), and secretory vesicles (Alvarez & Montero, 2002;Dickson, Duman, Moody, Chen, & Hille, 2012;Mitchell et al, 2001;Mitchell et al, 2003;Santodomingo et al, 2008).…”

“…Conversely, it would be pointless using it to monitor changes inside Ca 2þ stores, which are in the millimolar rangedfura-2 would remain saturated with Ca 2þ even if the store was 90% depleted. Therefore, investigators have used low-affinity Ca 2þ reporters for organellar Ca 2þ , be they chemical dyes such as mag-fura-2 (k d 53 mM) and fura-2FF (35 mM) (Hajnoczky & Thomas, 1997) or genetically encoded reporters such as cameleons (Miyawaki, 2011;Palmer & Tsien, 2006) or the newer CEPIAs (w600 mM) (Suzuki et al, 2014).…”