Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Abstract

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

HSPA5 Gene encoding Hsp70 chaperone BiP in the endoplasmic reticulum

“…Under physiological conditions, the Ca 2+ concentration in the ER ranges from 100 to 800µM, which is several orders of magnitude higher than that in the cytosol (~100 nM) (Samtleben et al, 2013). As one of the most abundant proteins in the ER, BiP serves as a high-capacity Ca 2+ binding protein to store Ca 2+ in the ER.…”

Monitoring of intra‐ER free Ca2+

“…The process of low‐affinity calcium indicator accumulation into the ER lumen was recently improved by the ER targeted‐esterase induced dye loading (TED) procedure . Transfection of living cells with a carboxylesterase expressing vector, targeted to the ER compartment, enhances and directs esterification of the AM‐ester form of the dyes in this organelle.…”

Targeted Esterase induced Dye loading supports Calcium Imaging in Eukaryotic Cell-Free Systems

“…Although some current evidences have been reported for electrophysiological experiments, no evidence on functionality assays with ion and voltage sensitive dyes is currently available. The concept of calcium imaging of eukaryotic cell-free translated channels revolves around the previously reported idea of Targeted Esterase induced Dye loading (TED) of the endoplasmic reticulum (ER) in mammalian cells (10,11). We use a mouse carboxylesterase 2 (mCES2) variant because, it is already well characterized for low affinity chemical fluorescent calcium dyes (10).…”

“…The concept of calcium imaging of eukaryotic cell-free translated channels revolves around the previously reported idea of Targeted Esterase induced Dye loading (TED) of the endoplasmic reticulum (ER) in mammalian cells (10,11). We use a mouse carboxylesterase 2 (mCES2) variant because, it is already well characterized for low affinity chemical fluorescent calcium dyes (10). We focus on studying calcium, being customarily a permeabilized ion through several families of ion channels and a second messenger in signal transduction in large number of cellular pathways.…”