title: 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.
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg.
authors.forename: Samira
authors.initials: S
authors.lastname: Samtleben
authors.forename: Juliane
authors.initials: J
authors.lastname: Jaepel
authors.forename: Caroline
authors.initials: C
authors.lastname: Fecher
authors.forename: Thomas
authors.initials: T
authors.lastname: Andreska
authors.forename: Markus
authors.initials: M
authors.lastname: Rehberg
authors.forename: Robert
authors.initials: R
authors.lastname: Blum
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D043182:Carboxylesterase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002540:Cerebral Cortex / Q000166:cytology; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D009457:Neuroglia / Q000737:chemistry / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D043182:Carboxylesterase; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein