Identification of Ca ²⁺ fluoro-probe loaded and GFP targeted compartments in single cells

Release of Ca ²⁺ from subcellular stores is important for coupling receptor activation to contraction in smooth muscle cells (SMC). Cell compartments of A7R5 SMC's were loaded with Ca ²⁺ sensitive fluoroprobes using AM dyes with subsequent cell permeabilization, a technique previously used to study Ca ²⁺ stores. Cell organelles that loaded with dye were compared to organelle specific markers by 3D imaging coupled with cross-correlation analysis of images from co-labeled cells. Most vesicles containing dye were identified as endosomes/ lysosomes (E/L) while little localized to either mitochondria or Golgi To obtain a specific ER marker, a gene construct containing the sequence for green fluorescent protein (GFP) preceded by the ER targeting sequence of growth hormone and followed by an engineered KDEL sequence was used to produce live cells with fluorescent ER Little Ca ²⁺ probe was found to be co-localized with GFP-targeted ER. Co-labeling with antibodies to Vacuolar(V) type-H ⁺-ATPase, and either SERCA2 or calreticulin indicated approximately 35% co-localization suggesting some organelles in the E/L pathway contain Ca ²⁺ store markers. However, many Ca ²⁺ storage vesicles (SERCA/CR positive) did not coincide with E/L compartments or targeted ER These findings suggest in this SMC: the E/L may store Ca ²⁺; Ca ²⁺ stores are not continuous with the ER lumen and calibration of probes in these compartments must consider their possible acidic nature.