A new fluorescence image-processing method to visualize Ca2 + -release and uptake Endoplasmatic Reticulum microdomains in cultured glia

Abstract

Astrocytes perinuclear zone is an intracellular region of great interest for its role in calcium intracellular dynamics regulation. Indeed IP3R and SERCA molecules, located on the membrane of the Endoplasmic Reticulum (ER) in the perinuclear region, show a clustering self-organizing capacity that influences intracellular calcium signaling. IP3Rs and SERCA pumps have respectively the role to release cal-cium ions into the cytoplasm and segregate them into the ER. In this work astrocytes fluorescence recordings were acquired with a confocal microscope and calcium activity was stimu-lated by the use of ionomycin. A fluorescence image processing method to study the organization, localization and dimension of these molecular clusters was developed. The method has been called DMSD and it leads to the visualization of a false-color map where the preferential behavior of each time series pixel is represented. In this way Ca2 + release and uptake ER membrane microdomains can be clearly identified