Real-Time Dynamics of Ca 2+, Phosphatidylserine, Caspase-3/7, and Morphological Changes in Apoptosis: Retinal Ganglion Cells Under Elevated Pressure

Abstract

Quantitative information on the dynamics of multiple molecular processes in individual live cells under controlled stress is central to the understanding of the cell behavior of interest and the establishment of reliable models. We report on the dynamics of the apoptosis regulator intracellular Ca2+, surface marker phosphatidylserine (PS), effector caspase-3/7, and morphological changes examined simultaneously in individual transformed retinal gangling cells undergoing apoptosis at elevated hydrostatic pressure. A custom-designed imaging platform that allows long-term real-time imaging of morphological and molecular-level physiological changes in large numbers of live cells (beyond the field-of-view of typical microscopy) under controlled pressure is employed.[1] Intracellular Ca2+ elevation and PS translocation to the outer leaflet of the plasma membrane at the early stages (typically 5 hours) is found. The data reveal a strong temporal correlation between the Ca2+ elevation, PS translocation, and morphological changes (neurite retraction and soma shrinkage) in the vast majority of the cells. This suggests that Ca2+ is likely responsible for the onset of PS translocation and apoptotic morphological changes. Moreover, the data show a significant cell-to-cell variation in the onset of caspase-3/7 activation, an inevitable consequence of the stochastic nature of the underlying biochemical reactions not captured by conventional assays based on population-averaged cellular responses. This study demonstrates that the approach of simultaneously imaging multiple intracellular events in large numbers of live cells provides statistically significant data to enable refinements and testing of models of signaling pathways, here apoptosis. [1] Lee JK, Lu S, Madhukar A (2010) Real-time Dynamics of Ca2+, Caspase-3/7, and Morphological Changes in Retinal Ganglion Cell Apoptosis under Elevated Pressure. PLoS ONE, In Press.