Multi-marker real-time optical imaging of live cell population under controlled stress: Apoptosis in retinal ganglion cells

In this dissertation, we introduced and followed the new paradigm of measuring simultaneously in real-time indicators / biomarkers of multiple processes in a statistically large number of individual live cells from the same population under controlled stress over prolonged times. Specifically, the mitochondria-mediated (i.e. intrinsic) pathway of apoptosis in the retinal ganglion cell (RGC) line differentiated RGC-5, induced by elevated hydrostatic pressure is examined via simultaneous imaging of five features: the time-dependant concentration of (1) intracellular Ca2+, (2) caspase-3/7 activation, (3) phosphatidylserine (PS) translocation, and the morphological changes of (4) cell body shrinkage and (5) neurite retraction. Simultaneous measurement of five features in apoptosis, and perhaps for any biological process, in a statistically large number of individual live cells under continuous stress is a first.;The acquisition of data on the dynamics of five features is not only a first, equally significant, the acquired Ca2+ data have enabled, for the first time, the modeling and analysis of the role of [Ca2+ ] in apoptosis. Although Ca2+ dynamics in normal healthy cells under physiological conditions has been measured, modeled, and analyzed for quite some time, the model proposed and analyzed here is the first one for [Ca2+] dynamics during apoptosis. The model provides a qualitative understanding of the [Ca2+] dynamics in apoptosis for reasonable values of the parameters involved.;As PS translocation gives rise to an increase in its concentration at the cell surface, we also carried out a unique study utilizing combined NSOM-AFM (Near Field Scanning Optical Microscopy - Atomic force microscopy) that allowed simultaneous imaging of the 3-dimensional topography of the cell surface and the local density of PS as manifest in the fluorescence intensity of Annexin-V conjugated to the PS measured at the nanoscale beyond the diffraction limit. A positive correlation (correlation coefficient 0.69) between the geometrical height and the density of the PS on the surface of the RGC-5 cell membrane is found. It suggests that this may be Nature's way of signaling for and enabling a means for effective binding of the associated receptors in phagocytes to the translocated PS in the apoptotic cell.;To investigate the very origin of RGC apoptosis under elevated pressure, we undertook examination of the neurotrophin deprivation hypothesis—namely that the blockage of neurotrophin uptake/transport through axon induces apoptosis in RGCs under elevated pressure. This was carried out employing dye as well as QD-tagged neurotrophins and for cells cultured in custom-designed microfluidic chips that enable isolation of the cell soma and processes. This ensures unambiguous measurements of spatially separated uptake regions and axonal transport of the neurotrophins. For the uptake studies, the cells employed are the differentiated RGC-5 cell line whereas for the axonal transport studies only the neuroblastoma NG108-15 cells were employed as these provide mature axons (unlike RGC-5 neurites which are not sufficiently mature). Reduction in the number of neurotrophin uptaken into the differentiated RGC-5 cells and more neurotrophins localized in the neurites were found under the elevated pressure of 100 mmHg compared to the cells under no elevated pressure. The neurotrophin transport in the neuroblastoma NG108-15 cells revealed a typical stop-and-go behavior. This suggests that the elevated pressure not only inhibits the overall uptake of the neurotrophins into RGCs but also potentially the transport through neurites in RGCs, thus providing the first evidence supporting the neurotrophin deprivation hypothesis of apoptosis. (Abstract shortened by UMI.).