DOI: 10.25549/usctheses-c127-662046
title: Multi-marker real-time optical imaging of live cell population under controlled stress: apoptosis in retinal ganglion cells

description: The response of live cells to external stress is regulated and processed through complex and highly-interconnected time-dependent signaling networks. The essential task for the understanding of the regulatory mechanism(s) is thus to determine the temporal and spatial quantitative map of the time-evolving molecular states of the signaling network to enable building a physically reasonable and biologically meaningful model that not only describes the acquired data but also can generate testable hypotheses. Conventional approaches in biological science and bioengineering rely almost invariably on population-averaged measurements of usually a single biomarker at a limited number of fixed time points. The true trajectories of the dynamics of multiple biomarkers of the cellular response and their cell-to-cell variation are thus averaged over and masked. In turn this limits the reliability and scope of the model(s) built upon such measurements. To overcome these limitations, 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. Such an approach, increasingly recognized in recent years as central to pursuing a systems perspective of biological processes, including those underlying disease, demanded the availability of appropriate experimental platform. Fortunately the Madhukar laboratory was embarked upon developing such a platform and this author could join Dr. Siyuan Lu in this effort. Thus a custom-designed high-throughput real-time live-cell imaging platform that allows optical imaging of multiple fluorescence biomarkers while also imaging morphological changes in a network of live cells was developed, tested, and applied to the study of programmed cell death called apoptosis. 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. It has enabled the following major findings for apoptosis: (1) resolving a controversy in the literature, the mechanism of the observed apoptotic morphological change of cell body shrinkage at the early stage can now be attributed to the ion channel activities regulating water efflux as evidenced by the strong temporal correlation between the early intracellular Ca2+ elevation and the cell body shrinkage; (2) the first quantitative evidence for significant cell-to-cell variation in the time interval between the onset of Ca2+ elevation at the early stage of apoptosis and the time of caspase-3/7 activation at the later stages of apoptosis and (3) the appearance of multiple stages in PS translocation from the inner to the outer leaflet of the plasma membrane, suggesting more than a single mechanism of PS translocation to be operative in RGC apoptosis under elevated pressure. ❧ 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 physical model proposed is guided by the seminal work of Boehning et al. [Nat. Cell Biol. 5 (2003)] who showed the significance of the cytochrome c released by the mitochondria for impacting the free cytosolic [Ca2+] via its binding to the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum (ER) membrane. Specifically, the impact of cytochrome c binding to IP3R is modeled as a change in the IP3R opening probability expression employed by Mak et al. [PNAS, 95 (1998)] in the modeling of [Ca2+] dynamics in non-apoptotic cells. 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.