| he first aim of this thesis was to study dynamic morphological changes during apoptosis in differentiated neuronal PC12 cells that die when nerve growth factor (NGF) is withdrawn. Previous morphological characterization of apoptosis before this thesis had predominantly focused on fixed or static tissues. To understand dynamic changes, a system was developed for long-term timelapse videomicroscopy. As observed using this system, the morphology of apoptotic neuronal PC12 cells was strikingly active, with the whole cytoplasm engaged in rapid extrusion and retraction of large blebs. The active nature suggested that there might be appreciable changes in cellular during apoptosis. Thus, a series of experiments examining cellular metabolism was performed and revealed, once the data were standardized relative to protein or cell number, that cellular metabolism was surprisingly well conserved. Protein and RNA synthesis were relatively unaffected until late in death of the culture, and overall metabolic activity (as assessed by 2-deoxyglucose uptake) was never significantly altered. Lactate production decreased soon after removing NGF and then increased significantly later, while ATP levels showed a slight, statistically significant decrease very early and then rebounded to control levels before eventually decreasing again. The dynamic blebbing also suggested that the cytoskeleton might be dramatically altered during apoptosis. Among the chief cytoskeletal proteins regulating cell shape are microtubules. Hence, the last aim of this thesis was to address the role microtubules and... |
