Student Theses and Dissertations

Patrick Bhola

Date of Award

2010

Document Type

Thesis

RU Laboratory

Simon Laboratory

Keywords

cell death, apoptosis signaling, pmDEVD reporter, cytochrome c

Abstract

Apoptosis is a form of programmed cell death that is necessary for development and organism homeostasis. The early stages of apoptosis are marked by the permeabilization of the mitochondrial outer membrane, the release of cytochrome c and Smac from mitochondria, and the activation of the caspase family of proteases. The permeabilization of the mitochondria outer membrane is thought to represent the commitment of a cell to an apoptotic fate, and the subsequent release of mitochondrial proteins is an “all or none” phenomenon that is completed in an individual cell within minutes. Although the cellular decision to commit to apoptosis is important for organism homeostasis, there is considerable variability in the onset of apoptosis between cells in culture, even in clonal populations. Here we developed a reporter (pmDEVD) for caspase activation in single cells. The pmDEVD reporter relocalizes from the plasma membrane to the cytoplasm after caspase activation. We find that the reporter has similar kinetic properties but better fold-increase properties than existing reporters. Using the pmDEVD reporter in single cells, we observe that the onset of caspase activity was tightly synchronized between nearby cells. This synchrony was not a consequence of secreted factors and was not correlated to the cell cycle. A similarity of apoptosis times was seen amongst sister and related cells and was lost over successive generations. The times of apoptosis also diverged within a generation, but this was blocked by inhibiting protein synthesis before triggering apoptosis. These results suggest that the cell-cell variability of apoptosis times is due to the divergence of the molecular composition of the cell, and that the decision to commit to apoptosis at the time of drug addition has a deterministic component. We used the deterministic nature of apoptosis in single cells to monitor the release of mitochondrial release at a rapid temporal resolution. At a rapid time resolution, we find that the release of cytochrome c propagates throughout the cell as a spatially coordinated wave. The permeability of the outer membrane to Smac propagates with the same spatial pattern as cytochrome c but lagging in time. This is followed by a wave of increased permeability of the inner membrane to protons. Only afterward do the mitochondria fission. The spatial dependence of the permeability wave was inhibited by thapsigargin, an inhibitor of the endoplasmic reticulum calcium pumps, but buffering cytosolic calcium had no effect. Altogether, these results demonstrate the organized dynamics of apoptosis. First the onset of apoptosis in a single cell has a deterministic component, and second, the trigger for apoptosis is spatially localized, initiating at one or only a few mitochondria and then propagating across the cell.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/375

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