The Investigation of Shrinkage in Apoptotic Bodies

By Ashley Ilinkoski and Michael Model

Objectives

Apoptosis is programmed cell death where the cell shrinks then eventually fragments and dies, also considered the point of no return. This method can possibly be controlled by putting cells in different media to ensure ions pouring into the cell rather than out. Shrinkage is one of the most universal and specific signs of apoptotic cell death. (Some data suggest that shrinkage due to water loss is the requirement for apoptosis). However, in some apoptotic systems shrinkage occurs late, after irreversible cell damage has occurred. Such systems provide a good model to investigate the exact role of shrinkage in apoptotic development. One specific hypothesis we wish to address is that shrinkage is necessary for cell fragmentation into “apoptotic bodies”.

Methods

Apoptosis is induced in HeLa cells with camptothecin or RNA polymerase actinomycin D. After the onset of irreversible changes (i.e., morphological blebbing, mitochondrial depolarization or cytochrome c release) treatments designed to inhibit cell water loss are applied. Aqua porin inhibitors are also being introduced to the cells to see if the porins close to sustain cell life by cutting off water transport. The results include formation of apoptotic bodies and DNA fragmentation. The loss of cell material is monitored by the TIE-TTD microscopy and DNA fragmentation by the TUNEL assay.

Results

Preliminary data indicates that high-potassium medium that opposes water loss prevents formation of apoptotic bodies. Conversely, high-sodium medium that activates water loss results in active cell fragmentation. Future work will clarify these results.