Apoptotic regulation of cellular extrusion

The purpose of this dissertation is to define how the pathways of apoptosis and extrusion connect. These studies employed MDCK and HBE in vitro cultured epithelia as model systems to explore the ability of different components of the apoptosis pathways to participate in the activation of the extrusion response. Because of the ease of their manipulation for genetic, pharmacological and cell biology analyses but most importantly because their ability to reproduce in vivo apoptotic cellular extrusion, cultured epithelia provide a powerful and efficient system to study and dissect the molecular basis of apoptotic cellular extrusion. In the first part of this dissertation, we established that both intrinsic and extrinsic apoptotic pathways activate cellular extrusion and that the contraction force that drives cellular extrusion requires caspase activity. Accordingly, we found that necrosis does not trigger the cellular extrusion response; nevertheless, necrotic cells are removed from epithelia by a passive mechanism that involves forces of the stochastic movement of epithelial cells. The second part of this dissertation is focused on the regulation of apoptotic cellular extrusion by the anti-apoptotic protein Bcl-2. Mutations that increase Bcl-2 protein levels result in resistance to cell death, which in turn lead to several malignances in the living organism. Here, as a way to complement our studies in the first part of this dissertation, iv we explore the effect of Bcl-2 on cell junctions, test the ability of HA14-1, a Bcl-2 inhibitor, to activate cell extrusion and finally we test the ability of this oncogene to regulate cellular extrusion in vivo. The studies reveal the connection between apoptosis and cellular extrusion: caspase activation, a conserved point in the apoptosis pathway where all known apoptotic signals converge. The discovery of the requirement of caspases for cellular extrusion reveals a new non-apoptotic role for caspases and sets caspase activity as an important checkpoint for epithelial cellular removal. These studies also reveal a new passive mechanism by which dead cells unable to trigger cellular extrusion are removed.