apoptotic and necrotic pathways
Necrosis and apoptosis represent two major forms of cellular death. Necrosis is associated with compromised plasma membrane integrity of the cell, which results in leakage of cellular components into the extracellular milieu. In the context of organism, necrosis can hence trigger sterile inflammation. Necrosis itself can be caused by agents that perforate cellular membrane, such as detergents and organic solvents, or by inflammatory agents. The major intracellular players involved in necrotic pathways are receptor interacting protein (RIP)-kinases.
Apoptosis, on the other hand, is the programmed way of cellular death where the intracellular proteins are degraded and the contents of the dying cell are packaged into apoptotic vesicles, which in living organisms are cleaved by phagocytosis. Apoptosis is associated with activation of a special family of intracellular proteases called caspases. At the late stages of apoptosis, the composition of the cellular membrane is also changed, and phosphatidylserine becomes exposed on the extracellular side of the plasma membrane. A multitude of synthetic compounds can cause apoptosis, including chemotherapy agents (e.g., doxorubicin or bortezomib), but also inhibitors of pro-survival enzymes (the latter include protein kinases Akt, CK2 and Pim) and compounds with wide profile of potential targets (e.g., staurosporine).
We investigate necrotic and apoptotic pathways induced by various toxins in primary and cultured cells using assays with membrane-impermeable dye Sytox Blue, FRET-based caspase sensors, or metabolism-affected dye Resazurin that is reduced to fluorescent Resorufin in viable cells.
Apoptosis, on the other hand, is the programmed way of cellular death where the intracellular proteins are degraded and the contents of the dying cell are packaged into apoptotic vesicles, which in living organisms are cleaved by phagocytosis. Apoptosis is associated with activation of a special family of intracellular proteases called caspases. At the late stages of apoptosis, the composition of the cellular membrane is also changed, and phosphatidylserine becomes exposed on the extracellular side of the plasma membrane. A multitude of synthetic compounds can cause apoptosis, including chemotherapy agents (e.g., doxorubicin or bortezomib), but also inhibitors of pro-survival enzymes (the latter include protein kinases Akt, CK2 and Pim) and compounds with wide profile of potential targets (e.g., staurosporine).
We investigate necrotic and apoptotic pathways induced by various toxins in primary and cultured cells using assays with membrane-impermeable dye Sytox Blue, FRET-based caspase sensors, or metabolism-affected dye Resazurin that is reduced to fluorescent Resorufin in viable cells.
Figure 1. Casper3-GR biosensor expressing Hela cells treated with staurosporine and imaged over 24 h. At first the apoptosis starts and the fluorescence intensity increases. When apoptosis finally kills the cell, it detatches from the plate which results in the decrease in the fluorescence intensity later in the esperiment.