Caspase Activity in Apoptotic Cells Cell Bio 220 Tenzin Dolkar Stephanie Kim Victoire Ndong Patricia Wong Caspases • Are families of proteases which are involved in initiating apoptotic events • Synthesized in the cell as precursorsprocaspase • Cleave proteins at aspartic acid residue sites • There are initiator caspase (procaspase-8) And executioner/effector caspase (procaspase-3) Caspase Structure • • • • • NH2-terminal domain Large subunit (~20kD) – (lavender) Small subunit (~10kD) – (gray) Red represents an inhibitor Thornberry and Lazebnik. Caspases: Enemies Within. 1998. p 1313 Caspase Role in Apoptosis • • • • • • • Cut off contact with surrounding cells Reorganize cytoskeleton Shut down DNA replication and repair Interrupt splicing Destroy DNA Disrupt nuclear structure Induce cell to display signals marking it for phagocytosis • Disintegrate cells into apoptotic bodies Initiator Caspase Activation • Initiator Caspases are activated by either internal (DNA damage) or external signals (ligand binding) • External: caspase-8, an initiator caspase is activated by binding to it’s specific cofactor, FADD (Fas-associated protein with death domain) Executioner/Effector Caspase Activation • Effector caspase are activated generally by other caspases. • Caspase activation results in proteolytic cascade which is self-amplifying and irreversible. • Caspase-3 is an effector caspase Our Experiment • MDCK cells treated with camptothecin (interferes with DNA replication and transcription) • Added “D2-R110,” a bis-L-aspartic acid amide of Rhodamine 110 • When the compound is cleaved by caspase, such as caspase-3, it fluoresces green Hypothesis/Goal • Cells treated with camptothecin will have a greater number of fluorescing cells, indicating a higher rate of caspase activity and the occurrence of apoptosis • To compare caspase activity in MDCK cells treated with camptothecin with control cells in order to determine the rate of caspase activity Sample A – Camptothecin Treated Average Percentage of Apoptotic Cells: 73.40% Sample B - Control Average Percentage of Apoptotic Cells: 2.95% Cellular Pathway Sources of Error and Uncertainty • Human errors • Direct causal relationship between caspase activity and fluorescence not proven Further Experimentation • Disable caspase-3 activation to see the extent of its importance in apoptosis • Disable caspase-9 to determine if apoptosis is a result of internal signaling • Compare caspase activity in cells undergoing apoptosis to those undergoing necrosis • Examine caspase activity at different time intervals