Apoptosis, or programmed cell death, occurs regularly throughout the body to maintain a homeostatic quantity of cells. Malfunctions in this process can result in numerous health complications including tumor development. Apoptosis occurs through two main pathways: extrinsic and intrinsic. The goal of this project is to monitor the temporal dynamics of caspase activity during the extrinsic pathway of apoptosis. The extrinsic pathway is instigated by an external stimulus which results in the activation of a destructive caspase cascade. The activity of several different caspases is monitored through fluorescence microscopy using microfluidic devices and targeted fluorogenic caspase probes derived from Rhodamine 110. The binding of the cells with the antibody, anti-CD95, coated in the microfluidic channel, will hold the cells in place during the experiment, while inducing apoptosis through FAS receptor binding. General caspase activation was visualized over a 9-hour period with the fluorogenic caspase probe L-bisaspartic acid Rhodamine 110 (D2R). Derivatives of the fluorogenic probe containing peptide sequences for specific caspases were used to monitor the first and last caspases to be activated in the apoptotic pathway. The derivative (IETD)2R was used to monitor the primary initiator caspase activation (caspase-8) and the (DEVD)2R for executioner caspase activation (caspase-3). The overall average caspase activity began 2.4 ± 0.6 hours post-induction and lasted 360 ± 60 minutes, caspase-8 activity was shown to start 1.8 ± 0.8 hours after induction and remain active for 120 ± 40 minutes, and caspase-3 activity started 5.2 ± 1.0 hours after induction, lasting 130 ± 60 minutes. Timing of caspase activation is an important field of study as it could be helpful in designing apoptosis-targeted therapies.

Analyzing the temporal dynamics of caspase activation in the extrinsic pathway of apoptosis

ACS Spring Meeting 2021

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