Question

1. There are a few forms of "apoptosis". All of these forms have the same end point, but they differ in how the cells get there. in particular some forms of apoptosis do NOT REQUIRE the activation of the caspases.

a. Given that the end point of apoptosis is the same, describe one way you would expect all of these forms of cell to be the same (Dead cells is NOT a sufficient answer).

You are studying apoptosis in a particular cell type and you would like to determine if caspase activation occurs during this apoptotic event.

b. Describe how caspases are normally activated during apoptosis.

c. design an experiment to test if caspases are active during your apoptotic event. This is just the experimental design, what you're going to do and NOT what will it show you.

d. Draw and label two figures with all of the appropriate controls. You do not need to write a figure legend, but your labeling should be sufficient enough to be able to interpret the figure.

i. A figure showing that caspases ARE activated by your apoptotic event.

ii. A figure showing that caspases ARE NOT activated by your apoptotic event.

Answer 1

a. Both the cases involve the proteases, which means there is final disintegration of cell membranes, DNA damage, paraptosis, excitotoxicity etc. leading to the cell death are the fates of cells in both caspase dependent and independent pathways.

b. Caspases are the inactive zymogens (family members of conserved cysteine proteases) present in the cell that after a series of catalytic reaction to get activated. In short they undergo a cascade of catalytic activation. They are of two types initiator and effectors. The initiator caspases gets activated by autocatalytic process, while the effector gots agtivated due to cleavage by initiator.

c. For this experiment we will use unique cell-permeable and non-cytotoxic reagent called Fluorochrome Inhibitor of Caspases (FLICA). It has specific binding activity with the caspases. This will help in the different caspases to be detected in different specificity with the reagent. So a cell with active caspase leading to apoptosis will be detected as FLICA positive and the rest will be not detected that suggests the inactivated form of caspases in the cells.

d. The diagram showing both apoptotic and non apoptotic cell death mechanism pathways are as follows: