Lecture 8 Apoptosis

Question 1

Why should cells die/be killed in a eukaryote? Name an example

Answer 1

Example:
During (embryonic)
development cells/
tissues need to be
removed (frog tail)

Question 2

How was apoptosis discovered?

Answer 2

Histology/microscopy:

“Apoptotic bodies” in
tissues from several
organisms (humans, rats) were found in 1972. Killed in a controlled way: no inflammation/sign of necrosis (=plaatselijk afsterven van weefsel).

Cells “packaged” for
removal?

Question 3

Why was C. Elegans used to research apoptosis? Was a big step

Answer 3

• Simple multicellular eukaryote
• Fixed no cells (959)
• Defined number of cells (~100) removed during
  embryogenesis

Question 4

What was the next big step in gaining information?

Answer 4

• Identifying mutants (gene sequencing techniques became available at that time).

Microscopy:
Mutants identified that
change location and survival
options during development.

Question 5

Where does apoptosis start? What is an apoptosis inducing agent and how is that made visible?

Answer 5

Starts in the mitochondria. Lymphocytes in cell culture were used as model (for staining).
Apoptosis inducing agents

=>
Change in mitochondrial
Proton motive force (it goes down)

Fluorescent dye indicating PMF

Question 6

Are there less enzymes in the mitochondria with the lower PMF? How to find out?

Answer 6

ATP synthase was labeled with fluorescent antibodies. However, No significant change in mitochondrial ATP synthase expression.

Question 7

Typical for a normal cell: Low PI staining (used as dead/alive marker), high PMF. Inducing apoptosis: what is impact on these factors? Which one changes first? How did they find out?

Answer 7

High PI, low PMF.
There are a lot of cells with a low PMF with PI staining still intact (low). No cells with PI staining high (not intact) and PMF also high. Meaning: PMF starts first.

Question 8

Next question: First DNA degradation, then PMF lower or other way around? How did they find out?

Answer 8

-> Took cells according to fluorescence intensity (PMF, cell integrity). Cells with high and low PMF (and thus high or low fluorescence)
-> tested for degradation of DNA.
-> Low PMF: degradation
-> high PMF: no degradation.
So, first PMF going down, then DNA degradation.

Question 9

ROS: lower or higher in apoptose-induced cells?

Answer 9

Higher

Question 10

What is the main signal coming from the mitochondria that initiates apoptosis?

Answer 10

Cytochrome C

Question 11

How was this discovered and what happens upon release of cytochrome C?

Answer 11

Cytochrome C of the mitochondria: main signal of initiating apoptosis.
In vitro cell-free system to investigate apoptosis: Test for caspase (CPP32) activation. = series of proteases that are executioners of apoptosis. Can activate each other. Marker protein is degraded (by the proteases I think) upon release of cytochrome C.

Question 12

Tested Cell-free extract (Nuclei, cytosol, ER), then add mitochondria. Add proteins of family Bcl-2. What happens?

Answer 12

Bcl-2 reduces degree of cytochrome c release. Bcl-2 decreases caspase activity and caspase-dependent proteolysis of the nuclear lamina.

Question 13

What can be a reason for cancer cells to have a decreased rate of apoptosis?

Answer 13

Warburg effect: Decreased use of oxidative phosphorylation, meaning less ROS creation, meaning less apoptosis

Question 14

What is a strategy against cancer cells?

Answer 14

Try to induce apoptosis:
A 14-33-like receptor in cancer cells looks like the one in plants, maybe fungus particle can also modulate this receptor and induce apoptosis (Just like the plant story). Seems to work!

Question 15

What is another way initiating apoptosis regarding cardiolipin?

Answer 15

Cardiolipin (lipid in
inner mitochondrial membrane)
covalently bound to cytochrome c.

Oxidation of cardiolipin releases cytochrome c
Mechanism of ROS triggering apoptosis?