Lecture 7: apoptosis Flashcards
For what is apoptosis or controlled/programmed cell death important?
- During embryonic developtment → cells / tissues need to be removed.
- Preventing cancer
How was apoptosis discovered?
Through histological and microscopy experiments, where apoptotic bodies were found in tissues from different organisms. The cells were different from living cells or even necrotic cells, where these cells were killed in an organized manner.
What is meant by the fact that apoptotic cells are killed in an organized manner?
Apoptotic cells are fragmented and cell content is released that can be re-used and taken up by phagocytosis.
After the discovery of the phenomenon of apoptosis, more insight was needed. What organism was used to identify genes important for apoptosis and why?
C. elegans. It has a known number of cells (959), so if cells are missing this is easily seen. Because of this, we also know about 100 cells are removed during embryogenesis.
How was C. elegans used to study gene identification and what was seen in this study?
A mutant of C. elegans was used, where the mutant showed a different pattern of apoptosis. This different pattern of apoptosis, naturally, has an affect on cell location and survival options during development.
The mutant cells that were identified in the mutants of C. elegans behaved in a different way than the wildtype. What was interesting about this?
That these mutant cells that were identified behaved like the parental line → mutantions lead to reiteration in the cell lineage of C. elegans (cells that behave like their parental line).
Next, C. elegans mutants with slowed-down apoptosis were used (easier to investigate) and treated with mutagenic chemicals. What was found in the new mutants?
They found new mutants with a decreased number of apoptotic cells (b in picture).
What was seen under the fluorescence microscope when the wild type of C. elegans was compared with the new mutants with decreased apoptosis?
More cells were found in the mutant C. elegans due to the decreased apoptotic rate (b in picture).
What do the mutations in C. elegans that lead to reiterations in the cell lineages of C. elegans say about this all?
That development may be suppressed by these kind of mutations and thus that these mutated genes might be involved in the transcription of developmental genes.
Why do we want to identify and map genes?
- Link developmental processes to defined gene locations
- Linking genes to protein function
- Linking developmental processes to defined gene locations.
What is flow cytometry?
It’s a technique that is used to detect and measure physical and chemical characteristics of a population of cells or particles.
The mitochrondrion seems to be the first player in apoptosis. How did they discover this?
Lymphocytes in cell culture were used to study mitochondria. Here, the mitochondria were stained with a fluorescent dye, indicating the proton motive force (PMF). When apoptosis inducing agents (DEX) were added to the lymphocytes, it caused changes (i.e. decrease) in the mitochondrial PMF.
This flow cytometry graph is a result of staining the mitochondria in lymphocytes and inducing apoptosis. What does this figure show (what’s on the X- and Y-axis etc.)?
- On the X-axis is fluorophore concentration (DiOC6) indicating PMF (high concentration = high PMF and vice versa) and on the Y-axis is the amount of cells that have absorped a certain amount of DiOC6.
- The graph observes the amount of PMF via DiOC6 absorption in lymphocytes from control (PBS) and the experimental group (DEX). It is seen that the PMF is lowered as an early step in apoptosis.
- mClCCP is an uncoupler of the PMF and acts as an control to show that the PMF can be decreased (19% compared to 81% in control).
What was thought to be the reason behind the lowering of the proton motive force (PMF)?
That there are changes in ATP synthase expression that cause the collapse in the proton motive force. This wasn’t the case, as can be seen in the picture.
So if it isn’t a change in expression of ATP synthase, what open question remains?
What is the cause of the lowering of the proton motive force?
- Is the PMF decreased due to dying cells
- or*
- Is the PMF decreased before cell death → does PMF decrease cause cell death?
What is seen when comparing the degraded DNA of cells with high or low proton motive force (PMF)?
In the cells with a high PMF, you don’t see any degradation products (right in picture). In the cells with a low PMF, some degradation products are observed (left in picture). So the PMF needs to go down, in order for DNA degradation to occur.
What is a decreased PMF related to?
To the production of reactive oxygen species (ROS).
- A high PMF correlates with low ROS
- A low PMF correlates with high ROS
Cytochrome C is a key component of the respiratory chain.
An in vitro cell-free system is now used to invesigate apoptosis and is tested for caspase (CPP32) activation. What happens to the CPP32 precursors when cytochrome C is added to this cell-free system?
Upon addition of cytochrome c, the precursor of CPP32 is cleaved and caspases are activated. This will lead to DNA fragmentation.
A specialized system is needed for Cytochrome C to leave the mitochondrion. To test what is needed for the release of cytochrome C, they compared apoptotic mitochondria with normal mitochondria (in a cell-free system). What was found?
It was found that Bcl-2, an anti-apoptotic protein, reduced the degree of cytochrome c release by decreasing caspase activity and caspase-dependent proteolysis of the nuclear lamina.
(Note: this Bcl-2 protein is part of a larger family of Bcl-2 proteins, where some are pro- and some are anti-apoptotic.)
How is cytochrome C released from the lipid in the inner mitochondrial membrane?
By oxidation of cardiolipin, this releases the cytochrome.
Fusicoccin-A (FC) is a natural compound that might be able to induce apoptosis in cancer cells. What was seen when normal cells compared to cancer cells were treated with this compound?
That there’s selective action against cancer cells. This is seen in the picture, where cancer cells are depicted on the left and normal cells on the right.
