- Discuss conditions that disrupt endoplasmic reticulum (ER) homeostasis leading to ER stress.
When the protein folding property of ER gets saturated it causes ER stress. The membrane that surrounds all cellular organelles is called the endoplasmic reticulum it is a large membrane and is only found in all eukaryotes. And it is a site for folding membrane, synthesis of lipid sterols, and secreted protein, which also works as storage for free calcium. And different stresses such as psychological, or pathological stress, for example, an increase in protein that is mutated and not able to properly fold in the ER, and this causes an imbalance between the demand and the storage of protein in ER for protein folding and this improper folding cause stress in ER. Some diseases that can cause ER stress are neurodegeneration, atherosclerosis, type 2 diabetes, liver diseases, and cancer. So basically, misfolded protein is the reason for ER stress (Westrate, et al., 2015).
- Calculate the average of viable, necrotic and apoptotic cells for untreated (control) cells, cells treated with the ER stress inducer
Tunicamycin and cells treated with a different ER stress inducer called Thapsigargin. Present your calculated averages in a new
table.
| Number of cells | Untreated (control) | average | Treated with Tunicamycin | average | Treated with Thapsigargin | average | ||||||
| 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | ||||
| Viable | 95 | 85 | 106 | 95.3 | 77 | 69 | 53 | 66.33 | 56 | 49 | 48 | 51 |
| Necrotic | 3 | 2 | 3 | 2.6 | 13 | 7 | 9 | 9.6 | 11 | 14 | 10 | 11.66 |
| Apoptotic | 2 | 1 | 3 | 2 | 25 | 19 | 18 | 20.6 | 30 | 25 | 33 | 29.33 |
- Calculate the percentage of cell death observed in untreated (control) cells, cells treated with Tunicamycin and cells treated with Thapsigargin. Show your calculations.
The percentage of cell death is shown below:
| Number of cells | Untreated (control) | % of cell death | Treated with Tunicamycin | % of cell death | Treated with Thapsigargin | % of cell death | ||||||
| 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | ||||
| Viable | 95 | 85 | 106 | 33.21 | 77 | 69 | 53 | 57 | 56 | 49 | 48 | 36.60 |
| Necrotic | 3 | 2 | 3 | 37.5 | 13 | 7 | 9 | 44.82 | 11 | 14 | 10 | 31.42 |
| Apoptotic | 2 | 1 | 3 | 33.33 | 25 | 19 | 18 | 40.32 | 30 | 25 | 33 | 51.72 |
(The above calculation for percentage is the result of all 3 conducted experiments under each given criteria and uses the average to calculate the final percentage for viable, necrotic, and apoptotic.)
- Present the percentages you calculated in point 3, in a suitable graph of your choice.
- Use basic statistics to explain which drug (Tunicamycin or Thapsigargin) was more toxic to the cells.
| Number of cells | Untreated (control) | average | Treated with Tunicamycin | average | Treated with Thapsigargin | average | ||||||
| 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | ||||
| Viable | 95 | 85 | 106 | 95.3 | 77 | 69 | 53 | 66.33 | 56 | 49 | 48 | 51 |
| Necrotic | 3 | 2 | 3 | 2.6 | 13 | 7 | 9 | 9.6 | 11 | 14 | 10 | 11.66 |
| Apoptotic | 2 | 1 | 3 | 2 | 25 | 19 | 18 | 20.6 | 30 | 25 | 33 | 29.33 |
With the help of the above-presented graph, we can see that viability of cells from tunicamycin is more than that of thapsigargin and the data in necrotic cells and apoptotic cell clearly shows that thapsigargin is much more toxic to the cells in comparison between this two.
- Explain the role of untreated (control) cells in the experiment.
In the performed experiment role of the untreated (control) cell is of providing the baseline for the experiment and helps us to check and to prove whether a variable can give baseline results. The test is conducted or observed under simulated surroundings which is very much similar to what a natural state could be. In short, this control experiment always has been used to compare the result of the different tests, and not only in this but it can be used in different experiments also, For needed purposes.
- Discuss the importance of examining necrotic and apoptotic cells and provide differences between the two modes of death.
Necrotic and apoptotic cells are two types of cell death that could occur in cells and cell death plays a variety of roles in the body which play important role in the development and many immunological defenses. And it is also one of the important elements in diseases plaguing modern society.
The difference between the two modes of death which are necrotic and apoptotic cells is as follows:
Necrotic cell death – in comparison to apoptotic cell death this is a more harmful way for a cell to die because this cell gets ruptured and all the organelles from inside get out in the tissue and produce swelling.
Apoptotic cell death – it is programmed cell death, and it is used in the elimination of cells at the early stage, for example, it helps in cancer prevention, and also in adults, it is used to rid the part of a body cell that is far from repair and is highly damaged (Walter, & Lingappa, 1986).
- Explain the mechanisms by which the two drugs used in this study (Tunicamycin and Thapsigargin) induce ER stress.
These two indeed are responsible for inducing ER stress by different means and this is explained below:
The mechanism by which thapsigargin induces ER stress is – it is considered one of the most popular contributors to ER stress. The process by which it causes ER stress is by depleting Ca which is stored in the ER and that leads to ER stress.
The mechanism by which tunicamycin induced ER stress is – it is an antibiotic and it occurs naturally and by inhibiting step one which is included in the biosynthesis of n-linked glycans in the protein which results in many misfolding in protein and resulting in ER stress.
- Why does ER stress threaten cell viability?
As we have already talked about ER stress (stress caused by the improper or misfolding of the protein in ER which causes problems between required and present protein in ER) and this unnatural event cause multiple effects in cells such as phosphorylates and elff2a, PERK is also activated. And this all results in selective induction of ATF4 and it leads protein CHOP and Noxa to flow downstream, and this cause the death of the cell, and because of this ER stress is not good for the viability of any cell.
Malfunctioning in ER is a serious problem as it stopped protein synthesis because there is no production of ribosomes and many different improper things cause cells to die (Walter, & Johnson, 1994).
- Discuss a human disorder that results from ER protein quality control failure.
ER is a protein folding factory of the main root. And as we know that all proteins are already destined for the plasma membrane, extra space in a cell, or another important area of the cell is undergone folding inside the ER. And ER performs a unique protein quality control and by that, it enables to modification of proteins such as glycosylation and disulfide bond formation and it is very essential for various folding and working of other proteins. And it also acts as the major checkpoint for the unfolded protein. And any failure in this system can cause serious diseases and problems inside the human body which can result in disorders. Some examples of the severe diseases that can be caused because of this are Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis disease (Hasnain, et al., 2016).
References
Hasnain, S. Z., Prins, J. B., & McGuckin, M. A. (2016). Oxidative and endoplasmic reticulum stress in b-cell dysfunction in diabetes. J Mol Endocrinol, 56(2), 33-54.
Walter, P., & Johnson, A. E. (1994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annual review of cell biology, 10(1), 87-119.
Walter, P., & Lingappa, V. R. (1986). Mechanism of protein translocation across the endoplasmic reticulum membrane. Annual review of cell biology, 2(1), 499-516.
Westrate, L. M., Lee, J. E., Prinz, W. A., & Voeltz, G. K. (2015). Form follows function: the importance of endoplasmic reticulum shape. Annu. Rev. Biochem, 84(1), 791-811.