Apoptosis and Cell Death Mechanisms Student PDF

Apoptosis and Cell Death Mechanisms November 20th, 2024 Learning Objectives Compare and contrast apoptosis, necrosis, and autophagy. Identify the process by understanding cellular and morphological changes. Explain why apoptosis is a normal and necessary part of life. Provide...

Apoptosis and Cell Death Mechanisms November 20th, 2024 Learning Objectives Compare and contrast apoptosis, necrosis, and autophagy. Identify the process by understanding cellular and morphological changes. Explain why apoptosis is a normal and necessary part of life. Provide examples. Explain the step-wise processes of intrinsic and extrinsic apoptosis. Explain the mechanisms by which pro- and anti-apoptotic proteins function. Describe how necrosis or dysregulation of apoptosis leads to disease. Provide examples of diseases where apoptosis is dysregulated. Disclaimer - Today’s lecture will be very different from other classes. Most of the time will be spent doing activities, which will facilitate your learning. Slides are provided to you, but I will spend much less time explaining the slides and expect your group members to use the slides to help you answer the questions in the activities. You will also need to use the assigned supplemental articles to answer questions. You will turn in your notes and answers to the activity questions for your homework assignment this week. In Canvas - Submit your class notes as a.pdf OR share your notes via google docs. Please make sure I have viewing access before you share a link.  Activity 1 - Tell me about the articles you read. (5 + 5 min) What was the purpose of each article? The purpose is to discuss the differences between different types of apoptosis. The articles focus on when each type would be used. It focuses on how apoptosis can benefit the cells. Who was the target audience in each? The first article caters more to the anyone interested in learning because it used more general description, while the second article is targeted towards anyone with a science background because it goes more into depth about the triggers and regulation. Was one more understandable than the other? Why do you think that? The first article is more understandable because the processes are described in easier to understand terms. (review Articles) What is a primary vs. a secondary literature article? Were these articles both primary, secondary, or neither? Primary is the original findings/ research while secondary is the summary of past research and experiments. Both articles are secondary because it references other articles. Is apoptosis normal? Explain. Apoptosis can be normal because it aides in developmental and it programed cell death. It can become abnormal depending on if it becomes uncontrolled or unmediated. Activity 2 - Apoptosis, Necrosis, and Autophagy (20 + 15 min) 1. Compare and contrast apoptosis, necrosis, and autophagy. Make sure that you define each process and explain when or why each process occurs. Give examples of when each process would be induced. Apoptosis:programmed cell death which can be a result after the cell has completed its function OR the cell is mispreforming, doesn’t cause inflammation, energy dependent, can act on neighboring cells (which can lead to uncontrolled), doesn’t release engulfed cell’s contents into outside tissues, ex) embryonic development, cancer Necrosis: cell death from injury by physical pain/ lack of oxygen, has physical symptoms (mitochondria ruptures), uncontrolled/passive, that affects large areas of cell, ex) burn victim Autophagy: cell digestion which allows it to self recycle but keeps the cytoskeletal intact, ides in development, ATP dependent, “cannibalizes itself”, induction → formation of a phagosomes → fusion w/ lysosome→ body breakdown/recycling, ex) nutrient deprivation → allows to kill off old, unfed cells to give growth to new,healthier cells 2. How can you identify each process using cellular and morphological features? Apoptosis: Morphological: cells smaller in size, cytoplasm is dense, organelles more densely packed,”blebbing”; Cellular: early in chromatin condensation the e- dense nuc material characteristically aggregates peripherally under nuc. Mem., chromatin condensation, fragmentation of nucleus Necrosis: Morphological: damage to organelles, cells swell which causes the internal features to be over spilled onto surrounding cells→ leads to inflammation response b/c there becomes damage to other surrounding cells, fragmentation of nucleus Autophagy: Morphological: cytoskeleton stays intact 3. Why is it important to distinguish between these processes? Each process has different outcomes, therefore it is important to know what process should be used for what.  Apoptosis: - EnergyRequired programmed all death , happens offer the all has performed its function - ready a dre Anduced: When there's domage hall bit becomes threat to the organism , like cone ↳ if the body ridies a all his gave rougue/ longe normal no features: programmed so it shrinks & , cytoplasmic "blebsh Chromatin Necrosis : Accidental all death , doesn't regare energy FeaturessCell torgonelles swell, call memb Isn't intact. eventualy all membul rupture - causy surround alls to have on deflommtoy response Can · deduce Inflommty Response effect , surround all Autophagy : self-digestion recycl component cytoskeleton stays ↳ When? - , don't have duss tonut. , So only way Intact "Self-Sacriprat 2 mak new , stuff to is degrodel recycle old stritt Phagolysosomes-fuse t degrade components + use else when Corryolysis : melty/culty of chromatin Corrgohexises. · happens in bothof them  Activity 2 - cont. 4. What processes were the authors trying to convey with this Satellite illustration? receptor Second & = they are trying to illustrate that cell death needs recognities - signa Messengers specific signals that are external. This causes intracellular pathways to activate enzymes and leads to cell death.They want to show how each cells need some sort of signals 5. Why did the authors use these specific objects in this illustration? The illustration shows a cellphone tower signaling to a satellite to transmit signals(?) The authors want the readers to envision everyday objects and correlate to how the cell goes through its processes. 6. This article was published in 1996. Today we call ICE-like proteases something else, what are they? Caspase-1  Inside · The all · Self-Eating · Double-mem structure-going to Lysosome double membrane structure Membrone of - Lysesome boily/blebbing membrane - of thing belg digested - Shrinkage Inside the all - Which process is Membrane shown above? disrupted Mito Matux potential , Mito plates ploy crucial pt Justify your of stimulanty Apoptosis response. Cleavage Apoptoseblebs"  Good Yoble 4 Comparing Apoptosis, Necrosis, and Autophagy Summonzes n From: Elmore, 2007. Toxicological Pathology.  How to quantifying apoptosis and cell death using flow cytometry - I’ll explain this slide PS flips to outer membrane. Annexin V-FITC binds to PS making cell green FACS plot Left upper Right upper quadrant- quadrant - Red only Dead Green + Red Right lower Left lower quadrant - quadrant - Green only no color Only dead cells take up PI Flow cytometry cont. Fluorescent intensity is quantified then plotted. PS flips to outer membrane. to Annexin V-FITC binds to PS making cell green Able be FACS plot Interpret At all is dead Left upper 2 beghwit canonytan se - Right upper quadrant- quadrant - Red only Dead Green + Red Right lower Left lower quadrant - quadrant - no color Live - Green only Only dead cells take up PI The picture below is an EM image showing organelles within a cell. Which of the processes is being shown in the figure? A) Apoptosis B) Autophagy: double membrane C) Necrosis D) Mitosis You are experimenting with a new chemotherapy drug. You treat some cells with saline solution and others with the chemotherapy drug, then stain the cells with FITC labeled Annexin V and Propidium iodide (PI). How would you interpret the results of your experiment, below? The panel on the left represents the control cells and the panel on the right represents the treated cells. Control More -chemotherapy right panel shows more cell death because there is a decrease in color in the lower left quadrant which is the live cells ina too -There are more early apoptotic cells in the chemotherapy panel More Less - -Increase in late apoptosis cells Dye - Moe cells PROVES CHEMOTHERAPY IS KILLING CELLS (could be good if they’re cancer cells and not healthy cells zota I In color ↓ representsSime alls - but fewer sells now BREAK  Compare and contrast intrinsic vs. extrinsic apoptosis. Explain the similarities and differences between the 2 pathways. Intrinsic: internal cellular stress like DNA damage or even oxidative stress Extrinsic: external cellular signaling on the plasma membraneImmune System Similarities: the use of caspases-3, have the same outcomes Differences: ○ Intrinsic: internal damage and relies on the mitochondria, caspase-9 specific ○ Extrinsic: uses a death receptor, and is caspase 8 specific, uses DISC formation What role do the following proteins/lipids play in the process? Are they pro- or anti-apoptotic? What would happen if there were mutations in these proteins? ○ Bcl-2: inhibits other membrane of mitochondria, prevents cyto C release and caspase activation, anti-proptotic, mutation means the damaged cell will survive ○ Bax: promotes outer membrane of mitochondria, allows cyto C release and caspase activation, pro-apoptotic, mutations can impair apoptosis ○ Bim: activator of BAX, pro apoptotic, mutations lead to the prevention of apoptosis ○ Cytochrome c: activate caspase 9, pro-apoptosis, mutations cause cancer ○ (Initiator caspases) Caspase 8, 9, 10: lead to caspase 3 activation which goes into the execution pathway, pro-apoptotic, mutations lead to the preventions of the cascade ○ (Executioner caspase)Caspase 3: cleaves cellular proteins and DNA repair, executing apoptosis. Mutations:incomplete apoptosis ○ FAS ligand: allows for extrinsic pathway, pro-apoptosis, mutations prevent the binding of to the receptor ○ IAP: inhibits caspases, anti-apoptotic, mutations contribute to cancer ○ Phosphatidylserine: signal to be broken down, mutations could lead to random cells being broken down or kept ○ (Pro apoptosis under stress) p53: stimulated when DNA is damaged. Mutation can lead to cancer Intrinsic Apopoanternal Signal External. Apop : external signa tells you to self kill, usually comes Prom Immune System  General apoptotic mechanism Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company Elmore, 2007. Toxicologic Pathology. Figure 3 Activating the Intrinsic Apoptotic Pathway Intrinsic Apoptosis Cascade Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company Extrinsic Apoptotic Cascade Mechanisms to block Apoptosis IAP = Inhibitor of Apoptosis Protein  P53 promotes cell cycle arrest or apoptosis p53 is stimulated when DNA is damaged. p53 activates transcription of p21, a cyklin-dependent kinase inhibitor. Activation of p21, leads to cell cycle arrest. If the DNA damage is not fixed, p53 stimulates apoptosis. Which answer choice has the correct sequence of events with respect to extrinsic apoptosis? 1. Caspase 3 activation 2. Caspase 8 activation 3. Cytoskeletal proteins cleaved 4. Fas ligand binds to death receptor 5. DISC complex formed A) 5, 4, 3, 2, 1 B) 4, 5 1, 2, 3 C) 4, 5, 2, 1, 3 D) 5, 4, 2, 1, 3 You are studying a new cell line and find that these cells are unable to cleave pro-caspase 9 to form active caspase 9. What effect does this have on the cells? A) Bcl-2 in not activated, thus the cells undergo extensive apoptosis. B) The apoptosome is not formed, thus cytochrome c remains in the mitochondria. C) The cells are unable to activate caspase 3, thus intrinsic apoptosis is blocked. D) The cells are unable to activate caspase 8, thus extrinsic apoptosis is blocked. You develop a new drug to treat an aggressive form of breast cancer. Which of the following mechanisms is the most likely explanation of how this drug works? A) Akt kinase is overactivated, phosphorylating Bad, promoting apoptosis. B) Bax is able to form dimers more easily, releasing additional cytochrome c into the cytoplasm C) MAP-kinase phosphorylates and activates Hid, which activates IAP proteins D) MTor protein activates Bcl2, which stimulates apoptosis BREAK Activity 4 - Dysregulated Apoptosis (20 + 10 min) A) Provide a detailed mechanism for a disease that explain how necrosis or excessive apoptosis leads to disease. You may use words or illustrations to explain. -Aids (kills helper T cells too soon) Parkinson’s, Alzheimer's -a lot of autoimmune -Type 1 diabetes B) Provide a detailed mechanism for a disease that explain how too little apoptosis leads to disease. You may use words or illustrations to explain. Cancer (Over expression of Bcl2) Excessive apoptosis and disease example Not enough apoptosis and disease example Muddiest point? What was the most difficult thing to understand today? -the intrinsic pathway description