BIOMG 1350 Notes Week 8
BIOMG 1350 Notes Week 8 BIOMG 1350
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This 12 page Class Notes was uploaded by genehan on Saturday September 3, 2016. The Class Notes belongs to BIOMG 1350 at Cornell University taught by Garcia-Garcia, M; Huffaker, T in Fall 2015. Since its upload, it has received 7 views. For similar materials see Introductory Biology: Cell and Developmental Biology in Molecular Biology and Genetics at Cornell University.
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Date Created: 09/03/16
BIOMG 1350 Professor Bretscher & GarciaGarcia Spring 2016 Week 8: Lecture 1 of 2 Wednesday, March 14, 2016 Lecture Title: The Cell Division Cycle Lecture Keywords: cell division cycle, M, G1, S, G2 phase, interphase, checkpoint, negative and positive regulator, activated and satisfied checkpoint, cuclindependent kinase CDK, CDK activating kinase CAK, CDK inhibitory kinase Wee1, activating phosphatase CDC25, poly ubiquitylation, proteasome, G0 phase, p53 gene, p21 1. Iclicker question – Lysosomal enzymes acquire a specific carbohydrate modification in the Golgi that is then recognized by the lysosomal enzyme receptor. There is a horrid disease called Icell disease in which the enzyme that generates the carbohydrate modification is defective, so lysosomal enzymes do not get the specific modification. In cells from a patient with lcell disease, which of the following scenarios would be correct? a. Lysosomal enzymes would accumulate in the TGN. (No, they would be secreted since they would not be diverted to the early endosome.) b. The lysosomal enzyme receptor would still cycle between the TGN and the early endosome. (No, the receptor needs to bind a lysosomal enzyme to leave the TGN.) c. The lysosome enzyme receptor would be stuck in the TGN. (Yes, the receptor needs to behind a lysosomal enzyme to leave the TGN.) d. The lysosomal enzyme receptor would be stuck in the early endosome. (No, they never get to the early endosome.) e. More than one answer is correct. 2. Early endosome is not a static structure. It is full of incoming vesicles that fuse to make a new early endosome and can collect lysosomal enzymes coming from the TGN. It can slowly mature into a late endosome and then eventually a lysosome with acidic pH. 3. The cell division cycle involves cell growth and chromosome replication, chromosome and organelle segregation, and then cell division to make two cells. a. Life is believed to have started with a common ancestral cell 3 billion years ago. i. It is likely that the mechanism is ancient and highly conserved. b. What are the challenges facing a cell that wants to give rise to two cells? i. It needs to accurately duplicate all its contents and find a way to divide them equally between the two daughter cells. c. What would happen if a cell tried to divide before its DNA had doubled? i. It would be catastrophic and at least one daughter cell would lack part of its genetic material. Cells have ways of checking steps in the cycle. d. What would happen if a cell would duplicate its DNA but then continually divide before all its other constituents had doubled? i. Daughter cells would lack some organelles and get smaller and smaller e. Cells have mechanism to precisely regulate cell growth. 4. The cell cycle is divided into 4 phases – M phase, G1 phase, S phase, and G2 phase. a. The M phase is nuclear (mitosis) and cytoplasmic division (cytokinesis). b. The other 3 cycles are known as interphase. S stands for the synthesis phase in which DNA synthesis and replication occurs. c. The length of a cell cycle can vary enormously. Under optimal nutritional conditions, bacteria can have a short cycle time of 10- 20 minutes whereas human liver cells take about a whole year. Most nerve cells never divide again i. The length is determined by the G1 phase, in which the cell decides whether to proceed with the whole cell cycle. Once past the G1 phase, the cell will undergo S, G2, and M phase. 5. The ordering of cell cycle events is controlled by checkpoints that are negative regulators. a. Analogy – When constructing a house, the walls of a house will not be put up until the foundation is checked for sturdiness. Then, the plumbing and electricity is done and checked. Then, furniture is brought in. Similarly, cells use checkpoints to make sure steps are occurring correctly. b. A checkpoint is a negative regulator because it delays the cycle. It is a molecular mechanism that delays the progression of the cell cycle to ensure that one process is completed before the next one is started. c. The first checkpoint is the G1 checkpoint. i. It checks the environment is favorable (enough food/nutrients) and once checked, it will proceed to the S and G2 phase. d. The G2 checkpoint will check if all the DNA is replicated and any DNA damage is repaired. e. The mitosis checkpoint checks if all the chromosomes properly attached to the mitotic spindle so that duplicated chromosomes can be pulled apart. f. When a checkpoint is activated, this means the cell cycle is delayed at that point. g. When a checkpoint is satisfied means that the process is okay and the cell cycle can proceed. 6. Cyclin-dependent kinases CDK (enzymes) are positive regulators that drive the cell cycle. (Phosphatases counteract kinases.) a. Cell cycle control depends on the cyclical activation of CDKs. b. Kinases are present throughout the cell cycle and its activity is dependent on cyclins. Without a cyclin, the CDK is useless. i. Cyclin concentration is varied throughout the cell cycle. ii. For example, M-cyclin concentration peaks during mitosis but drops at interphase. CDK concentration is constant but its activity requires the cyclin. c. S-cyclin will increase in G1 phase and would activate S-CDK which will phosphorylate through downstream pathways and then drive the cycle into S phase. d. Then, M-cyclin is synthesized and if the G2 checkpoint is satisfied, active M-CDK would result in M-phase substrates and drive the cell onto mitosis. 7. CDK has 2 phosphorylation sites – one inhibitory and one activation site a. CDK-activating kinase CAK puts the activating phosphate on. b. CDK-inhibitory kinase Wee1 places an inhibitory phosphate and turns all kinase activity off. c. The activating phosphatase CDC25 removes the inhibitory phosphate and the kinase activity is turned on. 8. Cyclin levels rise gradually but CDK activity rises abruptly. a. The activating phosphatase suddenly takes off all inhibitory phosphates. b. An inactive M-CDK has both the inhibitory and activating phosphate. At the G2 checkpoint, this partially activates CDC25 so only some of the M-CDK are activated. The few active M-CDK undergo positive feedback by phosphorylating inactive CDC25, which stimulates more active CDC25 activating M-CDK. Simultaneously, all M-CDK are active which then does feedback on the Wee1 by phosphorylation to stop the placement of the inactivating phosphate and further the cell cycle. i. The negative regulator keeps M-CDK inactive until the G2 checkpoint. The Wee1 kinase enforces the G2 checkpoint and this is reversed by CDC25 phosphatase. 9. Iclicker question – We just discussed Wee1 that phosphorylates inhibitory sites on CDK. Cells in which the gene for Wee1 is deleted can live. What do you think would happen to the cell cycle if you inhibited Wee1 kinase? a. Cell would not enter M phase. (No, M-CDK will be active and mitosis will occur.) b. Cell would get stuck in M phase. (No, entry into and exit from M- phase will occur.) c. Cells would go through M phase, but not enter S phase. (No, this will have no effect on S phase, as this is an M phase specific kinase.) d. Cells would go through M phase and arrest in G1. (No, this will have no effect on the rest of the cell cycle.) e. Cells would undergo M phase early and divide early. (Yes, this phosphorylation is a negative regulator of entry into the M phase. If you remove it, cells will enter M phase prematurely. Then, they divide early and as cell growth continues in G2, the cells get smaller. 10. Both cyclin levels and CDK activity drop abruptly. a. CDK inactivation is triggered by cyclin degradation. b. Ubiquitin is a small protein that can be covalently linked to proteins. i. Poly-ubiquitylation of a protein is a common signal in cells that marks the protein for destruction in a structure called the proteasome. c. Cyclin is ubiquitylated and this results in destruction of cyclin and thus inactive CDK. 11. Defects in checkpoints can cause cancer. a. The checkpoint is when cells decide whether to enter the cell cycle based on environmental signals. b. Growth factors signal the cell to pass through the G1 checkpoint and enter the cell cycle and when they commit to entering, they will complete the cell cycle. Thus, the decision-making is done at the G1 checkpoint. (time differences in the cell cycle length is due to the time spent at the G1 checkpoint. c. Cells that never divide again withdraw from G1 into a state called G0. d. In animal cells, the G1 checkpoint stops cells that have DNA damage. 12. DNA damage can activate the G1 checkpoint due to the p53 gene (the guardian of the genome). a. Without DNA damage, p53 is degraded in proteasome. b. If p53 is activated by DNA damage, it goes into the nucleus and activates transcription of a p21 gene. This makes p21 mRNA that goes into cytoplasm and makes p21, a CDK inhibitor protein. p21 inactivates S-CDK to stop mutations from continuing on into the S phase. c. P53 is a tumor suppressor mutated in about 50% of cancers. Loss of p53 function results in more rapid accumulation of mutations, it enhances the change from a normal to a cancer cell. BIOMG 1350 Professor Bretscher & GarciaGarcia Spring 2016 Week 8: Lecture 2 of 2 Wednesday, March 16, 2016 Lecture Title: Mitosis Lecture Keywords: centromere, cohesins, centrosome, mitosis, cytokinesis, mitotic spindle, kinetochores, aster microtubules, kinetochore microtubules, interpolar microtubules, unipolar/bipolar attachment, aurora B kinase, phosphatase, Anaphase Promoting Complex APC, separase, securing, Anaphase A, Anaphase B, kinesin 5, dynein, cytokinesis 1. Iclicker question If you mutate the inhibitory site in yeast CDK so that it cannot be phosphorylated, your mutant should be similar to: a. Cells lacking the CDK activating kinase CAK (No, cells are dead) b. Cells lacking the kinase wee1 (Yes, these cells can live and go through mitosis early and become smaller cells.) c. Cells lacking phosphatase CDC25 (No, cells are dead. Cells with reduced CDC25 activity that is not sufficient for life, can grow when Wee1 is also deleted. This is genetic evidence that CDC25 counteracts Wee1 but that CDC25 also does something else.) d. Cells lacking kinase CDK1 (No, cells are dead) e. None of the above as it will be dead (C is correct.) 2. Wee1 kinase puts an inhibitory phosphate keeps M-CDK inactive until the G2 checkpoint is satisfied. a. Then, CDC25 removes the inhibitory phosphate. b. Wee1 kinase enforces the G2 checkpoint. 3. S phase a. Chromosomal DNA is duplicated precisely, so that it is not over- duplicated. b. Once it’s duplicated, you get a duplicated chromosome in which chromatids are held together with a centromere. c. Cohesins hold the duplicated chromatids together. d. The single centrosome is duplicated to prepare for M phase and will separate in mitosis. 4. G2/M transition happens with the activation of M-CDK. a. Active M-CDK phosphorylates hundreds of proteins in the cell and flips the switch to activate mitosis. 5. M phase has two distinct but overlapping parts a. Mitosis – précise and equal segregation of the duplicated chromosomes by the microtubule-based mitotic spindle b. Cytokinesis – division of the cytoplasm in two halves to generate two cells by contraction of the microfilament-based contractile ring c. Plants have a different process. 6. The mitotic spindle is a machine to distribute the chromosomes equally with a built in quality control system – the checkpoint. a. Brief sequence of the mitosis phases: b. Prophase – centrosomes separate to make a spindle pole c. Prometaphase – chromosomes are captured by microtubules d. Metaphase – chromosomes attach to mitotic spindle e. Checkpoint in mitosis f. Anaphase – draw chromosomes towards phase g. Telophase – contractile ring forms h. Cytokinesis – cell pinches into two 7. Recall that microtubules are built from alpha and beta heterodimers. a. The minus end is associated with the centrosome and spindle pole. b. The plus end is favored for growth – the periphery. 8. Mitotic spindle Parts a. Kinetochores attach to microtubules during prometaphase and metaphase. i. They link the centromeric DNA to the microtubules. b. The spindle has 3 distinct classes of microtubules i. Aster microtubules orient the spindle within the cell. ii. Kinetochore microtubules come form the spindle pole and link to the kinetochore. iii. Interpolar microtubules overlap from the two spindle poles. 9. Kinetochores capture microtubules. a. Poles are stabilized when an aster microtubule attaches to a kinetochore to make a unipolar attachment to become a kinetochore microtubule. b. A free kinetochore attaches to microtubules from an opposite spindle pole to make a bipolar attachment. c. The correct attachment is selected by tension across the two kinetochores from the two poles pulling on the chromosomes. i. Aurora B kinase continually phosphorylates the kinetochore that makes attachment unstable. ii. Phophatase on kinetochore dephosphorylates making the microtubule attachment. iii. With tension, aurora B is unable to phosphorylate, making a stable bipolar attachment. iv. This is how individual duplicated chromosomes become bi- oriented. 10. Checkpoint at metaphase checks if all chromosomes are attached. a. Any unattached kinetochore sends a signal that there is a disaster, which activates the checkpoint in mitosis. b. Once all chromosomes are bioriented, the checkpoint is satisfied. c. Then, this activates the Anaphase Promoting Complex APC, which leads to the degradation of M-cyclin and cohesins holding chromatids together. This is the transition to anaphase. i. M-cyclin is degraded by ubiquitylation and so M-CDK is inactivated. ii. A protease called separase is usually held in an inactive state by a protein called securin. iii. Active APC activates separase, which then degrades cohesins that were holding chromosomes together. This starts anaphase. 11. Iclicker question – What do you think would happen over a period of time to a metaphase cell if you used a laser to keep cutting the kinetochore microtubules to just one chromosome? a. It would enhance progression in to anaphase. (No, you would activate the mitosis checkpoint so you would not progress into anaphase.) b. Anaphase would occur normally except that both copies of the chromosome would end up at one pole. (No, the checkpoint is activated.) c. It would delay progression into anaphase for a short time. (No, it would not progress into anaphase.) d. The cell would not progress into anaphase. (Yes!) e. None of the above. 12. Anaphase A is when chromosomes are pulled poleward. a. Kinetochores are attached to the plus ends through a sleeve around the microtubules. b. The plus end depolymerizes, stimulated by the kinetochores, resulting in curved microtubules. This forces the sleeve to move and draws the kinetochore to the pole. 13. Anaphase B is when poles are pushed and pulled apart by two forces. a. The sliding part occurs by kinesin 5 with heads on both ends going towards the plus ends. The net result is that the poles are pushed further apart. b. Dynein on the cell membrane interacts with a microtubule, pulling on it. This pulls the spindle apart, lengthening it. 14. Cytokinesis in animals occurs through a contractile ring of actin and myosin filaments in cleavage furrow. a. In plants, Golgi derived vesicles in the middle of the cell fuse with each other to form a new cell wall.
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