BMS100 Physiology Concepts 1.17 Cell Cycle Fall 2022 student PDF

Physiology Concepts 1.17 Cell cycle Dr. Hurnik BMS 100 Week 9 Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable...

Physiology Concepts 1.17 Cell cycle Dr. Hurnik BMS 100 Week 9 Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable conditions CKIs, RB, p53 Cell survival Post-learning 1. DNA synthesis 1 2. DNA synthesis 2 3. Mitosis Cell cycle - intro The orderly sequence of events by which a cell duplicates its contents and divides in two The cell cycle is subdivided into phases or stages § We will focus on mitosis The cell cycle is driven by specific molecular signals present in the cytoplasm Cell cycle – intro continued The cell cycle consists of § Mitotic (M) phase (mitosis and cytokinesis) § Interphase (cell growth and copying of chromosomes in preparation for cell division) Interphase (about 90% of the cell cycle) can be divided into subphases: § § § § G1 phase: first gap S phase: synthesis G2 phase: second gap G0 phase: resting phase, postmitotic quiescent Cell cycle - visual Mitosis Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: G1 G1: § First gap phase Preparatory growth phase prior to cell entering DNA synthesis phase Cell is metabolically active, § Many organelles are duplicated § Duration: variable (6-24hrs) § § § § Requires nutrients & growth factors RNA, protein, lipid and carbohydrate synthesis occurs § No DNA replication yet! § § Short in embryonic and cancer cells Rapid or non-existent in rapidly dividing cells Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: S DNA and chromosomal protein synthesis occurs Duration: Lasts approximately 7-8 hours in a typical mammalian cell with a 16 hour cycle Cell is now committed to cell division Growth factors are no longer needed at this phase DNA replication occurs here, creating two identical daughter genomes Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: G2 § G2 § Second growth phase § Interval between DNA synthesis (S phase) and mitosis (M phase) § Enzyme, protein and ATP synthesis occur § cell growth continues § Duration: lasts approximately 3 hours in a typical mammalian cell with a 16-hour cycle Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Mitosis Cell cycle phases: M M § Mitotic phase § Cell undergoes mitosis and then cytokinesis Mitosis – covered in post-learning video 3 § Duration: 1-2 hours Anatomy and Physiology (Betts et al). Figure 3:32. Retrieved from: https://openstax.org/books/anatomy-and-physiology/pages/3-5cell-growth-and-division Top right image: Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: G0 G0: § State of withdrawal from cell cycle § Cell is neither dividing nor preparing to divide § Instead, the cell is “doing its job” - performing it’s function within the tissue Common for differentiated cells § Examples of cells in G0: Hepatocytes, neurons Anatomy and Physiology (Betts et al). Figure 3:30. Retrieved from: https://openstax.org/books/anatomy-and-physiology/pages/3-5-cellgrowth-and-division Cell cycle progression In order to progress through the cell cycle, a variety of signals must be turned on § If the environment is not favourable or there are errors in DNA, the cell cycle can be “paused” at several main check points (aka “transitions”) Checkpoints are based on series of biochemical switches to initiate a specific cell-cycle events. § Called the cell cycle control system Features of the cell cycle control system Features of the biochemical switches § 1. Generally binary (on/off) to launch an event in a complete & irreversible fashion Why might this be important?? § 2. Robust & reliable Contains back up mechanisms to ensure efficacy under variable conditions & if some components fail § 3. Adaptable & modified to suit specific cell types Responds to specific intracellular or extracellular signals § Cyclin dependent kinases (Cdks) – more to come Cell cycle checkpoints continued Checkpoints aka “Transitions”: § Points in the eukaryotic cell division cycle where progress through the cycle can be halted until conditions are suitable for the cell to proceed to the next stage Checkpoints can be regulated by: § Factors within the cell, mostly controlled by the “health” or “state of preparation” of the cell § Factors from outside the cell – i.e. messages from other cells within the same tissue or distant cells Cell cycle Checkpoints Three major regulatory transitions at the: § 1. Start Transition (aka G1/S) § 2. G2/M transition § 3. Metaphase-to-anaphase transition (aka M-to-A) For most cells, the G1/S seems to be the ratelimiting and committing step of the cell cycle Cell cycle Checkpoints - visual G2/M checkpoint Metaphase-to-Anaphase checkpoint G1/S checkpoint Aka Start transition Adapted from: https://upload.wikimedia.org/wikiped ia/commons/3/38/0332_Cell_Cycle_ With_Cyclins_and_Checkpoints.jpg Cell cycle Checkpoints continued Specific signals MUST be present for a cell to pass through the following checkpoints § If cell detects problem inside or outside the cell it will block progression beyond the checkpoint Eg. If extracellular conditions are not appropriate for cell proliferation, central control system blocks progression through the start transition Eg. If there is a problem with completion of DNA replication, cell will be held at G2/M checkpoint Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-9. Page 968 Cell cycle control system Key to the cell cycle control system are cyclindependent kinases (Cdks) What do you suppose Cdks do biochemically? Cdks are responsible for cyclical changes in phosphorylation of intracellular proteins that initiate/ regulate the major events of the cell cycle Eg. Might activate important enzymes to DNA synthesis Cdks are controlled a group of proteins called cyclins Cyclin-Cdk complexes - intro Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin–Cdk complexes at specific stages of the cell cycle. Correct & functional cyclin-cdk complexes are needed to progress through a checkpoint § Exception: M-to-A checkpoint is a little different Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-16. Page 973 Cyclin classes There are 4 classes of cyclins that form specific complexes with Cdks § 1. G1 cyclins: Cyclin D Forms complex with Cdk4 or Cdk 6 Involved in G1 phase of cell cycle, needed for initiation of transcription of G1/S cyclins to help promote passage through start transition § 2. G1/S cyclins: Cyclin E Forms complex with Cdk2 Bind Cdk’s at the end of G1 & help trigger progression through start transition Levels decrease in S phase Cyclin classes continued There are 4 classes of cyclins that form specific complexes with Cdks § 3. S-cyclins: Cyclin A Forms complex with Cdk1 and Cdk2 Bind Cdks after progression through start transition & helps stimulate chromosome duplication during S phase Levels remain elevated until mitosis; contributes to control of some early mitotic events § 4. M cyclins: Cyclin B Forms complex with Cdk1 Bind CdKs to stimulate entry into mitosis at the G2/M transition Levels decrease in mid-mitosis Cyclin-cdk complex summary There are 4 classes of cyclins that form specific complexes with Cdks Cyclin-Cdk complex Cyclin Cdk Function G1-Cdk Cyclin D Cdk4 Cdk6 Needed for progression through the start transition G1/S-Cdk Cyclin E Cdk2 Trigger progression through Start Transition S-Cdk Cyclin A Cdk2 Cdk1 Stimulate chromosome duplication Involved in early mitotic events M-Cdk Cyclin B Cdk1 Stimulate progression through G2/M checkpoint Cell cycle Checkpoints - visual G2/M checkpoint Metaphase-to-Anaphase checkpoint G1/S checkpoint Aka Start transition Adapted from: https://upload.wikimedia.org/wikiped ia/commons/3/38/0332_Cell_Cycle_ With_Cyclins_and_Checkpoints.jpg Cyclin-Cdk complex How do cyclin-Cdk complexes work? § Cyclins function by activating the Cdk FYI – another protein, cyclin activating kinase (CAK) is needed for full activation Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-12. Page 970 Cyclin-Cdk complex continued How do cyclin-Cdk complexes work? § Cyclin protein does not simply activate its Cdk partner, but also directs it to a specific target protein § Note: A cyclin-CdK complex can induce different effects at different times in the cell cycle Since accessibility of CdK substrates change during the cell cycle § Eg. Proteins the function in mitosis may only become available for phosphorylation in G2 APC/C Progression through the Metaphase-to-anaphase checkpoint is a little different § Occurs via regulated proteolysis Where have we seen this before? § A complex called the anaphase promoting complex (APC/ C; aka cyclosome) is needed Member of ubiquitin ligase family of enzymes § Used to stimulate proteolytic destruction of specific regulatory proteins § APC/C polyubiquitinates specific target proteins for destruction in proteasomes. Target proteins: securin, M-cyclins, S-cyclins Full cell cycle Let’s take a look at the full cell cycle under favourable conditions Cell cycle phases: G1 Growth factors are required in the G1 phase § Growth factors bind to specific receptors to stimulate cellular growth and proliferation Turns on early response genes and delayed response genes § Early response genes – usually transcription factors What was an example of an early response gene we have learned already? Will induce the transcription of delayed response genes. § Delayed response gene - usually Cdks, cyclins, or other proteins needed for cell division Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Review: Ras à MapK Eg. Myc Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-47. Page 855 G1 continued In response to binding of growth factor, § Cyclin D and then E are transcribed and translated Cyclin D can form complexes with Cdk4 and Cdk 6 § Call the G1-cdk complex Cyclin E can form complexes with Cdk2 § Called the G1/S-cdk complex Active G1-cdk and G1/S-cdk complexes allows progression through the start checkpoint. G1/S checkpoint: a closer look Active G1-cdk (and G1/S-cdk) complex will target a protein called RB and phosphorylate it. § Review: G1-cdk complex is: G1/S-cdk complex is: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 G1/S checkpoint: a closer look continued RB functions as a transcription co-repressor § Hyperphosphorylation of RB will inactivate RB § Inactive RB then releases a transcription factor E2F, allowing transcription to proceed Transcription of cyclin E => G1/S-cdk complex forms and promotes passage through the start transition Transcription of cyclin A => S-cdk complex forms Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Putting it all together Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Cell cycle phases: S In early S phase, cyclin D (G1-cdk complex) and E (G1/S-cdk complex) are targeted for destruction § FYI – they are targeting for destruction by a protein called SCF § How do you suppose they are targeted for destruction? This also promotes progression through the S phase of the cell cycle Cell cycle phases: S continued Active S-cdk complex allows progression through the S phase of the cell cycle § What was the S-cdk complex? § What is occurring during the S phase of the cell cycle? Cell cycle phases: G2 S-Cdk complex levels are still high in G2 M-cyclin levels begin to rise § Form a M-Cdk complex Which cyclins and cdks are part of this complex? § M-Cdk complex is needed to pass through the G2/M checkpoint At the end of G2, the S-cyclins are destroyed § Targeted for proteolysis by APC/C M-Cdk complex control Take a look at this diagram again, what do you notice about when the M-cyclins transcribed? Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-16. Page 973 M-Cdk complex control We need to be able to control the activity of Mcyclins so that mitosis doesn’t start too soon! § Once the M-Cdk complex is assembled, it is immediately inhibited via phosphorylation FYI – inhibitory phosphorylation occurs by a kinase called Wee1 § When the cell is ready for mitosis to begin, the M-Cdk complex is de-phosphorylated § FYI - de-phosphorylation occurs by a phosphatase called Cdc25 Cell progresses through the G2/M checkpoint and mitosis begins Cell cycle phases: M phase Mitosis begins § M-cdk complex is needed for activation of various proteins needed in mitosis § Mitosis proceeds: Prophase à pro-metaphase à metaphase § Before progressing to anaphase and then to telophase, we reach our final checkpoint: Metaphase-to-anaphase (M-to-A) checkpoint Molecular Biology of the Cell (Alberts et al) 6th ed. Panel17-1. Page 980 Metaphase to Anaphase checkpoint Instead of a cyclin-cdk complex being used to progress through the M-to-A checkpoint, instead we used regulated proteolysis § APC/C complex targets a protein called securin by ubiquitylation for destruction by a proteosome Securin is an inhibitory protein that protects protein linkages that hold sister-chromatid pairs together in early mitosis Destruction of securin activates a protease that separates the sister chromatids allowing progression to anaphase Molecular Biology of the Cell (Alberts et al) 6th ed. Panel17-1. Page 980 FYI – For visualization only Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-38. Page 993 Cell cycle: M phase continued After anaphase the cell continues through: § Telophase à cytokinesis Molecular Biology of the Cell (Alberts et al) 6th ed. Panel17-1. Page 980 Cell cycle: M phase continued At the end of mitosis, the M-cyclins are also targeted for destruction by APC/C § Destroying these cyclin inactivates most Cdks in cell § Then, many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases in the anaphase cell Dephosphorylation of Cdk targets is required for the completion of M phase, including the telophase and then cytokinesis Putting it all together Fill in the table at home Phases G1 S G2 M Functions Cyclin-Cdk complexes involved Unfavourable conditions In unfavourable conditions, the cell cycle can be paused at any of the main checkpoints § Progression through G1 is delayed if: DNA is damaged by radiation, chemicals, or errors Absence of nutrients or growth factors Abnormal cell size § Entry into M is prevented when: DNA replication is not complete Presence of DNA damage Abnormal cell size § Progression through M-to-A is prevented if Chromosomes are not properly attached to mitotic spindle Let’s consider how this is done Two important molecular mechanisms: § Cdk inhibitory proteins (CKIs) § Proteins coded by tumour suppressor genes Contact inhibition CKIs Binding of Cdk inhibitory protein (CKI) § Inactivates cyclin-Cdk complex Binding stimulate rearrangement in structure of Cdk active site § Primarily used by cells to govern the activities of G1/Sand S-Cdks early in cell cycle Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-14. Page 971 CKI examples Three important CKIs are: § p16 inhibits CyclinD-cdk4 & CyclinD-cdk5 (G1-cdk complex) § p21 inhibits CyclinE-cdk2 (G1/S-cdk complex) CyclinA-cdk2 & CyclinA-cdk1 (S-cdk complex) Cyclin B-cdk1 (M-cdk complex) § p27 inhibits CyclinA-cdk2 & CyclinA-cdk1 (S-cdk complex) CyclinE-cdk2 (G1/S-cdk complex) Cyclin B-cdk1 (M-cdk complex CKIs Know: § p16 § p21 § P27 Rest are FYI Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 1.19. Page27 CKIs – review at home Complex Cyclin Cdk Function G1-Cdk Cyclin D Cdk 4 Cdk6 Needed for progression through the start transition G1/S-Cdk Cyclin E Cdk2 Trigger progression through Start Transition S-Cdk Cyclin A Cdk2 Cdk1 Stimulate chromosome duplication Involved in early mitotic events M-Cdk Cyclin B Cdk1 Stimulate progression through G2/M checkpoint Associated CKI Tumour suppressor genes Let’s looks at two key tumour suppressor genes: § p53 Recognizes and binds damaged DNA Unstressed cells have lower levels of p53 since it will be bound by a protein called Mdm2 and be degraded Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 § RB Generally found in active form § Hypo- or hyperphosphorylated? Can also recognize damaged DNA Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 p53 In the presence of DNA damage, p53 will be phosphorylated, releasing Mdm2 § p53 will not be degraded Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 p53 Active p53 binds DNA and promotes the transcription of p21 § Wait… what was p21 again? p21 binds the G1/S-cdk complex, inhibiting it. § An inactive G1/S-cdk complex will pause the cell cycle at the __?__ transition Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 RB In the presence of a growth suppressor signal or DNA damage § p16 is transcribed; p16 inhibits the G1-cdk complex, which was needed to inactivate RB § RB remains activated and bound to E2F No transcription of G1/Scyclins or S-cyclins Cell cycle is paused at start transition Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Page 294 Contact inhibition Contact inhibition – The cell cycle progression can also be inhibited due to contact with: Other cells § Aka density-dependent inhibition A basement membrane or other matrix component § anchorage dependence § Most animal cells must be attached to a substratum in order to divide § This is regulated with cadherins and Beta-catenin pathway More to come next class Extracellular signaling: survival We’ve discussed how extracellular signalling can promote cell proliferation or inhibit the cell cycle § Eg. Growth factors and mitogens can promote cell growth and mitosis Cells can also receive signals to promote survival § Survival signals promote the cell cycle and prevent apoptosis PI3K-Akt-mTOR C pathway Akt in the cell cycle PI3K-Akt-mTOR C pathway § We’ve already discussed this pathway in detail, go back to your notes from a few weeks ago to review it. § Akt can promote cell cycle progression by: Akt activates/increases: § Cyclin A à activation of CDK-1 § Cyclin D à activation of CDK-4/6 Akt decreases/inactivates: § p21 and p27 Study strategies – at home Draw out the 4 stages of cell cycle § In 1 bullet point describe the main function in each stage Add in the 3 main checkpoints § Add in the specific cyclin-cdk complexes needed to get through each checkpoint § Which checkpoint is not regulated by cyclin-cdk complexes What enzyme regulates this complex? How does it work? Add in p16, p21, p27. How do they affect the cell cycle? How does RB stop the cell cycle? How does p53 stop the cell cycle? References Alberts et al. Molecular Biology of the Cell. Garland Science. Betts et al. Anatomy and Physiology (2ed). OpenStax Pathologic Basis of Disease(Robbins and Cotran) 10th ed.