Cell Cycle Combined PDF

L18 - Intro to the cell cycle Date @December 3, 2024 Difficulty Progress Done Overview Early cell division in drosophila embryo Purpose of the cell cycle...

L18 - Intro to the cell cycle Date @December 3, 2024 Difficulty Progress Done Overview Early cell division in drosophila embryo Purpose of the cell cycle What needs to happen to carry out a cell cycle Basic cell cycle Chromosome, centrosome and organelle duplication (recap) What drives the cell cycle Cyclin levels oscillate to order cell cycle phases Additional Cdk regulators Discovery of Cdks Cell division cycle (cdc) mutants Discovery of cyclins What makes cyclin levels oscillate The APC/C How cell cycle fidelity is maintained Checkpoint G1-S transition / G1 checkpoint G2-M transition/ G2 checkpoint Discovery of the DNA damage checkpoint Metaphase-Anaphase transition If a checkpoint cannot be satisfied Disease due to defective checkpoints Summary Overview What events need to happen to allow a cell to reproduce itself The key regulatory steps in the cell cycle: Cyclin-dependent kinases as master regulators Cell cycle checkpoints L18 - Intro to the cell cycle 1  The conservation of cell cycle regulation and the importance of model organisms Early cell division in drosophila embryo Different types of division: Cloning cells of a given type to make tissues Making cells of different types (differentiation) – can involve asymmetric divisions Making cells with half normal DNA content – eggs and sperm (meiosis) Purpose of the cell cycle To allow a cell to reproduce Cell cycle importance: Required for growth, development and procreation High fidelity/ accuracy required to ensure stable inheritance of cell and organism characteristics Most be controlled to allow development and prevent disease What needs to happen to carry out a cell cycle Chromosomes need to be duplicated Other organelles need to copied Cells need to grow Chromosomes need to be segregated accurately L18 - Intro to the cell cycle 2  Cell needs to physically divide Basic cell cycle L18 - Intro to the cell cycle 3  G1, S and G2 are in interphase and M - in the cell cycle whereas G0 isnt in the cell cycle G1: Gap 1 Deciding if conditions are right for a full cell cycle Growing and preparing for DNA synthesis S: Synthesis Replicating DNA and centrosomes G2: Gap 2 Deciding if conditions are right for mitosis M: Mitosis Chromosome segregation and cytokinesis G0: resting state Cells not in the cell cycle Terminally differentiated cells Quiescent cells Senescent cells L18 - Intro to the cell cycle 4  Chromosome, centrosome and organelle duplication (recap) Chromosomes: chromosome condensation and segregation occur in mitosis phase Decondensation occurs in G1 DNA replication occirs in S SIster chromatid cohestion occurs in G2 Centrosomes: to organize microtubules and provide structure for the cell, as well as work to pull chromatids apart during cell division. L18 - Intro to the cell cycle 5  Golgi: Golgi ribbon STack disassembly Vesiculation What drives the cell cycle Cyclin-dependent kinases (Cdks) are important proteins that control the cell cycle Protein kinases that transfer a phosphate onto their substrates Act as “master regulators” Have multiple target proteins to control numerous processes in the cell cycle – activating 1 can act on 100s substrates Cdks have little activity by themselves, but they are activated by Cyclin proteins Cyclins also influence the substrate specificity of Cdks Cyclin levels oscillate to order cell cycle phases L18 - Intro to the cell cycle 6  Diff cyclins and cdks organize cell cycle in this way Additional Cdk regulators 2 Ways: 1. When Cdk first made it is inactivate due to inactivating phosphates which require phosphotases to remove them and activate them 2. Another mechanism - cell can make Cdk inhibitory proteins which can bind onto the cyclin-cdk complex and push into inacative complex Discovery of Cdks Work in model organisms has been critical to understand the cell cycle Yoshio Masui- Identified a cytoplasmic factor (MPF) that could induce cell division in frog oocytes Leland Hartwell - Conducted screens in budding yeast that identified Cell Division Cycle (cdc) mutants including Cdk1 (Cdc28) L18 - Intro to the cell cycle 7  Sir Paul Nurse - Identified and characterised Cdk1 (Cdc2) in fission yeast, and cloned human Cdk1 by complementation complementation - have defective yeast in protein can try put things back into yeast to complement defect – if put human cdk1 back into yeast will rescue the yeast that was defective in yeast Cdk1 Cell division cycle (cdc) mutants Yeast mutants that are defective in the cell cycle are important for studying the cell cycle The phenotype of the cell cycle mutant yeast would be effectively dead as they cannot grow and divide How can we study these cells: Temperature sensitive (ts) mutants - mutations that allow gene products to function at low temps but not high temps Track cell cycle by size and budding EXPLAINED: This selection explains how scientists study cell cycle mutations in yeast: L18 - Intro to the cell cycle 8  Yeast with cell cycle mutations are used as research tools These mutant cells would normally die since they can't grow or divide properly However, scientists can study them using two main methods: Using temperature-sensitive mutations that only disable the gene at high temperatures but work normally at low temperatures Observing the yeast's size and budding pattern to track cell cycle progression Discovery of cyclins Discovered by Sir Tim Hunt at Woods Hole Marine Biological Laboratory in 1982 In radiolabelled extracts of sea urchin eggs, a protein appeared after fertilisation and disappeared each time cells divided L18 - Intro to the cell cycle 9  What makes cyclin levels oscillate Cyclin synthesis is crucial to drive the cell cycle Mechanisms controlling synthesis include changes in transcription and translation rate, which vary depending on cell type Control of Cyclin destruction is also vital Important example: degradation of M-Cyclin is triggered by the APC/C The APC/C Signals degradation of M-cyclin to end mitosis and initiate cell division The APC/C is a ubiquitin ligase - it covalently attaches to the small protein ubiquitin to client proteins e.g. M cyclin ubiquitinatin is a tag for protein degradation by proteosome APC/C choses when to degrade the cyclin L18 - Intro to the cell cycle 10  How cell cycle fidelity is maintained Cyclin oscillations provide timing for the successive phases of the cell cycle The cells have cell cycle checkpoints so can check if anything is wrong Checkpoints are monitoring systems that check if conditions are right before allowing the next phase to occur If cell in G1 – is it in a good place to replicate DNA?? In g2 – check if environment favourable and check DNA is fully replicated before go into mitosis In mitosis – make sure all chromosomes attaches to spindle – monitor before go into next pahse Checkpoint They act by promoting Cdk activation or inactivation L18 - Intro to the cell cycle 11  Mitogen: promotes G1/S-cyclin synthesis DNA damage: inhibits cyclin activity by phosphoregulation or CKI Unattached chromosome: prevents M-cyclin destruction APC/C - stops M phase G1-S transition / G1 checkpoint aka START, restriction point or G1 checkpoint The checkpoint asks: Are nutritional conditions suitable? (particularly in single cell organisms) Is the cell receiving proliferation signals? (particularly in multicellular organisms) Has any DNA damage been repaired? Was the previous mitosis too long? – suggests theres a problem Once passed, the cell is committed to the entire cell cycle L18 - Intro to the cell cycle 12  G2-M transition/ G2 checkpoint CHeckpoint asks: Is DNA replication complete? – don’t want to separate chromosomes before replicated Has any DNA damage been repaired? Is the cell big enough (yeast)? L18 - Intro to the cell cycle 13  Discovery of the DNA damage checkpoint Budding yeast cells in G2 normally arrest if their DNA is damaged with X- rays Rad9 mutant yeast do not delay in G2 after DNA damage and they continue to proliferate with damaged DNA and eventually die So, are Rad9 mutant yeast defective in DNA damage repair? Experiment: if cells are chemically-blocked in G2 for a few hours, then the DNA damage can be repaired Conclusion: Rad9 is part of a checkpoint response, not part of the DNA repair response Metaphase-Anaphase transition AKA Mitotic or Spindle Assembly Checkpoint, SAC The checkpoint asks: L18 - Intro to the cell cycle 14  Are the chromosomes properly attached to the spindle Once the checkpoint is satisfied: The APC/C is activated to degrade M-Cyclin The cells exit metaphase into anaphase If a checkpoint cannot be satisfied If cell errors or damage can be fixed the cells will resume their cell cycle. However if cannot be fixed in a timely way due to extensive DNA damage or if trial and error correction of chromosome attachments takes too long. Then: Senescence - cells withdraw from the cell cycle Terminal exit from cell cycle Allows cell to remain part of tissue (may still be structural/ functional) but it will not proliferate L18 - Intro to the cell cycle 15  Cell doesnt immediately die Or Apoptosis - cells undergo programmed cell death Removes cell from organism Disease due to defective checkpoints Aberrant mitogen signalling can inappropriately drive cells through the G1 checkpoint into the cell cycle e.g. Cancer cells overexpressing EGF Receptors (EGFR, HER2), or with mutations in their signalling pathways (eg Ras mutants) Defects in the G2 checkpoint can allow proliferating cells to accumulate DNA damage e.g. p53 mutations Defects in the mitotic checkpoint can cause aneuploidy (wrong number of chromosomes) e.g. BubR1 mutation causing cancer predisposition syndrome (MVA) Summary The cell cycle: How cells reproduce themselves The cell cycle engine: L18 - Intro to the cell cycle 16  Successive waves of Cyclin-dependent kinase (Cdk) activation, driven by the synthesis and degradation of Cyclin proteins Cell cycle fidelity: Checkpoints can delay subsequent phases if the conditions are not right Cell cycle exit: If errors cannot be fixed, cells can withdraw from the cell cycle or die L18 - Intro to the cell cycle 17  L19 - Cell cycle entry and DNA replication Date @December 4, 2024 Difficulty Progress Done Overview Basic cell cycle Background Controlling the cell cycle - regulating the activity of Cdks G1-S transition How mitogens drive progression into S phase How does G1-Cdk drive progression into S phase What can prevent G1-Cdk drive progression into S G1-S transition is controlled by promoting Cdk activation or inactivation S phase Mechanism of DNA replication Initiation of replication in bacteria Initiation of replication in eukaryotes The DNA replication machine DNA polymerase only work from 5’ to 3’ DNA polymerases are accurate Replicative DNA polymerase needs RNA primers The end replication problem Telomeres DNA damage repair 1. Strand directed mis match repair 2. Base and nucleotide excision repair 3. DNA break repair G2 checkpoint Summary Overview What drives cells to enter the cell cycle and proliferate How cells accomplish DNA replication and DNA repair L19 - Cell cycle entry and DNA replication 1  The interphase checkpoints that govern G1-S and G2-M transitions Basic cell cycle Background What makes a cell decide to enter the cell cycle and proliferate? Some cells in adult humans are actively proliferating (i.e. in the cell cycle) – replaced constantly e.g. intestinal epithelial stem cells, lymphocytes during immune responses Most cells in adults are not proliferating Some are terminally differentiated (eg neurons) or senescent and will not proliferate Others are quiescent and can be stimulated to proliferate What will make a quiescent cell in G0 enter the cell cycle? What will make a cell in G1 carry out another round of the cell cycle? Controlling the cell cycle - regulating the activity of Cdks L19 - Cell cycle entry and DNA replication 2  The effect of mitogens and how that drives cell cycle G1-S transition The checkpoint asks: Are nutritional conditions suitable? – suitable time? (particularly in single cell organisms) Is the cell receiving proliferation signals? (particularly in multicellular organisms) Has any DNA damage been repaired? Was the previous mitosis too long? Once passed, the cell is committed to the entire cell cycle L19 - Cell cycle entry and DNA replication 3  How mitogens drive progression into S phase 1. Mitogen binds to cell surface receptor tyrosine kinase (extracellular receptor) 2. Ras-Raf-MAPK kinase signalling pathway is triggered - allowing signal from surface of cell to be transduced 3. “Immediate early” genes including Myc are expressed Myc is a transcription factor which upregulates genes including Cyclin D Cyclin D, together with Cdk4 or 6, forms G1-Cdk L19 - Cell cycle entry and DNA replication 4  some cancers overexpress EGFR EGF: A mitogen that activates the EGFR EGFR: A cell surface protein that binds to EGF, which induces cell proliferation How does G1-Cdk drive progression into S phase A key target of G1-Cdk is Retinoblastoma protein, Rb 1. In an early G1 cell, Rb binds to and inactivates the transcription factor E2F Rb prevents E2F doing its job as a transcription factor 2. Phosphorylation of Rb by G1-Cdk inactivates Rb - releasing E2F 3. E2F is now free to upregulate expression of genes including Cyclin E and Cyclin A 4. Cyclins E and A associate with Cdk2 to form G1/S-Cdk and S-Cdk L19 - Cell cycle entry and DNA replication 5  Cell cycle regulation positive feedback – help drive strong transitions – once triggered pathway the products feedback to make more e.g. active G1/s cdk feedback to beginning of pathway to help phosphorylate Rb – strongly in 1 direction only. DNA damage can prevent G1/S transition What can prevent G1-Cdk drive progression into S p53 is a central regulator of checkpoint responses to stress (Lecture 17) L19 - Cell cycle entry and DNA replication 6  p53 is normally maintained at low levels in cells by Mdm2-mediated degradation - when Mdm2 bound to p53 helps target it for ubiquitination so degraded DNA damage activates kinase signalling through ATM/ATR and Chk1/Chk2 Phosphorylation of p53 displaces Mdm2 therefore prevents binding and p53 is stabilised p53 acts as a transcription factor to turn on expression of CKIs such as p21 which inhibits G1/S-Cdk activity Even with mitogenic signal wont be sufficient enough to drive into S phase G1-S transition is controlled by promoting Cdk activation or inactivation L19 - Cell cycle entry and DNA replication 7  S phase The main tasks of S-phase: The replication of genomic DNA Duplication of the centrosomes What are the major concerns when we think about replicating DNA? It needs to: produce exactly one additional copy of each chromosome be high fidelity (almost mutation free; 1 nucleotide change per 1010 nucleotides per cell division) dont want to intro mutations when replicating DNA Mechanism of DNA replication Semi conservative Requires an origin of replication: initiation Involves replication forks: elongation L19 - Cell cycle entry and DNA replication 8  Circular piece of DNA - bacterial chromosome Initiation of replication in bacteria Circular bacterial chromosomes have a single origin of replication defined by DNA sequence Specific proteins form an Origin of Replication Complex (ORC) to initiate DNA synthesis only 1 origin so can only start in 1 place Allows regulated initiation to ensure one round of replication (in about 30 min) If a human chromosome was replicated this way, it would take about a month! – so cannot be the way it works L19 - Cell cycle entry and DNA replication 9  Initiation of replication in eukaryotes Eukaryotes with larger genomes have multiple origins of replication Linear in humans still unclear what defines human ORC Lisencing - how the cell ensures that DNA is only copied once Once fired are deactivated so cannot refire the origin ORCs can only recruit pre-replicative proteins to origins in G1 Origins can only “fire” DNA replication in S phase, and then are deactivated The DNA replication machine L19 - Cell cycle entry and DNA replication 10  image on right = replication fork in vitro - blob = protein replication machine Multiple components needed for DNA replication: Helicase to separate the DNA double helix Single-strand binding protein to maintain separation of single strands DNA primase to initiate DNA polymerization Two DNA polymerases to synthesise the two new strands of DNA one on each strand - adds nucleotides onto the primer A sliding clamp (PCNA) to keep polymerase on DNA Topoisomerases nick or cut and reseal DNA ahead of the replication fork to remove supercoils and tangles DNA polymerase only work from 5’ to 3’ DNA strands are anti-parallel The leading strand can be synthesised continuously The lagging strand must be synthesised non-continuously as Okazaki fragments L19 - Cell cycle entry and DNA replication 11  dsDNA on one side (leading strenad) straight forward to synthesis DNA as fork opens However on lagging strand – sysnthesises lots of small fragments which must be joied together by DNA ligase DNA polymerases are accurate The wrong nucleotide can sometimes be added, but replicative DNA polymerases have “proofreading” activity L19 - Cell cycle entry and DNA replication 12  2 sites within the enzyme – P site where adds nucleotides E site = editing site – can cut off incorrect nucleotide then continue Replicative DNA polymerase needs RNA primers Proofreading DNA polymerases need a perfectly base-paired nucleotide to add new nucleotides So, these polymerases cannot initiate new DNA synthesis They need “primers” from which to extend the new strand DNA primase creates these primers using RNA L19 - Cell cycle entry and DNA replication 13  Because the primers are RNA, they can be distinguished from DNA and removed later DNA ligase then joins the Okazaki fragment Because of proofreading activity need primer – perfectly based paired starting point DNA primase adds short RNA primer complementary to original strand to give perfectly base paired end The end replication problem The need for primers creates a problem: how to replicate the end of linear DNA? The answer is to have a special structure at the chromosome ends: telomeres In humans, these are repeating units of GGGTTA (approx 1000 repeats) These repeating units are produced by an enzyme called telomerase L19 - Cell cycle entry and DNA replication 14  Telomeres Telomerase extends the 3’ end of the chromosome so it can be back filled by lagging strand synthesis without losing genetic information lengthening the chromosome all the time Telomere binding proteins plus a T-loop structure protect the free end shields ends of the chromosome - tuck end of chromosomes into itself to stabalise the proteins L19 - Cell cycle entry and DNA replication 15  DNA damage repair There are many DNA damage repair pathways 1. Detection of nucleotide mis-incorporation during replication 2. Detection of damaged nucleotides or bases (e.g. from exogenous sources such as chemicals, UV exposure) 3. Detection of DNA breaks Where possible, the cell makes use of the information from the undamaged DNA strand to carry out the repair In some cases, the cell can even use the information in a sister chromosome to correct damage 1. Strand directed mis match repair Mis-incorporations during DNA synthesis are not always corrected by DNA polymerase proofreading Scanning proteins can detect mismatched nucleotides But, has to be a mechanism to correct only the new strand In eukaryotes, this happens because only the new nicked strands are repaired L19 - Cell cycle entry and DNA replication 16  2. Base and nucleotide excision repair 1. A number of enzymes recognize different types of damaged base 2. Other enzymes can recognize distortions in the double helix e.g. pyrimidine dimer induced by UV light L19 - Cell cycle entry and DNA replication 17  3. DNA break repair Double stranded breaks (DSBs) are a potentially catastrophic form of DNA damage Can be repaired fairly simply by recognizing and re-ligating the free ends together (NHEJ) but this is error-prone If cell is in S/G2, then homologous recombination (HR) can use the information in a sister chromosome to accurately repair the damage L19 - Cell cycle entry and DNA replication 18  Simple way to repair a break in G1 = stick together by NHEJ - effective but error protne IF in S or G2 then may have another copy of sister chromosome Cell has enzymes so can cororectly copy info from 1 sister to other G2 checkpoint Mitosis doesn’t normally occur with incompletely replicated or damaged DNA How does this checkpoint operate? 1. Inhibiting M-Cdk through p53 and p21, as described earlier for the G1 checkpoint L19 - Cell cycle entry and DNA replication 19  2. By inhibiting Cdc25, the phosphatase needed to activate M-Cdk DNA damage can be detected – kinase patheway triggered this can lead to phosphorylation of Cdc25 which isn important for the transition of inactivw M cdk to active – therefore if inactivate Cdc25 then can prevenet this transition Summary L19 - Cell cycle entry and DNA replication 20  Transition from G0/G1 into S-phase is a critical cell cycle checkpoint Mitogens drive G1-S transition by stimulating production of G1-Cdk G1-Cdk inactivates Rb to allow the production of G1/S-Cdk and S-Cdk to drive DNA synthesis Cell stress (e.g. DNA damage) can prevent G1-S transition, e.g. by the p53 pathway DNA synthesis Requires licencing, initiation, and elongation by RNA-primed DNA polymerases Special telomere structures allow the replication and protection of DNA ends DNA stability is maintained DNA polymerase proofreading and strand-directed mismatch repair during replication Damaged nucleotides and DNA breaks are scanned and repaired Detection of DNA damage triggers a G2 checkpoint that prevents transition into mitosis until repairs are carried out L19 - Cell cycle entry and DNA replication 21  L20 - Mitosis and meiosis Date @December 5, 2024 Difficulty Progress Done Overview Walther Flemming Chromosome basics Replicated chromosome basics Meiosis and mitosis During mitosis The stages of mitosis Entry into mitosis (prophase) Kinetochore assembly (prophase) Cohesion and cohesion release - the prophase pathway Chromosome condense (prophase) Prophase - summary Prometaphase The mitotic spindle Microtubules The mitotic spindle - MAPs and Motors The mitotic spindle - microtubule motors Mitotic spindle assembly Chromosomes bi-orient on the spindle Error correction Kinase Aurora B detects bi-orientation Metaphase Spindle checkpoint - Metaphase to anaphase transition Examples Securin and cohesion release Chromosomes separate - anaphse Chromosomes decondense - telophase Cell divides into 2 - cytokinesis Summary Overview The essential events of mitosis, including: L20 - Mitosis and meiosis 1  How chromosome structure changes Assembly of a bipolar spindle Bi-orientation of chromosomes Anaphase and cytokinesis The key regulatory steps in mitosis: Error correction to ensure bi-orientation The mitotic (spindle) checkpoint Walther Flemming Can be seen under light microscopy and fluorescent L20 - Mitosis and meiosis 2  Spindle elongates and drags the chromosomes to opposite poles Chromosome basics Human somatic cells 46 chromosomes 2 copies each of chromosomes 1 to 22 (autosomes) plus X + X, or X + Y (sex chromosomes) Cells with 2 copies of each chromosome are diploid 1 chromosome set from mother, and 1 from father Human gametes Maternal and paternal chromosome sets come from gametes (egg and sperm) Cells with 1 copy of each chromosome are haploid L20 - Mitosis and meiosis 3  Most cells are diploid other than sex cells Replicated chromosome basics 2 sister chromatids are held together by cohesion complex Allows repair mechanism and sorting processing in mitosis Meiosis and mitosis L20 - Mitosis and meiosis 4  During mitosis Chromosomes condense Chromosomes attach to spindle microtubules Allow chromosomes to be moved around Chromosomes align on the spindle Sister chromatids separated Allow chromosomes to be moved into correct daughter cells Chromosomes decondense and cell divides into two To produce two diploid G1 cells L20 - Mitosis and meiosis 5  The stages of mitosis Phophase Nuclear envelope still inact but chromosomes are beginning to condense Centrosome are starting to move appart to opposite pole Prometaphase L20 - Mitosis and meiosis 6  Centromere are recruiting proteins Nuclear envelope breaks down which allows spindle microtubules to bind onto chromomseoms – beginning to bind Metaphase – all chromosomes lines up – metaphase plate Key feature of mitosis – one sister attaches to 1 pole and the other attaches to the other - bi oriental Anaphase Cohesion between them and pulling to opposite poles Telophase Chromosome start to decondense again Cytokinesis Pinching of the cells – to produce 2 sep daughter cells Entry into mitosis (prophase) Underlying regulatory processes that control this - driving the cell into mitosis is dependent on M-Cdk Entry is driven by the “master regulator” of mitosis, M-Cdk (Cyclin B-Cdk1) M-Cdk: 1. directly phosphorylates key substrate proteins L20 - Mitosis and meiosis 7  2. regulates downstream mitotic kinases (eg Aurora and Polo kinases) which then phosphorylate additional substrates Positive feedback makes activation stronger Once CDK active then will directly phosphorylate key substrate Kinetochore assembly (prophase) The kinetochore: the microtubule binding site on a chromosome a large macromolecular complex that assembles on the centromere M-Cdk (Cyclin B-Cdk1) and Aurora B kinases are required to recruit kinetochore proteins in early mitosis Spindle elements form microtubule which binds onto chromosome Under control by Cdk1 Kinetochore – allows chromosomes to attach to microtubule Cohesion and cohesion release - the prophase pathway L20 - Mitosis and meiosis 8  Regulation of cohesion Cohesin – circular complex of proteins – pohysically links two bits of DNA therefore sister chromatids too. Cohesin in G1 – forms loops which is important for gene regulation – functions throughout cell cycle Need regulated release of cohesin to separate chromosomes Chromosome condense (prophase) Condensins I and II co-operate to condense chromosomes in mitosis Condensin 1 and 2 have similar structural properties Loops come together in way - miporatn for condensing DNA Prophase - summary Interphase chromosome structure is lost L20 - Mitosis and meiosis 9  Chromosomes condense Kinetochore assembly begins Microtubule dynamics change so that the spindle starts to form Prometaphase Nuclear envelope breaks down allows access of microtubules to chromosomes Spindle assembles Microtubules attach to chromosomes Microtubule adapter proteins and motor proteins become active allows chromosomes to be moved on the spindle need adapter proteins to bind to microtubules The mitotic spindle A microtubule-based machine required to align and segregate chromosomes L20 - Mitosis and meiosis 10  Structure of mitotic spindle Spindle poles = centrosomes Microtubules emanate from here Interpolar Kinetochore – actually bind to chromosome Microtubules Nucleated at the minus end Can grow and shrink at the plus end (dynamic instability) Tubulin dimers assemble to give rod shape microtubules Asymmetric Nucleotide at the minus end Grow out from – end from spindle pole and shrink – for search and capture The mitotic spindle - MAPs and Motors Microtubule adapter proteins (MAPs) Allow cell components to bind microtubules Modulate the stability of microtubules eg Ndc80/Nuf2 at kinetochores L20 - Mitosis and meiosis 11  Motors Allow cell components to move along microtubules eg Kinesin-5 (walks to plus ends) Dynein (walks to minus ends) The mitotic spindle - microtubule motors Kinesin motors use the energy from ATP to walk along microtubules - to plus end Can carry various “cargo” proteins Kinesin-5: forms dimers and cross-links microtubules (cargo is another kinesin-5 molecule) CENP-E: binds to kinetochores (cargo is a kinetochore) - moves chromosomes along the microtubule L20 - Mitosis and meiosis 12  kinesin toward the plus end and dynein toward the minus end Mitotic spindle assembly Nucleation of microtubules at centrosomes (minus ends) Formation of interpolar microtubules and sliding moves centrosomes apart Nuclear envelope breakdown allows microtubules to capture kinetochores Kinesin 5 separate the interdigitating microtubules Chromosomes bi-orient on the spindle Bi-polarity of the spindle and bi-orientation of chromosomes are vital Bipolarity = one chromosome attach to 1 pole and one attaches to the other L20 - Mitosis and meiosis 13  Two key processes: Making correct attachments (error correction does this) – detects if connections are incorrect Preventing cell cycle progression until suitable attachments are made (spindle checkpoint) Error correction A trial and error process Only chromosomes that are bi oriented are stably attached to microtubules Kinase Aurora B detects bi-orientation Aurora B kinase localizes to centromeres By a mechanism that it is not fully understood, it detects tension L20 - Mitosis and meiosis 14  The cell knows if all chromosomes are attached in a bioriental way intrinsically by aurora B which localises between centromeres When biorientally attached - microtubules effect force/ tension across centromeres which sends out a signal In absence of tension Aurora B phosphorylates Ndc80 to remove microtubules from kinetochores - this causes the microtubule to detachs and allows another attempt to get the correct biorientation Ndc80 - complex which forms the bridge between kinetochore and microtubule Metaphase Chromosomes are all bi-oriented As a consequence, they align on the “metaphase plate” The cell is in metaphase and ready for anaphase How does the cell “know” this? L20 - Mitosis and meiosis 15  Spindle checkpoint - Metaphase to anaphase transition If chromosomes are incorrectly attached to the spindle, error correction produces unattached kinetochores The spindle checkpoint detects unattached kinetochores - stops cell cycle prior to anaphase If no unattached kinetochores means cell can proceed in mitosis Unattached kinetochores produce the Mitotic Checkpoint Complex (MCC) The MCC inhibits the APC/C and so prevents M-cyclin (Cyclin B) degradation M cyclin degradation - allows the cell to exit mitosis and move into next cell cycle L20 - Mitosis and meiosis 16  This keeps cells in mitosis Once all kinetochores are occupied with microtubules, the MCC is no longer produced M-cyclin (Cyclin B) is degraded, and the cells “biochemically” exit mitosis Numerous mitotic processes are terminated Examples Mitosis in Xenopus S3 cell with late aligning chromosomes Mitosis after microinjection of antibody that partially interferes with chromosome alignment and the spindle checkpoint Securin and cohesion release M cyclin is not the only target of the APC/C L20 - Mitosis and meiosis 17  Co-ordinated degradation of M-Cyclin and Securin ensures that “biochemical” exit from mitosis and the onset of anaphase chromosome movements occur together Securin gets tagged by ubiquitin to be degraded when APC/C is activated Securin blocks separase - which keeps the chromosome together when APC/C active, securin is degraded so allows separase to separate the chromatids When not all microtubules attached then will produce MCC which inhibits APC/C therefore securin can block separase which stops chromosomes being separated Chromosomes separate - anaphse Anaphase A - chromosomes move towards spindle poles Driven by microtubule depolymerisation at the plus ends of kinetochore microtubules (important in human cells) Anaphase B - spindle poles move apart Driven largely by microtubule motors e.g. kinesin-5 Relative importance and timing of anaphase A and B varies between cell types and species L20 - Mitosis and meiosis 18  Chromosomes decondense - telophase Need to re-establish interphase nuclear and chromosome structure Nuclear envelope reforms, and nuclear pores inserted Chromosomes decondense Condensins dissociate Cohesins re-associate and enable the formation of chromosome looping structures needed for correct gene expression Cell divides into 2 - cytokinesis A contractile ring formed from actin and myosin drives cleavage furrow formation and physically pinching of the dividing cell into two The key problem is to define the time and place of cleavage L20 - Mitosis and meiosis 19  This appears to involve more than one mechanism, including signals from the: central spindle spindle poles chromosomes Summary Prophase: M-Cdk activity drives mitotic entry Microtubule dynamics change; centrosomes move apart Chromosomes condense (Cohesin lost, Condensins recruited) Kinetochores assemble Prometaphase: Nuclear envelope breaks down Microtubules bind to kinetochores. On tensionless kinetochores, error correction takes place creating unattached kinetochores Unattached kinetochores cause spindle checkpoint signalling Metaphase: L20 - Mitosis and meiosis 20  All chromosomes have bi-oriented and no unattached kinetochores remain Spindle checkpoint signalling ceases; APC/C becomes active M-Cyclin is degraded leading to “biochemical” exit from mitosis; Securin is degraded so Separase separates sister chromatids Anaphase: Chromosomes move to spindle poles; spindle poles move apart Telophase: Chromosomes decondense (Condensins lost, Cohesin recruited); interphase chromosome structure re-established Cytokinesis: Contractile ring causes cleavage furrow formation and cell is divided into two daughters L20 - Mitosis and meiosis 21  L21 - asymmetric cell division Date @December 5, 2024 Difficulty Progress Done Importance of asymmetric cell division in development Symmetric vs asymmetric Asymmetric cell division of stem cells support tissue homeostasis Asymmetric cell division of germ cells supports their immortality Types of asymmetric cell division History Studies in invertebrates have led to our understanding of ACD Polarisation C.elegans Polarisation of C elegans zygote by PAR proteins Stem cell (neuroblast) asymmetric division in drosophila Mitotic spindle orientation Posterior displacement of the mitotic spindle in C elegans zygote depends on PARs Identification of the mitotic spindle positioning machinery Mitotic spindle orientation in drosophila neuroblast Another way to position mitotic spindle - centrosome stereotypical positioning Cell fate determinants localisation Reaction-diffusion in the asymmetric localisation of cell fate determinants In the neuroblasts Cell fate specificiation through aPKC phosphorylation Cell division plane localisation Positioning the final cut - cytokinesis Coupling cell cycle progression and cell polarity Summary Importance of asymmetric cell division in development Asymmetric division are critical for multicellular organisms, which begin life as a single cell that eventually differentiates into many types of tissue L21 - asymmetric cell division 1  Symmetric vs asymmetric Symmetric - leads to 2 equal daughter cells which leads to expansion / proliferation Asymmetric - leads to 2 different daughter cells different components and proteins, RNAs, lipids Histone modifications, DNA methylation Organelles (old mitochondria) Cytoskeletal organization (centrosomes) Cell fate components Reactive species, protein aggregates Not assymetric separation of chromosomes as this leads to disease - this is a natural state that stem cells do. L21 - asymmetric cell division 2  Asymmetric cell division of stem cells support tissue homeostasis Divide asymmetruc which leads to a cell which can self renew and another that will differentiate NEEDED FOR HOMEOSTSIS Too much proliferation - cancer Too little proliferation - premature differentiation of tissues - inability of tissues to regenerate - ageing Therefore must be balanced Asymmetric cell division of germ cells supports their immortality Germ cells also divide asymmetrically - the only immprtal cell in body L21 - asymmetric cell division 3  Transfer genomic material generation throguh generation Critical to keep and rejuvenate the cell leading to sperm and oocytes pass genomic material from parents to offspring anything that could be detremental to a cell will be inherited to less fit cell (green) other = super healathy to protect genome Types of asymmetric cell division L21 - asymmetric cell division 4  Intrinsic - asymetric division starts prior to the division of the cell must coordinate asymetric segragation of the factors together with the orientation of the mitotic spindle so when cell divides is only inherited by 1 daugher cell and not the other Extrinsic - needs extraceullar signalling components to mediate the differntiation between the 2 daughter cells e.g. male germaline stem cell - cell that remains after division close to somatic cell that supports its pluripoentcy/ self renewing capacity - will maintain pluripotency whereas other extruded from envuronemtn eill recieve signals which wil dictate its differentiation Both occur - some in comination History Ed Conklin - 1905 - The first observation of a asymetruc segregating determinant L21 - asymmetric cell division 5  first seen as a yellow factor in the cytoplasm which segregates with the muscle cell lineage of the Acidian Many years later the first identification of the molecular nature of an asymetriucally segragated factor was found - Found in Drosophila - external sensory organ which leads to 4 cell types absence or pver expression lead to different aberant cell types Numb is a regulating facor - regulates differentiation of these cell types - intrinsically Studies in invertebrates have led to our understanding of ACD Core mechanisms of asymmetric cell division have been first described in C. elegans and Drosophila 3 step process that drive ASD: 1. During interphase there is a need to Establishment of polarity axis - depends on par proteins 2. Mitosis - Use polarity axis for orientation of mitotic spindle and segregation of cell fate determinants 3. Telophase - Coordination between spindle orientation and cell fate determinant positioning to ensure that only one of the daughter cells L21 - asymmetric cell division 6  will inherit the cell fate determinant Polarisation Cell polarisation depends on the asymmetric localisation of polarity regulators every cell in our body is polarised - the ability of a cell to organise components and organelles along the polarised axis L21 - asymmetric cell division 7  leads to the functional differentiation of domains in the cell e.g. in epithelial cell apical polarity is in gut (absorbsion of nutrients so dev microvilla) C.elegans Great model system for the study of cell polarity and asymmetric division as the first division of the embryo is asymmetric leads to 2 cells of diff size and fate - 1 starts the somatic lineage and the other the germ-line embryo divides - the first division is asymetric - this is where the factors important were identified L21 - asymmetric cell division 8  Can do highthroughput genetic screens and through this can identify networks of genes and proteins involved in process (e.g. cell polarity) Have ability to spatiotemporally control the polarity regulators found in this process - gain insight into protein function bring a protein to a particular cell location or inactivate/activate it at particular time to see what happens Polarisation of C elegans zygote by PAR proteins 1. initially the embryo isnt polarised 2. Have the female and male pronucleus - male brings in centrosomes which makes up the spindle - key in polarisation 3. eventualyy wil start a signal which will indue the polarisation of the embryo Acummulation of PAR proteins is driven by signals from the centrosomes which leads to changes in the actomysoin networks L21 - asymmetric cell division 9  constantly assembling and disassembling -creates highly dynmic network where actomysoin foci are interconnected - under strong tension when centrosomes contact the posterior cortex - actomysoin network will flow towards the anterior due to relaxation of network - this flow is what is sensed by PAR proteins PARS not acc in direct contact but they are being advected Advection = transport without direct contact between proteins creeated by movement of actomyosin network whcih through friction is translated to the cytoplasm which will be moved in the same way towards the anterior - PARS can sense this. follow flow PAR protein clusters are being moved to anterior part L21 - asymmetric cell division 10  POSTERIOR PARS: Microtubules eminating from the sperm centrosome is to protect and allow posterior PARS to be accumulated at the membrane They prevent the function of PKC-3 (kinase) - bottom right mutual antagonism at bottom L21 - asymmetric cell division 11  No boundary in this cell type - so 2 domains being establised with no physical boudary - unlike the tight junctions in the epithelial cell. Therefore this is maintained by mutual antagonism and positive feedback loops Leads to 2 domains - anterior and posterior - then signals to mediate posterior displacement of mitotic spindle and differential segragation of cell fate determinants leading to ASD Stem cell (neuroblast) asymmetric division in drosophila Relies more on extrinsic factor In drosophila embryo there is a ventral neuroectoderm from where the neuroblasts are L21 - asymmetric cell division 12  this neuroblast inherit part of the polarity of the epithelium so PARs are now apical and through apical localisation - will mediate segragation of cell type deteminant factors to oposite end and orient mitotic spindle - lead to asymmetric cell division leads to self renewal cell and a cell whcih will differentiate into 2 neurones Other neuroblasts are present in the brain of the larva - here differentiation occurs in a diff manner 1. In the brain - Polarisation is given by the previous division cell knew orientation of division prior to its generation neuroplast - maintainig the way it divides leads to nice organisation of cell arrays similar to previosuly mentioned - clusters of PARS sensse s flow and become accumulated to apical end - nice array of cells seen in A and C L21 - asymmetric cell division 13  B- when this is perturbed (polarity signals are broken or cell doesnt remember where prior division taken place) - mess of structure and differentiation of cell therefore key to keep components tight. Once polarity is established how is that translated to orientation of mitotic spindle - dependednt on conserved molecular machinery. Mitotic spindle orientation How cells transduce cortical polarity to spindle positioning? Evolutionary conserved machinery involved in mitotic spindle positioning Posterior displacement of the mitotic spindle in C elegans zygote depends on PARs Mitotic spindle prior to anaphase pulls closer to the posterior - therefore must be pulling forces driving movement of spindle pole L21 - asymmetric cell division 14  Researchers did laser ablation experiments: Ablate central spindel - breaks MT interactions - measure speed of spindle poles arrows reflect magnitude of speed - posterior pole subjected to stronger pulling forces than the anterior = asymmetric positioning of mitotic spindle Did the same experiment but depleted PAR proteins - when deplete anterior pars - spindle pulls have high velpocity when deplete posterior PARS - lower speeds therefore weaker pulling forces Posterior PARs are stronger? Identification of the mitotic spindle positioning machinery L21 - asymmetric cell division 15  Found set of proteins where dynein motor that anchors to the membrane indirectly throguh the activation of these proteins NUMA, LGN and this hetrotrimeric G protein - which is relayed and associated to the membrane Dynein being anchored and walkign along microtubules - minus end directed pulls towards the membranes when walks along these components are regulated by PARs e.g. LGN promoted by posterior PARs so stonger at posterior - APKC phosphorylates NUMA hence reducing function of complex also regulated by cell cycle components e.g. Aurora A which promotes complex formation pulling forces will only start in anaphase Mitotic spindle orientation in drosophila neuroblast Same molecular machinery invovled In neuroblast - PARs are positioned apically - this recruits LGN protein which will dock rest of the components that bring dyenin motor to the membrane - mitotic spindle in organised by apical cap of PARs L21 - asymmetric cell division 16  coexists with another pathway which invovles a kinesin whcih works oposite - towards the plus end of the microtubules - known to be a spindle capturing complex which can associate to the membrane and recruit microtubules balancing act between force generating complex and a spindle capturing one - this is what positions the mitotic spindle in relation to the apical cap Another way to position mitotic spindle - centrosome stereotypical positioning Prior mitotic spindle formation neuroblast in sucessive divisions will have a centrosome which will remain attached to apical side when it duplicates - the daughter centrosome will remain attached to apical - keep its microtubule capacity so is active whereas the mother centrosome will release from PCM so will have less capability to produce MT through this action will already be in the right orientation to start to generate the mitotic spindle This asymmetic segegation is used to segregate other components e.g. RNAs, protein aggregates ect L21 - asymmetric cell division 17  Cell fate determinants localisation How cell polatiry is transferred for asymmetric segragation of cell fate determinants/ cell factors How cells transduce cortical polarity to cell fate determinant asymmetric segregation? Kinase reactions regulating molecular dynamics of the cytoplasm. Reaction-diffusion in the asymmetric localisation of cell fate determinants Have PARs cortically localised in an asymmetric manner Blue component = MEX5 - involved in degradation of proteins which favour germ line lineage - inherited in anterior daughter cells to protect somatic lineage Asymmetry of MEX5 is genrated through PAR1 kinase action PAR1 phosphorylates MEX5 - leads to fast movement of MEX5 in cyrtoplasm counteracting phosphotase - in anterior of embryo where doent have PAR1 - prevail dephosphorylated state of MEx5 which slows L21 - asymmetric cell division 18  down the protein (possibly through intersction with the ER) differences in dynamic lead to a net flux of MEX5 from posterior to anterior creating this gradient Another mechanism that involves a gradient is the accumulation of the granules at the posterior - contain condensates of proteins and RNA which are inherited by the germ lineage. Able to come out of cytoplasm without the need of a membrane through liquid phase separation like what occurs when draw oil from water Without a membrane Can have accumulation of proteins and RNA in given part of cell At the anterior the grandules dissolute due to MEX5 as MEX5 competes with protiwins of P granules for their interactions with the RNA - the disollution of P granules at the anterior and condensation of P granules at posterior leads to flux No cytoskeleton in use - diff mechanism whee kinase reaction can control dynamics of proteins in cytoplasm and control gradients which can mediate these asymmetries. In the neuroblasts L21 - asymmetric cell division 19  Cell factors become localised to basal which now depends on kinase aPKC - targets diff substrates like Numb and miranda to promote cell differentiation aPKC targets promote differentiation and inhibit cell renewal in different ways. Numb – inhibitor of Notch signaling Miranda – directs the localization of : Staufen –mRNA binding protein Prospero - transcription factor Brat – protein translation inhibitor Cell fate specificiation through aPKC phosphorylation L21 - asymmetric cell division 20  Apical PARs containing aPKC - this avtivity release numb from the apical hence note sigannling pathways on leading to poliferation whereas in basal - prescence of numb will promote differentiation APKc phosphorylates miranda so a scaffolding protein will recruit other components which will define the differentiated cell. Cell division plane localisation How cells transduce cortical polarity to the cell division plane? Spindle dependent and independent processes Positioning the final cut - cytokinesis Requires the formation of actomyosin ring which contracts - through this contraction of the myosin motors which slide actin filaments lead to contraction of ring - therefore invaginatio nof membrane and final cut of cell leading to separation of daughter cells Depends on position of mitotic spindle therefore the polarity can regulate where this cut is placed Astral MT prevent accumulation of myosin at the poles - prevent contractivity at the sites L21 - asymmetric cell division 21  on the other hand the central spindle with the antiparallel MT will recruit components that will promote formation of contractile ring L21 - asymmetric cell division 22  Central spindle components are fascoured by central part of mitotic spindle whcih will promote activity of factors that regulate this role e.g. GTPase which leads to different pathways to formation of cytokinetic furrow PARS by positioning the mitotic spoindle also help position where division will take place cell cycle regulation components are also involved in this process component of PARs whcih inhibits central spindle which needs to be blocked by Aurora B for this to take place Coupling cell cycle progression and cell polarity L21 - asymmetric cell division 23  How is asymmetric cell division coordinated in time with other events of the cell cycle? Are there surveillance mechanisms ensuring that a cell divides only when polarity is established? cell wont divide until cell polarity is properly established Links are starting to appear: Red - cell cycle regulators Green - cell fate determinants Blue - cell polarity factors kinases heavily involved in regulation of PAR proteins finding from drosophila neuroblasts and c. elegans are contradictary as in 1 they promote and in the other they inhibit (c. elegans) Kinases whebn actyivated promote mitotic entry and progression - in term are regulating cell fate determinants - however these are inhibiting cell cycle progression as are promoting differentiation in c elegans evidence that PAR proteins may be regulating cell cycle progression through the regulation of replication Summary L21 - asymmetric cell division 24  Asymmetric cell division (ACD) is at the heart of the generation of the plethora of cell types needed to form an organism. There is a tight balance between self-renewal and differentiation - critical In ACD the orientation of the mitotic spindle needs to be coordinated with the differential segregation of cell fate determinants (or cellular components) for these to be differentially inherited by the daughter cells relies on 3 steps We are only starting to understand the cross regulation between polarity and cell cycle regulation L21 - asymmetric cell division 25 L21 - asymmetric cell division 26  L22 - Cell cycle and disease Date @December 6, 2024 Difficulty Progress Done Cell proliferation during development Surveillance mechanisms ensure cell cycle exit upon DNA damage or inappropriate chromosome segregation Cancer - what happens when cell cannot exit the cell cycle Chromosome instabilities (CIN) in cancer Aneuploidy How do cancer cells tolerate aneupliody ABL protoncogene activation Chromosome translocations BCR- ABL oncogene Translocations and identification of genes involved in cancer Rb tumour supressor inhibition Rb canonical mechanism of action Rb loss and retinoblastoma Loss of Rb can compromise genome integrity (non canonical pathways) Difficulty to target Rb related cancers (most cancers) Positive side - looking for broad impact therapeutic approaches Summary Cell proliferation during development Proliferration v high during development - balance between proliferation and differentiation (whether remain or leave cell cycle) L22 - Cell cycle and disease 1  In adulthood - millions of cells that divide everyday to support growth and replacement of damaged tissue not every cell divides e.g. Mature muscles (cardiac muscle cells) and nerve cells never divide Many cells remain in the G0 state - no proliferating but can do if necessary e.g. skin fibroblast, smooth muscle cells, endothelial cells from blood vessels, epithelial cells (liver, pancreas, kidney, lung, prostate, breast) Stem cells in tissues that need a continuous cell renewal e.g. blood cells, intestinal epithelial cells All of these are subjected to control Surveillance mechanisms ensure cell cycle exit upon DNA damage or inappropriate chromosome segregation If damage in DNA then cycle will stop. 3 main checkpoints DNA damage checkpoint acts in diff phases of cell cycle - damage to chromosomes stops cell cycle - can be stopped prior entry, prior replication or prior entry to mitosis inhibits CDKs Replication stress checkpoint chromosomal abnormalities lead to replication fork faults which need to be repaired - if remain stuck then prevents entry to mitosis The spindle assembly checkpoint during mitosis - chromosomes not properly aligned and not segregate appropriately then chekpoint is activated so blocks exit of mitosis if problems not resolved - cell can enter senescence (irreversible ageing stage of cell) or quiescence (reversible) or apoptosis if damage too bad L22 - Cell cycle and disease 2  In cancer these mechanisms are bypassed. Cancer - what happens when cell cannot exit the cell cycle Cell keeps dividing even when cell cycle controls havent been met Driven by alteration in protooncogenes - drivers of cell cycle in a normal cell but when mutated turn into oncogenes (ABL) so cannot stop cell cycle - leading to extreme cell growth and division a stuck accelerator, stimulating aberrant cellular growth and division Tumour supressor genes (e.g.Rb and p53) can also be mutated - mutation leads to inactivation - uncontrolled progression in cell cycle The cell doesnt exit the cell when required upon DNA damage so the loss of tumour supressors leads to a defecive break. L22 - Cell cycle and disease 3  Any mutations or genomic instability is propagated so cell keeps dividing with these problems - trademark of cancer Numerical and structural chromosomal abnormalities in cancer cells were observed more than a century ago Are very common in cancer i.e. 70% of solid human tumours present aneuploidies (abnormal number of chromosomes in a cell) Current research has shown that structural or segmental rearrangements in chromosomes can have an impact on tumor development through activation of oncogenes and the inactivation of tumour supressors Oncogen activastion and tumour supressir inactivation leads to genomic instability as problems of genome can be passed into next cells - not only this but genome instablilities inturn promote more mutations in oncogens ect = positive feedback loop - tissue develops cancer - progressive degeneration of cell mutations allowing cancer cells to proliferate and metastasise are selected and grow L22 - Cell cycle and disease 4  Chromosome instabilities (CIN) in cancer Generation of abnormal chromosome numbers (aneuploidy) in mitosis through mis-segregation of chromosomes. Can arise in diff ways: Aberrant kinetochore MT attachments - when cell dividing chromosomes dont separate properly which leads to lagging chromosomes - randomly inherrited by 1 daughter cell - leads to cell with more and a cell with less chromosomes. even if goes into right cell - segregation is delayed so cannot be wrapped by nuclear envelope in time - gets extruded into micornuclei which are less protective (so chromosome can be shattered and stiched in random organisation) = chromothripsis (extreme chromosome rearrangement) Translocations - chromosome rearrangement - fragements of a chromosome can stick from 1 part to another - in the same chromosome, in diff chromosomes or interchaged between chromosomes (reciprocal chromosomes - ABL oncogenes are made by this) Arise due to problems in DSB - fusion of wrong ends L22 - Cell cycle and disease 5  Other cancer mutations - deletions, amplifications, insertions, inversions, point mutations - occur throughout the cell cycle as the cell divides Aneuploidy Causes in mitosis: 1. Inappropriate kinetochore MT attachments e.g. too strong - merotelic interactions - chromosomes kinetochore is attched to microtubules eminating from both poles so gets stuck in the middle - can also be caused by: 2. Compromised SAC - spindle assembly checkpoint (rare in cancer) - normally prevents cells progression and problem is resolved however in this mutation then will keep going. L22 - Cell cycle and disease 6  3. Supernumary centrosomes - centrosome overduplication (cancer and microcephaly) - cancer cells can resolve this by clustering the centrosomes to 1 end to create a pull a. still not efficient at recruiting chromosomes - leads to lagging chromosomes 4. Problems in chromosome cohesion - start separating even tho chromosome struggling with segragation - also leads to lagging chromosomes All lead to a lagging chromosomes - aneuploidy 5. Tetraploidy (4n)- complete duplication of chromosomes in cell - occur through cytokinesis failure, cell fusion, endoreduplication (G1-S-G2-G1-S- G1) (cycles of S phase which dont divide the cell) pre state of cancer cells - important in tumour development more copies of genes means more likely to get a mutation Hard to live with aneuploidy - Organism wide aneupliody is usually leathal however: Downs Syndrome (Chr21 trisomy) Rare patients with mosaic trisomy (12,18,21) L22 - Cell cycle and disease 7  At a cellular level aneuploidy leads to substantial fitness loss impared proliferation metabolic alterations defective stress responses However aneupliody occurs in: Hepatocytes neural progenitors neurones Highly present in cancer How do cancer cells tolerate aneupliody Aneupolidy has been reported to be detrimental also to cancer but cancer cells might be able to tolerate aneupolidy by: Lowering DNA damage responses e.g. p53 mutations mistakes in cell division frequently lead to p53 activation (i.e. chromosome mis segregation, aneuploidies, tetraploidides) p53 mutation is one of the most frequent events in tumourigenesis and allows cancer cells to tolerate a broad range of insults, including chromosome instability (CIN) L22 - Cell cycle and disease 8  Increase replication stress tolerance - when have stalled replication forks can remain in replication phase for a long time to try and resolve them Prolonged mitosis (SAC activation) - keep cell in mitosis for longer to lead to proper segregation of chromosomes DNA replication stres and SAC checkpoints are vital for cancer cells to prevent catastrophic levels of DNA damage resulting from replication stress or incomplete spindle assembly these are weraknesses of cancer cells that researches trying to expoit as areas of new therapy Certain level of CIN can be beneficial to cancer but too much can be harmful Certain aneuploidies might favor tumorigenesis Trisomy Chr12 is assocaited with increase proliferation and tumorigenicity of hESC In a colorectal cancer cell line single trisomies confer a selective advantage and increased tumorigenic behavior upon stress (starvation, hypoxia) Provides genetic diversity, substrate for tumor evolution: Certain aneuploidies correlate with metastasis, promoting EMT Large chromosomal changes affect hundreds of genes (or more) at once, making it difficult to identify the gene/s that drive cancer. most likely there is a requirement for multiple altered genes to act cooperatively in cancer. L22 - Cell cycle and disease 9  Due to the presence of more chromosomes over time can mutate and evolve increases capability for cells to proliferate, transform, progress into tissues and lead to metastasis accumulative mutations can be selected - hard to find genes involved as have whole chromosome rearrangement. ABL protoncogene activation What happens when a cell is forced into cell cycle Acts in the G1-S transition in response to mitogens ABL is a tyrosine kinase that become aberrantly activated as a result of a reciprocal translocation BCR-ABL chimeric protein is the cause of Chronic Myeloid Leukemia (CML) L22 - Cell cycle and disease 10  Chromosome translocations Arise from aberrant double strand breaks (DSB) repair in interphase Agents that promote DSB and might increase risk of translocations: DNA topoisomerase II poisons Radiation CIN The DSB repair is miss guided by: Sequence homology at the chromosome breakpoint The 3D chromosomal organisation in interphase In interphase due to organisation - sequences that are usually far apart can get closer and if a DSB is introduced then can pair with a gene that is far from original locus can happen within or between chromosomes (diff chromatin compartments) L22 - Cell cycle and disease 11  staining locus of ABL and BCR - in nucleus these genes are close to eachother which leads to recipriocal translocation - fuses 5’ of BCR to 3’ of ABL ABL kinase usually presents negative regulations due to conformations but when BCR translocation occurs these inhibitions are released - kinase is activated and cytoplasmic and interaction with BCR promotes oligomerisation and autophosphorylation (all promote kinase activity). BCR- ABL oncogene L22 - Cell cycle and disease 12  Aberrant activation of signalling pathways e.g. RAS-MAPK AKT JAK-STAT RAC GTPase signalling (cell proliferation) Production of MYC protooncogenes and the anti-apoptotic proteins BCL-2 and BCL-X. ALL of which will produce an overproduction of the MYC gene (protooncogene) and will promotoes anti apoptotic proteins BCL2 and X Promotes cell proliferation and survival L22 - Cell cycle and disease 13  This mutation occurs in hematopoetic stem cells v early on - leading to progressive proliferation and loss of apoptotic activity Dont occur in isolation - other protoncogenes and tumour supressors are altered e.g. p53 eventually leads to leukemic stem cell which if undergoes hyperblast stage will lead to CML. Effective treatment - Inhibition of ABL1 - imatinib finding the point of translocation and genes involved were v important Translocations and identification of genes involved in cancer Research has focused on the breakpoints in the translocations, as they can point to cancer-relevant genes L22 - Cell cycle and disease 14  328 gene fusions have been identified in malignant disorders 306 genes in the breakpoint have been found to be deregulated Chromosome rearrangements lead to: Gene fusions leading to a hybrid/ chimeric gene frequently targetting transcription factors and tyrosine kinase (i.e. BCR-ABL). Observed in hematological disorders and solid tumours majority of translocations occur in TFs or tyrosine kinases which lead to hyperactivation Alterations in gene expression Over expression of the MYC gene under immunoglobulin gene enhancers that relocalise to the proximity of the MYC gene upon translocation. Observed in lymphoid leukemias and in lymphomas of B and T cell Promoter swapping also observed in solid tumors Rb tumour supressor inhibition What happens when a cell cannot exit the cell cycle Rb (retinoblastoma protein) first tumor supressor identified Operated in the G1-S transition - cell cycle start Most recently other cell cycle roles for Rb have been identified Deletions and frameshifts (premature stop) that lead to loss of Rb function are observed in children retinal tumors - retinal blastomas Rb inactivation predisposes patients to many types of cancer. Patients typically exhibit defects in other pathways affecting cell growth and division e.g. p