Cell Division Lecturer: O ffice: Dr. S tephen El ledge T303 Ph one: 798-5040 Requ ired Re adin g Chapters 17 an d 18 in Molecular Biology of.
Download ReportTranscript Cell Division Lecturer: O ffice: Dr. S tephen El ledge T303 Ph one: 798-5040 Requ ired Re adin g Chapters 17 an d 18 in Molecular Biology of.
Cell Division Lecturer: O ffice: Dr. S tephen El ledge T303 Ph one: 798-5040 Requ ired Re adin g Chapters 17 an d 18 in Molecular Biology of the C el l, Th ird Edi tion Bardi n AJ, Visi nti n R, Amon A (2000) A me chan ism for cou pling e xit from m itosis to partition ing of the nucleu s. C el l 102:21-31. Cell Cycle Regulation + + + Continuous Duplication 2 1 0 Quantum Duplication 2 1 0 Continuous Duplication Cytoplasmic Cycle 2 Cell Growth Cytokinesis 1 0 Quantum Duplication Chromosome Cycle DNA Replication 2 1 0 Mitosis The Basic Problem S phase G1 G2 Mitosis Cell Cycle Stages G1(G0) - S - G2 - M How do we determine which stage of the cycle a cell is in? 1. FACS analysis Fluorescence Activated Cell Sorting 2. Incorporation of radioactive or epitope tagged nucleotides (BrdU) 3. Landmarks Nuclear envelope breakdown Condensed chromosomes Spindle elongation Determining G0 vs G1 can be difficult. G1 4 Cells sense their environment - nutritional - geometrical (cell-cell contact) - physical (size am I big enough) - regulatory (growth factors) If all is well, the cell will commit to a new cell cycle and pass START in yeast or the R-point (restriction) in mammals. The later stages of G1 involve preparation for S phase and mitosis. These include induction of gene expression and duplication of centrosomes (MTOC). S phase 5 Cells initiate DNA synthesis. They fire early origins early and late origins late. Early Late Cell growth continues. During S phase cells must have a mechanism to prevent activation of mitosis until DNA replication is completed. In addition, chromosomes must be replicated once and only once per S phase. Origins fire only once except in special cases. G2 No major cytological events Probably a sensing period for accumulation of information leading to the commitment to Mitosis. Proteins needed for Mitosis are synthesized in G2. 6 Mitosis Seven parts. Prophase Prometaphase Metaphase Anaphase A Anaphase B Telophase Cytokinesis Each of these is carefully regulated so as to occur in the proper order. Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis Interphase Metaphase Early Prophase Anaphase A Late Prophase Anaphase B Prometaphase Telophase Cell Cycle Transitions State A State B Cell Cycle Transitions State A State B Cell Cycle Transitions State A State B Metastable States Mutual Incompatibility Cell Cycle Transitions State A State B Inhibitory Barriers State C Cell Cycle Transitions State A State B Cell Cycle Transitions Cdc Mutants State A X State B Cell Cycle Transitions State A X State B X A Checkpoint Pathway Creates a Dependency Relationship Cell Cycle Transitions State A State B Self-Reinforcement Cell Cycle Transitions State A State B Self-Reinforcement Cell Cycle Transitions Mitosis G1 S G2 Meta Ana Telo Cell Cycle Transitions G1 S Cdk1 SCF Meta G2 Cdk1 SCF Ana Esp1 APC Telo APC Cell Cycle Transitions G1 S Meta Cdk Cdk Esp1 Telo APC/C CKI Wee1 Pds1 Cdk1 G2 Ana Cell Cycle Genetics YEAST The genetic system. Advantages 1. Eukaryotic cell cycle 2. Haploid---Diploid 3. Transformable 4. Reverse genetics CDC Mutants 1. Conditional lethal mutants 2. Arrest the cell cycle at a unique position. Can be used for four purposes 1) Make a molecular map of the order of function of cdc protein and landmarkevents. cdc mutant 24°C 2) Make a determination of whether certain processes are dependent or not. 3) Identify genes important for a particular process. 4) Identify other genes in the process by reversion analysis. 37°C Cdk1 = cdc2 + Cdc28 cdc2 was identified in S. pombe as a mutant that arrests in G2 and gives rise to long cells. A critical experiment identified a dominant allele of cdc2 that cause the cell cycle to accelerate rather than stop, yielding shorter cells. Why is this so important? CDC28 was identified in S. cerevisiae -protein kinase Two stop points G1 and G2 (the original allele had only one arrest point in G1) How does one protein regulate two completely different processes? The Relationship Between Cyclins and MPF mitosis Cyclin A Cyclin B RNR interphase mitosis interphase Cyclins Cyclins are periodically accumulated during the cycle, then rapidly destroyed. I M 1 I M 2 I M MPF 3 MPF activity peaked with cyclin levels. Cyclins are a regulatory component of MPF. Cyclins are the regulatory subunit of cyclin-dependent kinases (Cdk) Cyclins bind to Cdks and activate the kinase and, in some circumstances, control their substrate specificity Activation of Cdks controls certain cell cycle transitions G1 Cyclin/Cdk G1 G1 Cdk ON S phase Cyclins/Cdk S Mitotic Cyclins/Cdk G2 S Cdk ON M Cdk ON M Mitotic Exit All Cdks OFF S. cerevisiae Cyclin/Cdk Activity 2 Classes of Cyclins G1 Cyclins S phase Mitotic CLN1 CLB5 CLN3 CLN2 CLB6 CLB3 CLB1 CLB4 CLB2 CLN Cln1, 2, 3 CLB Clb5, 6 CLB Clb, 1, 2, 3, 4, Redundancy among cyclins G1 S G2 Deletion of one Cln or Clb has little effect Dcln1, 2, 3, ------ G1 Arrest Dclb1, 2, 3, 4 ------ G2 Arrest Cyclins gain functions later in the cycle. Clbs can carry out the function of Clns in some circumstances Clb1-4 can carryout the functions of Clb5,6 but Clb5,6 cannot function in place of Clb1-4. M The Start of START Auto-activation Loop Cell size ? ? Cln3 Cdc28 (Swi4/6) Sic1 ? MBF Nutrients uORF SBF ? CLN3 Cln1/2 Cdc28 Clb5/6 Cdc28 Auto-activation Loop What exactly is START? S phase The end of START and the start of S phase Budding + Centrosome Duplication SBF Cln1/2 Cdc28 SCF Sic1 Clb5/6 Cdc28 MBF Nutrients Sic1 is made during the previous mitosis G1 Clb1-4 Cdc28 S G2 M Sic1 ensures that S is dependent upon G1 cyclins Ubiquitin Conjugation Cascade E1 S Ub Ub Activating Enzyme Ub Conjugating Enzyme Ub Ligase E2 S E3 Ub Ub n substrate Ub n substrate Destruction by the 26S Proteasome Sic1 degradation through the SCFCdc4 pathway Signal Rbx1 Cdc34 E2 Cdc53 P Clb5 Cdc28 + SCFCdc4 Sic1 Sic1 P Skp1 Cln/Cdc28 Clb5 Cdc28 Cdc4 E1-S-Ub Rbx1 Cdc34 Cdc53 Skp1 Ub N S-Ub P Ub P Cdc4 Sic1 P Clb5 Cdc28 Sic1 Ub N P Proteasome Clb5 Cdc28 S phase The F-box Hypothesis Rbx1 Substrate Cdc34 Cdc53 F Skp1 F Function Sic1 Cdk Inhibitor Cln2 Cyclin Cdc4 Grr1 F Other F-box Proteins Unknown Targets Tumorigenesis Cell Cycle Control G1 cyclins (Cln1) Cdk Inhibitors (Sic1) Bud site selection p27 Cyclin E DNA replication (Cdc6) Grr1 Skp2 Fbw7 Cdc4 UFO Circadian rhythms in plants FKF1 F-box Proteins Sel-10 Cell Fate (Notch) Dactylin Limb development Hedgehog (Ci) Development TIR1 Plant flowering Auxin response in plants Met30 b-TRCP Wnt Pathway (b-catenin) NFkB Activation (IkB) Aminoacid biosynthesis (Met4) Signaling/ Transcription RING-Finger Based Ubiquitin Ligases E1 Rbx1 Ub Apc11 Cdc34 Ub Cdc53/ Cul1 Skp1 Ub n substrate P E1 E2 Apc2 P F-box Proteins BC-box Proteins 5 different Cullins Ub E2 Ub n substrate RING F SCF, VCB Ub Anaphase Promoting Complex Cdc20 Cdh1 Simple RING-E3s MDM2 Cbl BRCA1 Parkin Two Major Classes of E3s HECT Family E6-AP (Angelman’s Syndrome) Smurf1 (Smad destruction) Itch (Notch destruction) Rsp5 (membrane protein endocytosis) RING Superfamily SCFs APC Simple RING E3s Cell Cycle Logic Making the cycle go forward: SCFGrr1 SBF (Swi4/6) MBF Cln1/2 Cdc28 SCFCdc4 Sic1 SCFCdc4 Clb5/6 Cdc28 S phase Time Mitotic Entry Activation of Mitotic Cyclin-Dependent Kinases Cdk Regulation Synthesis Destruction SCF Complexes Phosphatase Kap1 Kinases cyclin T161 CAK Civ Synthesis Destruction Cdk Cks SCF Complexes T14 Y15 Kinases Phosphatases Wee1 Cdc25 Mik1 Myt1 Pyp3 * * CKIs Cdk Inhibitors Sic1 Far1 Rum1 p21, p27, p57 p16, p15, p18, p19 Mitotic Entry in S. pombe and Mammals Phosphorylation Regulation of Cdc2 during Mitosis (Cdc13) cyclin B Cdc2 (Cdk1) cyclin B/Cdc2 CAK Tyrosine Wee1 Kinases Mik1 - + cyclin B/Cdc2 (T160-P) ACTIVE KINASE cyclin B/Cdc2 Y-P (Inactive Y15-P) Phosphotyrosine Cdc25 Phosphatase + cyclin B/Cdc2 ACTIVE KINASE G2 M Cell Cycle Logic Autoactivation of Cdc2 makes mitosis irreversible (Cdc13) cyclin B Cdc2 (Cdk1) cyclin B/Cdc2 CAK Tyrosine Wee1 Kinases Mik1 - - + cyclin B/Cdc2 (T160-P) cyclin B/Cdc2 Y-P (Inactive Y15-P) Phosphotyrosine Cdc25 Phosphatase + cyclin B/Cdc2 (active) G2 + M APC/Cyclosome The APC is a complex ubiquitin ligase that is required for anaphase entry and mitotic exit. Like the SCF, it has substrate specificity components called Cdc20 and Cdh1/Hct1, 2 WD40 repeat proteins. The regulation of these specificity components is critical. Anaphase Entry and Exit Chromosomes OK ? APC Cdc20 Clb5 Clb2* APC Cdh1 Pds1 Cohesion Factors Clbs Cdk1 Mitotic Exit Anaphase Chromosome Cohesion Cdc20 APC Pds1 Securin Esp1 Separin Cohesin Pds1 Ub Ub Ub Destruction by the 26S Proteosome Pds1 has a destruction box which allows it to be recognized by the APC Esp1 Separin Anaphase Securin = Pds1 Separin = Esp1 Cohesin = Scc1 + APC Cohesion in Mammals Separin (Esp1) Mitotic Exit After anaphase is complete, in order to exit mitosis and initiate cytokinesis, cells must inactivate B-type cyclin/Cdks. Mitosis High Cyclin B/Cdk Activity Low Cdc14 Phosphatase Mitotic Exit Low Cyclin B/Cdk Activity High Cdc14 Phosphatase Cytokinesis & G1 Entry Mitotic Exit After anaphase is complete, in order to exit mitosis and initiate cytokinesis, cells must inactivate B-type cyclin/Cdks. This involves activation of the Cdh1 form of the APC. Cdh1 Clb Cdk1 Cdh1 P Inactive Cdc14 (Phosphatase) Cdh1 Active Cdh1 Cdc14 Inactive Swi5 Transcription Factor MEN Clb Cdk1 APC Cdc14 Active Swi5 P Clb/Cdk1 Cdc14 Cytoplasmic Inactive Swi5 Nuclear Active Sic1 Mitotic Exit Cdc14 Activation for Mitotic Exit The activation of Cdc14 is the key event in execution of mitotic exit. During S, G2 and Pre-anaphase, Cdc14 is held tethered in an inactive complex in the nucleolus. When Anaphase is executed, Cdc14 is released and goes throughout the nucleus and cytoplasm to dephosphorylate key Cdk1 substrates. Spindle Nucleolar Cdc14 MEN Cfi1 (Net1) Cdc14 Cfi1 (Net1) Cdc14 Inactive Active The mitotic exit network (MEN) consists of several protein kinases and a G-protein Tem1. How it MEN regulated is not known. How Mitotic Exit is Coupled to Anaphase Spindle Pole Body Tem1-GDP Lte1 Bfa1/Bub2 (GEF) (GAP) Tem1-GTP Cdc15 Mitotic Exit Network Dbf2, Mob1 Clb2 Cdc14 Cdc14 (Nucleolus) (Released) Mitotic Exit Sic1 The Tem1-bearing SPB migrates into the daughter cell to encounter Lte1 Tem1 Lte1 D Lte1 Tem1 Tem1 M Lte1 in Red Tem1 in Red Spindle in Green Mitotic Exit Summary 1. When anaphase occurs, Tem1 on the SPB is thrust into the daughter cell where it encounters the GEF, Lte1, Tem1 is converted to the active GTP form. 2. Active Tem1 activates Cdc15 and MEN, which causes the release of the Cdc14 phosphatase from the nucleolus where it is inhibited. 3. Cdc14 dephosphorylates Cdh1 to activate the APC to destroy Clbs, it also activates the synthesis of Sic1, a Cdk inhibitor. 4. Together, the APC and SIC1 turn off Cdk activity to initiate mitotic exit. Cell move from high CDK, low CDC14 state to a Low CDK, high Cdc14 state. To re-enter the next cell cycle they need to turn off Cdc14 to re-establish the null state, CDK off, Cdc14 off, APC off, making cells permissive for Clb activation of S phase. How does the cycle move forward? - Positive amplification loops - Feedback inhibition 1) Clns activate their own transcription 2) Once Clns provide sufficient activity to pass START, they activate a ubiquitin proteolysis pathway that destroys an inhibitor of Clb kinase activity, Sic1. 3) Clb/Cdc28 kinase activate Clb transcription and repress Cln transcription. 4) Clb/kinase activate S phase. 5) Once S phase is complete, Clb kinases activate mitosis. 6) Once chromosomes are properly aligned at the metaphase plate, a ubiquitin proteolysis pathway is activated that destroys Clbs but not Clns and resets the cycle. 7)Clb destruction allows PRC complexes to form. 8) Cln kinase activity is required to shut off the Clb proteolysis pathway to allow S entry in the next cell cycle. This allows Pds1 to be synthesized again which recruits Esp1 into the nucleus. In Mammals Cyclin B/Cdc2 can help activate itself by turning on an activating phosphatase and turning off an inhibitory kinase The Rao and Johnson Cell Fusion Experiments Cell Cycle Regulation M M cells + G1, S, or G2 cells - Mitotic state is dominant. S cells + G1 G1 cells enter S S cells + G2 G2 cells do not enter S, but do not enter mitosis until the S-phase nucleus has entered G2. - Block to re-replication - Inhibitor of mitosis produced by S phase cells G1 cells + G2 Like S above. - G1 cells also block mitosis Cell Cycle Checkpoints Definition: "A checkpoint is a biochemical pathway that ensures dependence of one process on another process that is otherwise biochemically unrelated." B A C Intrinsic Mechanism E D Damage Extrinsic Mechanism Why are checkpoints important? Checkpoints control the order and timing of events. In some cases the natural timing of events can allow the proper order of events in the absence of a checkpoint. However, the fidelity is often compromised. The accumulation of errors, whether due to entering DNA replication in the presence of damage, or missegregating a chromosome is deleterious to the reproductive fitness of unicellular organisms, and in multicellular organisms may lead to uncontrolled cell proliferation and cancer. Checkpoints in S. c. DNA Damage Checkpoints Spindle Assembly Checkpoints S phase Checkpoints Size Checkpoints G1/M Checkpoint Morphology Checkpoint Meiotic Checkpoints Checkpoints are defined by loss of function mutations that relieve the dependency of two events. cdc13 ts mutants cdc13 rad9 mutants The Spindle Assembly Checkpoint The proper assembly of a spindle is sensed by a group of proteins called Mad or Bub located on the kinetochore. These proteins send a signal to inhibit the APC. Mutant Hunt - benomyl sensitive mutants that continue to cycle in the presence of benomyl. ben WT ben mad or bub mutants Metaphase Misaligned Chromosomes Mps1 Mad1,2,3 Bub1,2,3 Cdc20 APC Scc1 Esp1 Pds1 Anaphase A/B The Spindle Assembly Checkpoint What is being sensed? Kinetochore - Microtubule Attachment Tension and bipolar attachment When tension is not present at sister chromatids, a Mad/Bubdependent phosphorylation occurs on the kinetochore. This is thought to be part of the signal used to turn off the APC. Signal Transduction Signal Sensor Transducer Effector DNA Damage Response Pathways SIGNALS Conserved Families PCNA- & RFC-like Proteins SENSORS Mediators (BRCT proteins, Mrc1/Claspin) Kinases: PIK ATM + ATR PK CHK1 and CHK2 TRANSDUCERS EFFECTORS STOP Cell Cycle Arrest Apoptosis Transcription DNA Repair The DNA Damage Response in Humans Hus1 RFC Rad17 P Rad1 P ATRIP Rad9 PC P Claspin P P P Chk2 p53 p21 G1 P Chk1 P P BRCA1 BLM NBS1 Repair Proteins P Cdc25 DNA replication proteins? S Cdc2/Cyclin B G2 M DNA Damage Checkpoints - Sensing Damage P P ATRIP RFC Rad17 P ATP Hus1 RFC Rad17 Hus1 Rad1 Rad1 Rad9 Rad9 ATRIP P ATR and RC-PC Engagement Activates Checkpoint Hus1 RFC P Hus1 Rad1 Rad17 P ATRIP Rad1 Rad9 P P Rad9 P P P Mediators Chk1 Chk2 BRCA1 Nbs1 Checkpoint Responses P G1 Arrest in Mammals Cdk activity is rate limiting for S phase entry and is the target for checkpoint control. DNA Damage Chk1,2 Cdc25A ATM/ATR ? Chk2 G1 Cyclin Mdm2 p21 p53 p53* Cdks or Apoptosis p53 levels increase in response to DNA damage and activate transcription of p21 How is p53 activated? - Relief of repression. MDM2 binds p53 and targets it for ubiquitin-mediated proteolysis. p53 transcriptionally regulates MDM2 to make a feedback loop. Mdm2 RING Finger Ubiquitin Ligase p53 Mdm2 transcription In response to DNA damage, both p53 and Mdm2 are phosphorylated, causing a disruption in Mdm2 binding, thereby allowing p53 to both increase in abundance and become transcriptionally active. During activation, p53 increases the amount of Mdm2 protein to return to low p53 levels when the signal is eventually turned off. This also explains why p53 levels are so high in tumors in which p53 is mutant, no Mdm2 is made. G2 Arrest in Mammals Cdc25 is regulated by Chk1 phosphorylation DNA Damage Active Ser216 Cdc25C ON Nuclear Mitosis A TR Chk1 Inactive Ser216 14-3-3 P Cdc25C OFF Cytoplasmic S/G2 G2 Arrest in S. pombe + Mammals Cdk activity is rate limiting for entry into Mitosis and is the target for checkpoint control. DNA Damage Chk1 Chk2 OFF 14-3-3 Cdc25 P Cytoplasm OFF ATM or ATR Chk1* Nucleus Chk2* Cdc25 ON Wee1 ON cyclin B cyclin B Cdc2 Y-P Cdc2 OFF ON p53* p21 Mitosis Mechanism of pre-anaphase arrest in response to DNA damage S. pombe Mammals S. cerevisiae rad3 rad26 crb2 * ATR MEC1 DDC2 Mediator chk1 Chk Kinases cdc25 Effectors CHK1 RAD9 * PDS1 cdc2 ESP1 Mitotic Entry Anaphase Entry Chk1 phosphorylation of Pds1 protects it from degradation by the APCCdc20 RAD53 CDK Mitotic Exit Overall Organization of the Cell Cycle Replication Checkpoint G1 G2 S S Cdk Pds1 SCF APC ON B/Cdk1 OFF SCF AnaB Spindle Checkpoint APC OFF B/Cdk1 ON Tele M Cdk M Cdk Sic1 G1 Cdk AnaA Meta ? APC Cdc20 Cdc14 B/Cdk1 APC Cdh1 Mitosis Cdh1 Cdc20 APC ON APC ON APC ON B/Cdk1 OFF General Points Cells need to do only a few things absolutely right 1. They must duplicate their chromosomes precisely, i.e. completely but only once per cycle. 2. They must segregate their chromosomes precisely. 3. They must divide their cell in two. General Properties of Cell Cycle Transitions 1. Amplification mechanisms. 2. Out with the old, in with the new. 3. Overcoming inhibitory barriers- Checkpoints. Checkpoints allow the coordination of events.