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Flow cytometry - FACS
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Cell Cycle (reminder) Life is made of cells Cells come from cells Life/Cells reproduce
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Reproduction of cells require correct duplication and segregation M G1 S G2
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What else must be replicated & segregated to the daughter cells ? Cytoplasm Cytoskeleton Plasma membrane Organelles Mitochondria Golgi Centrosomes Everything…..
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The basic cell cycle control system A clock, or timer, that turns on each event at a specific time, providing a fixed amount of time for the completion of each event. A mechanism for initiating events in the correct order (for example, M only after S). A mechanism to ensure that each event is triggered only once per cycle. Binary (on/off) switches that trigger events in a complete, irreversible fashion.
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Cell Cycle (reminder)
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Figure 17-6 Molecular Biology of the Cell (© Garland Science 2008) Temperature sensitive mutants used to identify critical components of cell cycle control
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Cleavage division of Xenopus embryos
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MPF - Maturation promoting factor (-Cdk+Cyclin) purified from oocytes
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Cyclin dependent Kinase (CDK) is the central component controlling the cell cycle
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The basic cell cycle control system A clock, or timer, that turns on each event at a specific time, providing a fixed amount of time for the completion of each event. A mechanism for initiating events in the correct order (for example, M only after S). A mechanism to ensure that each event is triggered only once per cycle. Binary (on/off) switches that trigger events in a complete, irreversible fashion.
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The basic cell cycle control system
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The CDK-Cyclin complex activates events of the cell cycle
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Since different Cyclins drive different events, they must contribute to substrate specificity
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Cyclins are degraded in a cell cycle dependent manner
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APC complex has E3 Ubiquitin ligase activity APC activity is controlled by specific activating proteins: CDC20 in anaphase Cdh1 in G1 Regulation of CDK by proteolysis – The APC complex
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APC complex has E3 Ubiquitin ligase activity APC activity is controlled by specific activating proteins: CDC20 in anaphase Cdh1 in G1 APC becomes activated by M-CDKs, Active APC degrades M –cyclins and promotes anaphase Regulation of CDK by proteolysis
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APC complex has E3 Ubiquitin ligase activity APC activity is controlled by specific activating proteins: CDC20 in anaphase Cdh1 in G1 APC becomes activated by M-CDKs, Active APC degrades M –cyclins Loss of M-CDK result in reduced APC phosphorylation and disociation of CDC20
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The CDK-Cyclin complex activates events of the cell cycle
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APC activity is controlled by specific activating proteins: CDC20 in anaphase Cdh1 in G1 APC becomes activated by M-CDKs, Active APC degrades M –cyclins and promotes anaphase Loss of M-CDK result in reduced APC phosphorylation and dissociation of CDC20 In G1 Cdh1 is activating APC Not all cells have G1 Regulation of CDK by proteolysis
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M-CDK phosphorylate APC and activate APC-Cdc20 but inhibit APC-Hct1(Cdh1) activity. APC-Cdc20 degrades M-Cyclin -> M-CDK activity drops APC-Hct1 activity increases In yeast Sic1 (a CDKi) inhibits S & M CDK activity Additional control at transcription level (Cyclins, Sic1…) Regulation of proteolysis by CDK - Regulation of CDK by proteolysis
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Regulation of CDK by proteolysis – SCF complex
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Regulation of CDK by proteolysis SCF complexes are present throughout the cell cycle Complexes can harbor various F-box subunits. Act mostly on phosphorylated target Example: CycE (G1/S) becomes phosphorylated by CycA-CDK (S) and it subsequently degraded Many more F-Box subunits (unrelated to cell cycle)
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Modulation of the basic cell cycle control system Checkpoint control system – cell cycle is controlled by STOP & GO – process is assessed & possibly halted – signals indicate if key cellular processes have been completed correctly
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Regulation of CDK by CDK inhibitors
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Regulation of CDKi by proteolysis SCF complexes are present throughout the cell cycle Complexes can harbor various F-box subunits. Act only on phosphorylated target Example 1: CycE (G1/S) becomes phosphorylated by CycA-CDK (S) an degraded Example 2: p27 (CDKi) is recognized by SCF (with a different F box protein) when phosphorylated
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How DNA damage arrests the cell cycle? E3 Ub ligase
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The CDK active site is exposed only when bound to Cyclin (and with T160 phosphorylation by CAK/CDK7+CycH)
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Substrate ATP
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Regulation of CDK by inhibitory phosphorylation (Kinase)(Phosphatase) Inactive Active Inactive
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Summary of CDK regulation mechanisms
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Checkpoint control is achieved by CDK regulation
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FACS machine Fluorescence Activated Cell Sorter can analyze and sort cells
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FACS machine Cell cycle analysis using FACS and DNA fluorescence dye Fluorescence Intensity (amount of DNA) # of Events G1 S G2
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FACS machine Cell cycle analysis using EACS and DNA fluorescence dye DNA stained with propidium iodide DNA Content G1 S Phase G2/M
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FACS machine S phase block G1 S G2 T=0 T=24
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FACS machine Nocodazole M phase block G1 S G2 T=0 T=24
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G1: APC-Hct1(Cdh1) active CDKi present and active Rb is active and inhibits activity of E2F TFs Transcription of G1/S,S cyclins is repressed Activation of G1-CDK causes Rb phosphorylation and activation of E2Fs Positive feedback loop enhances E2F activity: E2F activate E2F transcription G1/S CDK enhances phosphorylation of Rb, and CDKis Start of Cell cycle
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G1: APC-Hct1(Cdh1) active CDKi present and active Rb is active and inhibits activity of E2F TFs Transcription of G1/S,S cyclins is repressed Activation of G1-CDK causes Rb phosphorylation and activation of E2Fs Positive feedback loop enhances E2F activity: E2F activate E2F transcription G1/S CDK enhances phosphorylation of Rb, and CDKis Start of Cell cycle
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Rb inhibits E2F function Blocks recruitment of transcription activators
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E2F transcription factor family: repressors and activators repressors activators E2F7,8 Trimarchi and Lees, 2002 NR MCB 3:11
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E2F transcription factors activate many S-phase associated genes From Knudsen NRC 2008
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Feedback loops in the G1-S transition E2F activates CycE, A and CDK2 - positive FB CycE/CDK2 inactivates p27 - positive FB CycA/CDK2 inactivates E2F- Posphorylates CycE negative FB SCF
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