Flow cytometry - FACS
Cell Cycle (reminder) Life is made of cells Cells come from cells Life/Cells reproduce
Reproduction of cells require correct duplication and segregation M G1 S G2
What else must be replicated & segregated to the daughter cells ? Cytoplasm Cytoskeleton Plasma membrane Organelles Mitochondria Golgi Centrosomes Everything…..
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.
Cell Cycle (reminder)
Figure 17-6 Molecular Biology of the Cell (© Garland Science 2008) Temperature sensitive mutants used to identify critical components of cell cycle control
Cleavage division of Xenopus embryos
MPF - Maturation promoting factor (-Cdk+Cyclin) purified from oocytes
Cyclin dependent Kinase (CDK) is the central component controlling the cell cycle
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.
The basic cell cycle control system
The CDK-Cyclin complex activates events of the cell cycle
Since different Cyclins drive different events, they must contribute to substrate specificity
Cyclins are degraded in a cell cycle dependent manner
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
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
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
The CDK-Cyclin complex activates events of the cell cycle
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
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
Regulation of CDK by proteolysis – SCF complex
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)
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
Regulation of CDK by CDK inhibitors
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
How DNA damage arrests the cell cycle? E3 Ub ligase
The CDK active site is exposed only when bound to Cyclin (and with T160 phosphorylation by CAK/CDK7+CycH)
Substrate ATP
Regulation of CDK by inhibitory phosphorylation (Kinase)(Phosphatase) Inactive Active Inactive
Summary of CDK regulation mechanisms
Checkpoint control is achieved by CDK regulation
FACS machine Fluorescence Activated Cell Sorter can analyze and sort cells
FACS machine Cell cycle analysis using FACS and DNA fluorescence dye Fluorescence Intensity (amount of DNA) # of Events G1 S G2
FACS machine Cell cycle analysis using EACS and DNA fluorescence dye DNA stained with propidium iodide DNA Content G1 S Phase G2/M
FACS machine S phase block G1 S G2 T=0 T=24
FACS machine Nocodazole M phase block G1 S G2 T=0 T=24
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
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
Rb inhibits E2F function Blocks recruitment of transcription activators
E2F transcription factor family: repressors and activators repressors activators E2F7,8 Trimarchi and Lees, 2002 NR MCB 3:11
E2F transcription factors activate many S-phase associated genes From Knudsen NRC 2008
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