Cell cycle oscillations

Transcription

1 Positive and negative feedback produce a cell cycle Fast Slow Cell cycle oscillations Active Cdk1-Cyclin Inactive Cdk1-Cyclin Active APC 1

2 Ubiquitin mediated proteolysis Glycine Isopeptide bond Lysine Similarities between phosphorylation and ubiquitination Reversible reaction? Demodifying enzymes Regulates Number of human modifying enzymes Protein Phosphorylation No Protein phosphatases Protein activity, location, stability 500 Ubiquitination No Deubiquitinases Protein activity, location, stability >500 2

3 Different cyclins appear at different times in the standard cell cycle G1 cyclins overcome cell cycle roadblocks Cyclin-dependent kinase inhibitors (CKI) 3

4 NORMAL Cancer cells lack G1 roadblocks Standard Cycle Cdk Inhibitors Lost Cyclin-dependent kinase inhibitors CANCER OR Too much G1 cyclin Cyclin-dependent kinase Xinhibitors CANCER Cyclin-dependent kinase inhibitors Cell cycle checkpoints a. Cell cycle arrests b. Damage repair 4

5 Partial DNA replication breaks chromosomes DNA damage stops the cell cycle Damaged DNA DNA damage checkpoint DNA repair Cell Death (Apoptosis) 5

6 Apoptosis kills damaged or unwanted cells 6

7 Life Sciences 1a: Section 3B. Mitosis Objectives Understand the steps of mitosis Understand the difference between equilibrium and non-equilibrium polymers Understand how GTP hydrolysis explains how microtubules grow, and shrink Understand how exploration with selection captures chromosomes Understand that exploration with selection is a general biological principle Introduction Mitosis segregates chromosomes Prophase -> metaphase -> anaphase -> cytokinesis -> interphase DNA replication is templated, spindle assembly is not Cdk1-cyclin complexes induce massive reorganization of the cell Nuclear lamina phosphorylation induces nuclear breakdown Phosphorylation induces chromosome condensation (compaction) Microtubules become more dynamic The cytoskeleton The scaffolding and roadways of the cell Dynamic structures which are constantly assembled and disassembled Actin filaments (5 nm diameter), intermediate filaments (10 nm), microtubules (25 nm) Actin filaments and microtubules: polar, non-equilibrium, motors carry cargo in both directions Microtubules Microtubules are composed of 13 protofilaments They are nucleated at microtubule organizing centers (centrosomes in animal cells) Equilibrium polymers must all shrink or all grow But with non-equilibrium polymers, some polymers can grow whilst others shrink Tubulin exchanges GTP for GDP as a free subunit and hydrolyzes the GTP when it polymerizes Growing microtubules are mostly GDP-tubulin, but have a cap of GTP-tubulin If the subunits of the cap are bound to GTP, the microtubule grows If the subunits of the cap are bound to GDP, the microtubule shrinks (protofilaments splay apart) Chance fluctuations can convert a GTP cap into a GDP cap initiating rapid shrinkage (catastrophe) Chance fluctuations can convert a GDP cap into a GDP cap initiating growth (rescue) At the level of single molecules, the same reaction doesn t always happen at the same rate Microtubules and chromosome capture In mitosis more microtubules are nucleated, but increased catastrophe rates make them shorter The centrosome nucleates microtubules equally in all directions Microtubules that interact with chromosome arms or centromeres are protected from catastrophe Their increased stability converts an initially uniform distribution of microtubules into a bipolar spindle Exploration with selection (also known as search and capture) Stabilize a subset of random explorations from a starting point The next round of random exploration starts from these stabilized points Repeating this process is an efficient way to find very non-random solutions to biological problems This is a fundamental biological principle over a range of sizes and times; Protein folding: <10 nm, 1ms 10 min Spindle assembly: 10 µm, 1 min 1 hour Nervous system: 1 µm 1 m, 1 day 1 year Evolution: 0.1 nm 100 m, 1 month 10 9 years Reading: Alberts: pp (microtubules) (mitosis)

8 Different cyclins appear at different times in the standard cell cycle G1 cyclins overcome cell cycle roadblocks Cyclin-dependent kinase inhibitors (CKI) 1

9 NORMAL Cancer cells lack G1 roadblocks Standard Cycle Cdk Inhibitors Lost Cyclin-dependent kinase inhibitors CANCER OR Too much G1 cyclin Cyclin-dependent kinase Xinhibitors CANCER Cyclin-dependent kinase inhibitors Cell cycle checkpoints a. Cell cycle arrests b. Damage repair 2

10 Partial DNA replication breaks chromosomes DNA damage stops the cell cycle Damaged DNA DNA damage checkpoint DNA repair Cell Death (Apoptosis) 3

11 Apoptosis kills damaged or unwanted cells Mitosis and the cytoskeleton a. Phosphorylation controls cellular architecture b. The cytoskeleton: roadways & scaffolding c. Microtubules are dynamically unstable d. Self assembly by exploration with selection: Chromosome capture by exploration with selection 4

12 Mitosis segregates chromosomes Mitosis: the movies Aaron Straight 5

13 Templates versus self assembly DNA Replication + Spindle Assembly? Events of mitosis Nuclear breakdown Chromosome condensation Cdk1-cyclin Microtubules more dynamic 6

14 Filament systems of the cytoskeleton Diameter Polarity Dynamics Actin filaments 5 nm Non-equilibrium (ATP hydrolysis) Intermediate filaments 10 nm Equilibrium Microtubules 25 nm Non-equilibrium (GTP hydrolysis) Monomers (entire proteins) are held together by non-covalent bonds Microtubules are polymers Protofilament 7

15 Microtubules are nucleated at organizing centers Spontaneous nucleation + Templated nucleation Polymerization Equilibrium polymers can grow OR shrink M n + M M n+1 Growing (high [monomer]) Shrinking (low [monomer]) M n + M M n+1 M n + M M n+1 8

16 Finding polymer equilibrium M n + M M n+1 Growth = k on [monomer][ polymer _ ends] Shrinkage = k off [ polymer _ ends] At equilibrium Shrinkage = Growth k off [ polymer _ ends] = k on [ polymer _ ends][monomer] eq k off = k on [monomer] eq k off k on = [monomer] eq Non-equilibrium polymers can grow AND shrink M n + M M n+1 M n + M M n+1 k off k off = [monomer] eq = [monomer] eq k on k on 9

17 Microtubules can grow AND shrink Tubulin hydrolyzes GTP GTP GDP Free GDP On microtubules GTP P 10

18 Microtubules can grow AND shrink Microtubule growth, shrinkage, catastrophe, & rescue GTP-tubulin GDP-tubulin Add (0.2 ms -1 ) Hydrolyze (0.05 ms -1 ) (Catastrophe) Add (0.03 ms -1 ) (Rescue) Lose (0.3 ms -1 ) time, ms time, ms 11

19 Microtubules are more dynamic in mitosis Interphase Mitosis Increased nucleation Increased catastrophe Spindle assembly: Exploration with selection 12

20 Exploration with selection is a general principle Process Distance Time Protein Folding nm 1 ms - 10 min Spindle Assembly Nervous system development Evolution 10 µm 1 µm - 1 m 0.1 nm m 1 min - 1 hour 1 day - 1 year 1 month yr 13