Section 10. Junaid Malek, M.D.

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1 Section 10 Junaid Malek, M.D.

2 Cell Division Make sure you understand: How do cells know when to divide? (What drives the cell cycle? Why is it important to regulate this?) How is DNA replication regulated? What limits number of mistakes made during DNA replication? How are chromosome segregation and cell division ensured? How are cell growth and proliferation coordinated to regulate cell size? How do cells make sure one task is finished before beginning the next?

3 Cell Division G0 Cells rest in G0 G1: gap between M and S S: synthesis- cell replicates its DNA G2: interval between S and M M : mitosis- nucleus and cytoplasm divide Interphase = G1+S+G2

4 Question 1 Q: Why might the cell need two gap phases? A: G1 and G2 provide additional time for the cell to grow and duplicate its cytoplasmic organelles. They give the cell time needed to double its mass before it divides

5 Phases of Cell Cycle During interphase (G1, S, and G2), cells continue to transcribe genes, synthesize proteins and grown in mass Most cells are neither growing nor dividing but are resting in G0, which is reached from G1 Embryonic vs Normal Cell Cycle: Embryonic Cell Cycle Often extremely rapid-early frog embryos have cell cycle time of 30 minutes Often growth and division uncoupled Does not require nucleus Does not require DNA Requires post-translational modifications Requires protein degradation Normal Cell Cycle Not as fast for adult mammals normal cell cycle around 24 hours Growth and division normally coupled Requires nucleus Requires DNA and proper replication and segregation of chromosomes Requires post-translational modifications Requires protein degradation

6 Question 2 Q: Why do you suppose that cells have evolved a special G0 state to exit the cell cycle rather than just stopping in G1? A: See p. 624 Alberts 18-5 for answer

7 Question 3 Q: What would be the consequences if a cell did not finish replicating the DNA before dividing? A: Incomplete replication would lead to chromosome loss and breakage Q: Would slowing down DNA replication or mitosis affect the cell cycle in frog embryos? in human stem cells? A: In frog embryos, no. In human stem cells, yes.

8 Cyclin and the Cell Cycle Fast Slow Cyclin accumulates in interphase (newly synthesized) and is destroyed at the end of mitosis Cyclin binds to and activates cyclin dependent kinase 1 (Cdk1) Engine oscillates because of positive and negative feedbacks (reaction kinetics and topology matters)

9 Positive Feedback Positive feedback is fast Cdk1 alone - inactive Initial Cdk1-cyclin complexes - low activity Active Cdk1-cyclin complexes stimulate reactions that turn the inactive Cdk1-cyclin complexes into active Cdk1-cyclin complexes

10 Negative Feedback Negative feedback is slow With a delay, active Cdk1-cyclin complexes turn on the cyclin destruction machinery

11 Question 3 Q: Does all of the Cdk1 need to be newly synthesized for each round of the cell cycle? A: No, it is not synthesized and degraded in the same way as cyclin only its activity is changed

12 Protein Phosphorylation and Regulation of Protein Activity Protein kinases add phosphate from ATP to hydroxyl groups on serine, threonine, or tyrosine What do the sidechains of these three residues have in common? Phosphatases remove the phosphate allowing independent regulation of phosphate addition and removal Phosphorylation regulates protein activity, localization, binding, and stability

13 Question 4 Q: Why can phosphorylation have such a dramatic effect on the activity and function of a protein? A: Changing a polar OH group into a negatively charged group can effect the interactions that the protein can make. Could also affect its three dimensional structure

14 Protein Degradation and Biological Regulation The amount of protein in a cell depends not only on how many new copies are made but also on how long they survive just like any population Breakdown of proteins by cells is a way of regulating the amount of protein present at a given time Some proteins like structural proteins may last for months or years. Other proteins like those that regulate the cell cycle or signaling may last only for minutes or seconds Most proteins degraded in cytosol of eukaryotic cells are broken down by the proteasome. The proteasome is a cylinder with protease active sites inside the chamber; the ends of the cylinder are capped by other proteins Proteasome primarily acts on proteins that have been marked for destruction by the addition of a small protein called ubiquitin on one of their lysine residues Ubiquitination is like phosphorylation: irreversible, diverse, regulates protein activity, location, and stability

15 Cyclin Proteolysis Cyclin is degraded by ubiquitin-mediated proteolysis C terminus of ubiquitin is coupled to lysine side chains of cyclin Anaphase promoting complex (APC) is the final coupling enzyme and is activated by Cdk1-cyclin

16 Question 5 Q: How might you create a mutant version of a protein that is more stable? A: Mutate the lysine residues responsible for ubiquitination so that the protein cannot be marked for destruction by the proteasome

17 Cell Cycle and Genomic Integrity Note that cell cycle checkpoints protect the integrity of the genome Standard cell cycles have temporally separate G1 (D), S phase (A, E) and mitotic (B) cyclins G1 cyclins overcome a cell cycle roadblock set by inhibitors of cyclindependent kinases Cancer mutations remove the roadblock or make enough G1 cyclins to over-ride it. Checkpoints monitor completion and induce arrest and repair Not finishing tasks damages chromosomes: incomplete replication leads to chromosome loss & breakage Normally damaged DNA prevents Cdk1-cyclin B activation, induces DNA damage repair, and can induce cell death Apoptosis: a program of cell death to remove damaged or unwanted cells

18 Mitosis Prophase chromosomes condensing, centrosomes beginning migration Prometaphase nuclear envelope breakdown, chromosomes begin attaching to spindle Metaphase chromosomes aligned at equator of the spindle Anaphase paired chromosomes are pulled apart towards each spindle pole Telophase chromosomes arrive at spindle poles, nuclear envelope begins reforming, division of cytoplasm commences. Cytokinesis cytoplasm is divided and the membrane is pinched off to form two daughter cells. Interphase all the time before and after mitosis

19 Mitosis Microtubules = Green DNA = Blue

20 Mitosis Role of CDK1-Cyclin: phosphorylates nuclear lamina facilitates nuclear envelope breakdown Phosphorylation of proteins initiates chromosome condensation This allows microtubules to become even more dynamic

21 Equilibrium vs. Non-Equilibrium Polymers At equilibrium no net change in average lengths of a population of polymers. Each individual polymer exists at approximately the same average length. Non-equilibrium polymers no net change in average lengths of a population of polymers. However the individual polymers can be of extremely different lengths. Example of microtubules will be discussed

shrinkage is occurring for the microtubules.

22 Microtubule Growth and Disassembly Microtubules are composed of 13 protofilaments and nucleated at microtubule organizing centers (MTOC, centrosomes in animal cells) If a population of microtubules is at equilibrium with their monomer units, no net growth or shrinkage is occurring for the microtubules. However microtubules are not at equilibrium, but rather exist in a steady state (energy is released in both directions, and the microtubule assembly reaction is different from the microtubule disassembly reaction).

23 Microtubule Growth and Disassembly Assembly: GTP capped protofilament adds on a GTP bound tubulin monomer Disassembly: GDP capped protofilament falls apart releasing many GDP bound tubulin monomers

24 Microtubule Growth and Disassembly 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 GTP cap initiating growth (rescue) At the level of single molecules, the same reaction doesn t always happen at the same rate

25 Question 6 Q: How would the average length of microtubules in a cell be affected if the hydrolysis rate of GTP to GDP in microtubules increased? A: It would decrease Q: How would the length be affected if the hydrolysis rate decreased? A: It would increase Q: How would the length be affected if the concentration of GTP-tubulin monomers increased inside the cell? A: It would increase

26 Exploration and Chromosome Capture Microtubules are nucleated at the centrosome and grow outwards in all directions This growth in all directions is the exploration phase If a microtubule reaches a centromere or chromosome arm, it is rescued from catastrophe (selection) All other microtubules are subject to catastrophe Leads to the formation of a bipolar spindle A brief exploration phase is conducted, where the microtubules grow outwards in all directions, but they do not grow very far Those that reach a chromosome are rescued, while all others fall apart This allows for the spindle to search a much smaller space more quickly than if each microtubule were allowed to grow for an indefinite period of time

Cell cycle oscillations

Positive and negative feedback produce a cell cycle Fast Slow Cell cycle oscillations Active Cdk1-Cyclin Inactive Cdk1-Cyclin Active APC 1 Ubiquitin mediated proteolysis Glycine Isopeptide bond Lysine