Overview of the Control of the Cell Cycle Pages 1053-1067

The Cell Cycle -Cell cycle control system – a network of regulatory proteins -The cell monitors: 1. Events inside the cell 2. Conditions outside the cell

Phases of the Cell Cycle G1 – Cell growth and doubling of organelles S – Duplication of DNA G2 – Preparation of division M – Mitosis, Separation of chromosomes Cytokinesis – Division into 2 daughter cells Interphase = G1 + S + G2

Stages of Mitosis Defined as structure and location of DNA

Control Points of the Cell Cycle -Commitment points in the cell cycle are known as Start (in yeast) or restriction point (in mammalian cells) or checkpoints -The cell monitors internal and external conditions -Main Checkpoints are before S and M phases

Cell Cycle Control in Eucaryotes -Time to complete the cell cycle can vary greatly between cell types within the same organism -All eucaryotes used similar machinery and control mechanisms in the cell cycle Model systems used to study the cell cycle 1. Yeast 2. Animal embryos 3. Cell-free systems 4. Cell culture

Yeast Model System Advantages include: -Reproduce rapidly -Small genome, 1% of a mammal -Can proliferate in a haploid state -Nuclear envelope remains intact -Budding yeast does not have a normal G2 pahse

Temperature-Sensitive Mutants Cell-division-cell (Cdc) genes – genes important in controling the cell cycle A mutant that cannot complete the cell cycle cannot grow and divide

Temperature-Sensitive Mutants Normal Population Cdc15 Ts mutant (high temp)

Animal Embryo Model System Xenopus -Fertilized eggs are large (1mm) -Large amounts of cell division proteins -Rapid sequence of cell divisions after fertilization -1st division takes 90 min -Next 11 divisions takes 30 min each

Xenopus Animal Model During early embryonic cycles the basic processes are occurring: DNA duplication and separation Very little cell cycle control Mid-blastula transition

Cell-Free Model System -Cell-free of in vitro system -Advantage -Manipulate cell-cycle events under controlled conditions

Cell Culture Model System -Normal cells can be cultured for 25-40 divisions before undergoing replicative cell senescence -Immortalized cells can proliferate indefinitely Advantage: -Unlimited supply of genetically homogeneous cells

Studying the Cell Cycle Staining cells -DNA-binding fluorescent dyes -Antibodies to specific molecules 2. BrdU (bromo-deoxyuridine) incorporation 3. Tritiated thymidine incorporation 4. Flow cytometer

BrdU Labeling

Flow Cytometry -Measures DNA content -Thousands of cells per minute

Control of the Cell Cycle -The proteins that control the cell cycle are different from the proteins that are involved in the process -Series of checkpoints -Each checkpoint serves as a biochemical switch

Control of the Cell Cycle The control system: 1. Swithes are binary: “On or Off” and once started the process continues to completion 2. Reliable due to back-up systems 3. Adaptable so it can be modified to different cell types or conditions

Key Control Components 1. Cyclin-dependent kinases (Cdk) -Activities vary throughout the cell cycle -The targets of phosphorylation then varies 2. Cyclins -Control the activity of Cdk’s -Undergo synthesis and degradation in each cell cycle Cycling of the cyclins creates the cycles of the cyclin/Cdk complex allowing for the varying activities resulting in progression through the cell cycle

Cyclin-Cdk Complexes 4 classes of cyclins G1-cyclin 4 classes of cyclins -All eucaryotes require 3 cyclins (G1/S, S, M)

Yeast – 1 Cdk Vertebrates – 4 Cdks Cyclin – activates the Cdk and helps direct it to the specific target

Cdk Activation Cdk fullly active 1. Cyclin bound 2. Phosphorylated at the active site

Regulation of Cdk Activity -Cdk activity can be fine tuned through 2 phosphorylations at the top of the active site -Important in control of M-Cdk activity

Inhibition of Cyclin-Cdk Complexes -Binding of inhibitors alters the structure of the active site -Cdk inhibitors are utilized more early in the cell cycle

Cyclin-Cdk Complexes APC/C Anaphase-Promoting Complex, or Cyclosome G1-cyclin APC/C Anaphase-Promoting Complex, or Cyclosome -Regulated by protein destruction

Marking Proteins by Ubiquitin

Ubiquitination Process I

Ubiquitination Process II There are about 300 different E2-E3 each recognizing a different degradation signal. Therefore subsets of proteins can be regulated as a group.

Control of Proteolysis of APC/C -Degrading S and M-cyclins stops the Cdk activity and the Cdk’s targets become dephosphorylated and inactive or Cdh1 or S-cyclin -APC/C is active in G1 keeping Cdks inactive

Control of Proteolysis by SCF -Degrades Cdk Inhibitors of S-Cdks and allows S phase to occur -F-box protein is constant through the cell cycle and is used to recognize the target

Activation of a Ubiquitin Ligase

Activation of a Degradation Signal

Cell-Cycle Control Overview 10% of yeast genes encode mRNAs which oscillate in the cell cycle