A briefe history of the cell cycle 18xx Interphase-Mitosis 1951 Discovery of a distinct S-phase 1970 Cell fusion experiments 1971 Discovery of MPF 197x Yeast cdc mutants 1983 Discovery of the first cyclins 1988 Cyclin B+cdc2 = MPF 199x Ubiquitin and the cell cycle 1999 Sister chromatid separation

The most important role of cell cycle control is to ensure accurate chromosome segregation M G1 S G2

General principles and methods G1 - To cycle or not to cycle S- Replicating the genome M-Segregating the genome Cytokinesis - Making two cells

ONCE and ONLY ONCE

Rao and Johnson 1970

Fluorescent Activated Cell Scan/Sorter (FACS)

A typical FACS analysis

Methods for cell synchronisation

Model organisms I - Budding yeast (S.cerevisiae) L. Hartwell

Model organisms II - Fission yeast (S.pombe) P. Nurse

Identification of cell cycle ts mutants

Generation of temperature sensitive budding yeast mutants

Analysis of ts cell cycle mutants G1 S M M M M

Maturation promoting factor

Oscillations of MPF

Cyclins and cyclin dependent kinases (cdks) - in Budding yeast Cdk1 (cdc28) (S.p. cdc2) Cln1,2,3 G1 Clb5,6 S Clb3,4 G2 Clb1,2 M

Cyclins and cyclin dependent kinases (cdks) - in multi-cellular organisms Cdk1 (S.p. cdc2) Cyclin D1,2,3G1Cyclin E Cyclin A S Cdk4,6 Cdk2 Cyclin A Cyclin B1,2,3 M

Cdc2/28 (=cdk1) is regulated by: 1. Binding of a cyclin 2. Inhibitory phosphorylation 3. Activating phosophorylation 4. Binding of CKI 5. Cellular localization

G1 - To cycle or not to cycle S- Replicating the genome M-Segregating the genome Cytokinesis - Making two cells

P Cyc D cdk4 pRB E2F P P P P Cyc E cdk2

G1 - To cycle or not to cycle S- Replicating the genome M-Segregating the genome Cytokinesis - Making two cells

The human genome is encoded by 3,000,000,000 bp (3x10 9 ) which must all be replicated within a few hours with the highest fidelity. The loss of a bp is irreversible and a single mistake could potentially lead to the death of the organism.

Analogy One page contains about 3000 letters. Imagine that you have to copy one million pages in a few hours and have to bind them into 23 volumes. You can use as many typist as you like but you are allowed no mistakes -no missing letter, no letter twice, and no substitutions.

G1 - To cycle or not to cycle S- Replicating the genome M-Segregating the genome Cytokinesis - Making two cells

The story of the two blind men Two blind men go into a shop and buy each five different pairs of socks. Unfortunately all ten pairs were packed in the same bag. How can the two men separate the socks so that each of them will get five pairs ?

The three factors that enable sister chromatid separation The glue - cohesins The pulling force - the mitotic spindle The timing mechanism - spindle checkpoint

The three factors that enable sister chromatid separation The glue - cohesins The pulling force - the mitotic spindle The timing mechanism - spindle checkpoint

Sister chromatid separation from prometaphase to anaphase Securin Separase Cohesin metaphase Securin Separase Cohesin prometaphase APC Check point Separase APC Securin Separase anaphase Check point

Non-degradable securin does not prevent cytokinesis but interferes with complete chromatide separation histon-GFPHoechst HeLa securin KAA-DM securin KAA-DM NIH3T3

The three factors that enable sister chromatid separation The glue - cohesins The pulling force - the mitotic spindle The timing mechanism - spindle checkpoint

Anaphase Promoting Complex/ Cyclosome

APC Checkpoint cdk1 Cyclin B Fzy Checkpoint Fzr Polo Prophase

APC cdk1 Cyclin B Fzy Checkpoint Fzr Polo Metaphase

APC cdk1 Cyclin B Fzr Fzy Fzr Fzy Anaphase Polo cdc14 Metaphase

Yes (chrom. movement) Metaphase Chrom. alignment on metaphase plate Xkid ?TelophaseMitosisE2 of the APC/CUbcH10 No Telophase - G1 MetaphaseAPC/C activatorFzy Yes (cyclin A+B degr.) PrometaphaseG2, prophaseKinaseNek2A NoG1SReplicationCdc6 ? Telophase - G1 MitosisMitotic kinasePlk1 Yes (cytokinesis) MetaphaseProphase- metaphase Mitotic kinaseCyclin B1 Yes (chrom. separation) MetaphaseS-metaphaseChromatid cohesion maintenance Securin Yes (metaphase) PrometaphaseS, G2, prophase Kinase (+cdk1/2)Cyclin A2 Degrad. essential Degradation initiated ActiveRoleSubstrate