You may not believe it but by the end of the semester This will make sense! Hanahan and Weinberg, Cell 100:57-70 (2000)

You may not believe it but by the end of the semesterThis will make sense!

Hanahan and Weinberg, Cell 100:57-70 (2000)

Cell cycle and its control

Cells must be able to proliferate

- during development- wound healing- stem cells in blood, small intestine, immune system

For cells to copy themselves they need to:

- Grow; make more stuff; e.g. proteins, lipids

- Copy their genetic material

- Segregate contents to daughter cells, especially…

- Segregate replicated chromosomes to daughter cells

Many of the images in the cell cycle part of the course are taken from The Cell Cycle , by David O Morgan (New Science Press)

Interphasecells duplicate chromosomes Mitosiscells segregate duplicated chromosomes into two daughter cells

The Cell Cycle

Interphase has 3 periods: G1, S, G2

G1: cells decide whether to divide or not:- Have I grown big enough to enter the cell cycle?- Am I OK?

Restriction Point / START

S: chromosomes are duplicated

G2: cell prepare to enter mitosis by asking:- Have I completed DNA synthesis properly?- Am I OK?

Execution of these decisions commits a cell to complete a full division cycle

The main jobs of the cell cycle:

1. To accurately transmit the genetic information!2. To maintain normal ploidy; i.e. diploidy!

Regulatory mechanisms:- Accuracy in the “assembly line” (e.g. DNA

polymerase)- Extrinsic regulatory mechanisms (all processes

follow a correct order)

Let’s remind ourselves some basic stuff

Starting with the S phase

Early G1 Pre-replicative complex (origin licensing)

Early SActivation of helicase;Assembly of pre-initiation complex

Helicase

DNA does not come naked

It is packed into chromatin

Mainly, histone proteins

Thus, duplicating chromosome = duplicating DNA and duplicating histones

In addition, we need to repack the duplicated DNA

Histone synthesis increases sharply during the S phase

Increase in transcription, in processing, and in stability

Chromatin Inheritance

- TelomeresCis-elements: sequences recruiting proteins that

modify histones

- Centromere Epigenetic mechanisms, not clearly understood

Reproducing chromatin organization during the S phase

Mitosis

During the S phase, the duplicated DNA is rearranged through cohesion to form two sister-chromatids attached to each other by cohesins

Gradually, the cohesins will be removed to allow sister-chromatid separation

- Sister-chromatids condense

- Centrosomes move to opposite poles of the cell, nucleating microtubules (MTs)

- Nuclear envelope breakdown

Prophase

Prometaphase

- Nuclear envelope breakdown is completed

- The centrosomes nucleate MTs towards each other, forming the spindle MTs

- The growing (+) ends of the MTs capture the chromosomes at the site of the centromere through a protein complex called the kinteochore

Kerry Bloom

Kinetochore

Microtubule

Microtubule

KinetochoreCentromere

Ted Salmon

Prometaphase

- Nuclear envelope breakdown is completed

- The centrosomes nucleate MTs towards each other, forming the spindle MTs

- The growing (+) ends of the MTs capture the chromosomes at the site of the centromere through a protein complex called the kinteochore

At the end of the day: Metaphase

Now, we are ready for Anaphase

Anaphase (A+ B)

Salmon lab

Silverman-Gavrila lab

MitosisProphaseChromatid condensationPrometaphaseKinetochore-MTs bindingSpindle assemblyMetaphaseChromosomes align at the midline

Telophase and CytokinesisBirth of two daughter cells

AnaphaseSegregation of sister-chromatids

Cell cycle is controlled

Cells can be fused

- Fuse S phase cell with G1 cell: The G1 nucleus enters S phase

Rao and Johnson (1970)Cell fusion experiments

- Fuse M phase cell with interphase cell: Interphase nucleus enters M

Cell cycle has a clock, regulated by promoting factors and checkpoints

For example, anaphase-metaphase transition will take place only if ALL the kinetochores are attached to MTs

If the checkpoint regulators are compromised, unattached chromosome might be lagging behind, resulting in aneuploidy

G1

Cyclin Dependent Kinases Regulate the Cell Cycle

Experimental Systems Important for Cell Cycle Studies

Arbacia punctulata

Xenopus laevisSchizosaccharomyces pombe

Saccharomyces cerevisiae

Budding Yeast: Saccharomyces cerevisiae

Lee Hartwell

Hartwell was interested in the protein synthesis machinery

Budding Yeast: a genetic eukaryotic model organism

Let’s look for mutants that cannot synthesize proteins

Isolating temperature sensitive mutants in haploid yeast

Lee Hartwell

Budding Yeast: Saccharomyces cerevisiae

Serendipity, our old friendBrian Reid, an undergrad, needs to look at a microscope to follow a mutant. They realize that bud size stores information about the cell cycle

Brian Reid

Permissive (low) temperature(mixed population of cells in different

stages of the cell cycle)

Restrictive (high) temperature

An assay for isolating cdc mutantscdc: cell division cycle mutants

cdc mutant growingat permissive temp

cdc mutant growth arrested after 6 hrs at restrictive temp

Temperature sensitive cdc mutant

Genetic and descriptive analysis discover the interactions between the mutants

DNA

How to clone cdc genes in yeast?

Let’s say you have a candidate sequence

cdc28 (-)

If the candidate sequence complements (rescues) the mutated phenotype: that’s your gene!

WT

How to Clone cdc Genes in Yeast

Gene Z

Many of the cdc genes encode proteins needed for DNA replication

cdc28 gene encodes a kinase

Sir Paul Nurse

Fission yeast: Schizosaccharomyces pombe

cdc genes encode proteins needed for the G2-M transition: studies in s. pombe

cdc2D = gain of function mutant

Cloning cdc2The same approach used in budding yeasts:

complementation by a library

Only using a budding yeast library

START/Restriction Point

Cdc2 (fission)

Cdc2 (fission)Cdc28 (budding)

This is all great

Yeast are really cute and interesting

Can we really learn something from that about humans?

Schizosaccharomyces pombe

Sir Paul Nurse

Crazy idea

Let’s try to complement (rescue) the cdc2 (-) mutant of pombe with a human cDNA library

It worked for us with budding yeast genes. Why not try human genes?

Human cdc2 rescues cdc2 mutants

Elongated cdc2 mutants, failing to undergo mitosis

cdc2 mutants, complemented by a

human cdc2 gene

Melanie Lee

Summary

- A genetic approach in fission and budding yeasts reveal genes that are essential in promoting the cells through the cell cycle

- These genes encode kinases proteins and are called CDKs for Cyclin-Dependent Kinases

Cdk1 = the protein encoded by cdc2/CDC28

Tim Hunt

Woods Hole Marine Biological Laboratory

can be stimulated to lay lots of eggs

Sea urchins

The summer project: to follow protein synthesis upon fertilization by following incorporation of S35 - Met and getting samples every 10’

Proteins X,Y,Z are synthesized only in unfertilized eggs Proteins A,B,C are synthesized upon fertilization

Protein A disappears 10’ before completion of mitosis

mitosis mitosis mitosis

In clams two proteins, A and B, express this cyclic behavior

Cyclins are synthesized and degraded in a cyclic manner and with correlation to the cell cycle

ProteinLevel

Time

cyclin A cyclin B

M M M

Something needs to go away in order for the cell cycle to proceed

Yeast genetics Needed for promoting cells through the cell cycle

CDK

Biochemistry in sea urchinAppear in correlation with the cell cycle

Cyclin

Time to bring them together