The Eukaryotic cell cycle 1: LECTURE 2

(basic principles of master regulators

Steps for proliferating cells

• cell growth

• chromosome segregation

• cell division

But these events must be

• in the correct order

• non-overlapping temporal compartments

How they figured out that DNA synthesis occurred in discrete interval in interphase

• studied populations of meristmatic cells

• for P32 uptake in the nucelus

  results:

  1. DNA synthesis began 12 hours from end of division

  2. synthesis took 6 hours

  3. entry into prophase another 8 hours

Conclusion from this

• Cell cycle consists of

  1. Pre-S-phase (Gap1)

  2. S phase (period of DNA synthesis)

  3. pre-mitotic (Gap 2)

  4. cell division

HOw do human cells proliferate

• duplicate in 24 hours

• last 1 hour is for M phase

  • segregates chromsomes

  • contractile cytokinesis ring

Cell growth vs stepwise processes

• Cell growth is continuous

  • syntehsis of new ribosomes

  • membranes

  • ER

  • most cellular proteins

• Step wise processes

  • only happen once

  • DNA replication, centrosome duplication, chromosome segregation

The eukaryotic cell cycle

Growth 1:

• Gap 1

Chromosomal replication:

• S phase

Growth 2:

• Gap2

Chromosomal segregation:

• M phase

Why are there two growth phases inbetween

Growth phases are in between the S and the M to prevent the clashes and make sure the growth is a continuous process

How is cell cycle controlled?

Respond to

• External cues:

  • signals, nutrients

• Internal cues

  • ‘ has critical size been reached?’

  • ‘are chromosomes fully replicated and intact?’

Can also exit and return to different signals

• exit: terminally differentiated cells

• exit but return: repair tissue damage or replenish a specific cell pool

Research 1 into finding info about the cell cycle regulation:

Cell fusion experiments

Outline of the experiments

• Cell at different stages of the cell cycle

  • pre-synchronised

• fused together

• to form heterokayrones

• see what cycle phase they went into next

Experiment 1: Mitotic cell + any interphase cell

Observation:

• heterokayons interphase nuceli forced into nuclear envelope breakdown

• condense their chromsomes

Conclusion:

• M phase state was dominant over all other states

  • must contain active factor

  • which direct cells to go straight into mitotic state

Experiment 2: S phase and G1 phase cell

Observation 1:

• G1 nucleus began DNA replication

Conclusion:

• Presence of S phase inducer

• shared by the S phase cell upon fusion

Observation 2:

• heterokayron could not enter M phase until G1 nucleus completed replication

Conclusion:

• feedback control that prevents a cell engaged in ongoing DNA replication to proceed into M

Experiment 3: S phase + G2 cells

Observation:

• S phase nucleus continued replication

• G2 could not induced S nucleus to start DNA replication

Conclusion:

• must be a block to re-replication

  • after completetion of DNA replication in the given cycle

Limitation with this research

• Hinted at a molcular factor that induced things going into mitosis

but

• provide no way forward in finding these molecules that were repponsible

• no insight into the coordination between continuous stepwise processes

Conclusion of this research

• mitotic state is dominant over all interphase states

• Dependency relationships restrict events to occur only once per cell cycle

To help get a deeper dive into the molecular control- look at different cell cyles

• Early embryonic cell cycle

• Somatic cell cycle

Early emrbyoninc cell cycle features

• specialised for rapid and synchronous cell division without cell growth

  • rounds of DNA replication alternate with rounds of chromosome segregation

• what cell it forms?:

  • smaller cells in successive cell cycles

• Stop points?

  • natural arrest points and synchronous division

    • may be exploited

How can they do this?

• massive maternal stockpiles of cellular transcripts and factors

  • promote fast cell divisions

  • cleaving embryo

Popular model for embryonic cells

• frog eggs

• marine invetebrate eggs

• fruit fly syncytial embryo

The somatic cell cycle features

• Canonical cell cycle: G1, S, G2, M

• Control?

  • prominent control of these stages

  • can be synchronised by drugs

• What cells form?

  • cell size homeostasis is reached

  • due to cells cooridanted growth and division

Models for somatic cell division:

• Most other cells other than embryonic

• Also:

  • fission and budding yeast

INn fission and budding yeast

Morphology may be correlated with cell cycle position

(right = fission yeast)

Left = fission yeast= bud emergence is an important landmark

TO distinguish stages of the animal cell cycle

New markers developed in recent years:

• distinguish cells through interphase

• not morphological markers

These two cell cycles can be used to investigate different things

1. Biochemical approach

   • identify activities that correlate with M-phase state

2. Genetic approach

   • mutations that block the cell cycle without affecting cell growth

Both used to ultimately find the master regulator of the eukaryotic cell cycle

Wht to use for the Biochemical approach

Embryonic Cell Cycle:

Why?

• specialised for rapid and synchronous division

• (exhibiting natural blocks)

• Easy to purify

• east to characterise

• can be induced to divide syncrhonously

• can easily be microinjected

How used?

• time course analysis of extract for activities driving cells into M phase

  • easy to characterise and purify

What cycle type if used for the genetic approach

Somatic cell cycle:

Why?

• genetically tractable

• cells displaying prominent control points

• so lots of control points to explore and find the genetics/ proteins invovled!

How used?

• screen for mutants that disable transit across control points

• isolate corresponding genes

  • by complementation

  • quite easy!

Research 2: Biochemical experiments with Frog eggs- how do the cells divide?

INn meiosis

Finding the activity that correlate with the M-phase state

Experiment

• transferred from cytoplasmic contents from mature oocytes into immature oocyctes

  • the mature oocyctes has matured coz they were injected with progesterone

Observation:

• drove maturation of the immature oocytes

Conclusion:

• a factor from the donor oocyte forced the transition into meiosis

• even without the progesterone!

But just to check you havnt just tranferred the injected progestrone from before:

• Repeat many times to more immature oocytes

• RESULT: still matured the cells!

Overall conclusion

• component that promotes maturation was from donor egg

  • NOT from carry-over of progestrone

• This activity was called maturation promoting factor

Maturation promoting factor (MPF)

• induces oocyte maturation (meiodid) even if protein synthesis is blcked

• ALSO induces mitosis in fertilised eggs

  • DESPITE being blocked in interphase by protein synthesis inhibitors

Link between past cell fusion experiments?

MPF dominant activity similiar to the ‘dominant state’ of the M Phase from fusion experiments

What was needed to assay the MPF activity

cell-free system was developed

• possible to undertake its purifation and eludicate its composition!

Research 3: Trying to find the components of the MPF

Experiment:

• Fertilized eggs + 35S-methionine

• sampled at set intervals

• Anaylsed the patterns of radiolabbeled proteins being made

Results:

• Most proteins increased continuously

  • consistent with continuous growth

• BUT: ‘cyclin’ behaves differently

  • disappreaed at the end of each mitosis

  • reappeared during the next interphase A

Conclusion:

• Matched the activity of MPF

• cyclin must be a component or activator of MPF

• destruction of cyclin leads to M phase exit

Research 4: Actually trying to find thse components: Genetic approach

Using somatic cells!

Features of yeast somatic cell cycles for this research

Budding yeast:

• critical size: at G1

Fission yeast

• Critical size at G2

These features can be used to easily find know which stage the yeast is at in the cycle.

Why budding yeast is useful to use

• easy to isolate mutants

  • easily find corresponding genes

• model for somatic cell cycle control

• morphology (see last slide)

  • diagnostic of the position in cell cycle

what is the START of the cell cycle in late G1 of budding yeast?

Decide between proliferation or arrest

• point of no return in the cell cycle

  (conditions only evaluated again in the subsequent cycle at START)

Dependent on?

• Various cues

• Critical size reached

What is triggers?

• Bud emergence

  • convenient morphological marker

  • indicating the cells have gone past this control point

Various cues: divide mitotically

• Nutrients

• size

  → divide mitotically

Various cues: stop dividing and mate

• Mating partners

  • if diploid

→ stop dividing and mate

Various cues: enter meiosis and sporulation

• non-fermentable carbon source

• no Nitrogen source

  • if diploid!

→ enter meiosis and sporolation

Why are isolated mutants used to find out about the cell cycle

• Essential functions can be identified genetically

• By screening for conditional mutants

  • e.g temperature sensitive mutants

What temperature sensitive mutants do

• generally encode gene products that are

  • Active @→ permissive temperature

  • Inactive @→ restrictive temperature

cdc mutants

cell division cycle mutants

• isolated and characterised independently first in

  • first in budding yeast

  • then in fisssion yeast (pombe)

Why are cdc mutants useful

Arrest with uniform morphology at restrictive temperature

• i.e can see exactly at which part of the cell cycle the mutant is affecting

e.g cdc20ts

1. at first asynchronous proliferation at

   • at permissive temperature

2. shift to restrictive temperature

3. arrest with uniform morphology

   • each cell reaching the point of the block

   • due to mutant (e.g cdc20ts)

   • unable to to proceed past it

4. really useful just to change temperature to see a change

   • easy to see that the protein the gene encode is for

(research 4): How used to find out about START

1. many different cdc mutants characterised

2. functional map of which parts of the cycle they affect

3. found that cdc28^ts→ Prevents cells from proceeding past START

   • at the restrictive temperature

4. What this means?: the CDC28 gene product must be needed for START

Finding the protein once the gene was found (CDC28ts)

1. CDC28 cloned by complementation

   • bit like DNA fingerprinting?? or probe thingy?

2. Found it codes for ~34kDa protein

   • similar to protein kinases

   • Ser/Thr Kinase activity was demonstrated

How CDC28 isolated by complementation

1. Take the cells which have the mutants

2. These cannot proliferate at restrictive temperature

3. Transform with a yeast genomic library

   • plasmid mix with random inserts of yeast genomic DNA

4. Grow on restrictive temperature

5. The ones that can grow must be the ones that have receieved a CDC28 plasmid (which we don’t really know yet!)

6. Take these cells and analysis which plasmid they got

7. Sooo now we know which gene the mutant had come from

8. Can now try figure out what this gene encodes

So now we know a bit more about the START

How are fission yeast useful for studying the cell cycle

• rod-shaped cells

  • grow in length

  • divide symmentrically

• BUT: no morpholigcal marker to show START progression

• must reach critical size instead at M phase

  → so very useful in finding out about the M phase transition point!

Two mutants found to affect M phase transition

1. wee1

2. cdc2^ts

What does wee1 tell us?

• Mutant→ cells divide at smaller size

  • So M phase must be starting sooner

  • Before it has grown to critical size

  • incorrect timing

THEREFORE:

• wee1 gene must code for something that ensures the cell waits till grown

  • i.e stops the M phase progressing

What does the cdc2ts mutant tell us

@ restrictive temperature

• M phase is blocked

• BUT

• Continues to grow

  • Become very long cells

THEREFORE: cdc2 is essential for progression into M phase

• protein is essential for mitotic entry

Interaction of cdc2 and wee1

• Wee1 is an inhibitor of cdc2

  • coz cdc2 wants M phase

  • but wee1 stops it

• In wee1- mutant: cdc activation is advanced

  • cells really want to go into M phase

What about the START in fission yeast (pombe?)

• Found that cdc2+ in pombe corresponds to the CDC28 in cerevisiae (budding)

THEREFORE:

1. cdc2+ is needed for START and M phase in fission yesat

2. cdc2+ and CDC28 and functional homolgoes

   • can replace CDC28 with cdc2+ and vise versa

What about in humans?

CDC2Hs

• Human homologue

• identified by screening a human cDNA expression library

• in an S.pombe cdc2ts mutant

What this shows

• Budding and fission yeast are as evolutionary apart from eachother

• as much as yeast from humans

OVERALL protein p34 from both these genes

A key cell cycle regulator

• CONSERVED throughout evolution

NOW: how to link these gene protein products to the MPF we have already found (linking biochemical and genetic approaches)

1. Cdc2

   • Antibodies raised against cdc2 cross-react MPF

   • Found: crosses with the 32kDa subunit of MPF

     • therefore cdc2→ 32kDa polypeptide

2. Cyclin B

   • corresponded to 46kDa polypetites

What have we overall found about MPF

1. Prototype of an evolutionary conseved family of:

   • Ser/Thr protein kinases (‘cyclin dependent kinase (CDK)’)

2. CDK are made of

   • Cdk: catalytic component

   • Cyclin: regulatory component

THERFORE: MPF = CDK (cyclin-dependent Kinase)

How do Cdk and cyclin interact

1. cyclin binds to the Cdk

   • cyclin activates cdk

   • controls its temporal and spatial function

2. forms a kinase

3. phosphorylates the stuff for M phase

   • S and T residues within the [S/T]PX[K/R sites

   • this substrate specificity is controlled too!

4. cyclin is phosphorylated with phospatases

5. Cyclin GONE→ no more M phase

THEREFORE: regulation

How does cyclin regulate cdk

• activates cdk

• controls temporal and spatial function

• influences CDK substrate specificity!