SYNCHRONISATION + TEMPERATURE-SENSITIVE MUTANTS

What is synchronisation in cell cycle studies?

A method to force a population of cells into the same cell cycle stage

Why do scientists synchronise cells?

To study how proteins

Name one use of yeast synchronisation

Measuring fluctuations in protein expression

Name another use of yeast synchronisation

Mapping phosphorylation timing or protein localisation

How can synchronisation reveal protein interactions?

Cells arrested at specific stages can be immunoprecipitated and analysed

What major organism is used for synchronisation research?

S. cerevisiae (budding yeast)

Why is budding yeast useful?

Easy to culture

Name three methods of yeast synchronisation

Centrifugal elutriation

What is centrifugal elutriation?

Cell separation by size using spinning and fluid flow

Why does elutriation synchronise yeast?

Smaller newborn daughter cells are enriched and stall in G1

What is a disadvantage of elutriation?

Requires special equipment and gives only short synchrony windows

What defines temperature-sensitive mutations?

Alleles that function normally at permissive temperatures but lose function at restrictive temperatures

Why are ts mutants valuable?

They allow reversible

What happens at permissive temperature?

Mutant proteins behave normally

What happens at restrictive temperature?

Proteins misfold or lose activity

Name one commonly used ts mutant

cdc28(ts)

Why does cdc28(ts) block the cycle?

Cdc28 is the primary CDK; without function no cell cycle event occurs

What phase does cdc28(ts) arrest in?

G1

What processes fail in cdc28(ts) mutants?

Budding

Do cdc28(ts) cells still respond to mating pheromone?

Yes

What happens to haploid cdc28(ts) mutants?

They can mate even though they cannot enter G1 properly

What happens to diploid cdc28(ts) mutants?

They can sporulate under starvation because meiosis bypasses G1 demands

Why is that surprising?

Shows meiosis and G0 entry decisions branch before START

What happens to cdc7(ts) mutants?

Cells cannot execute DNA replication and cannot enter G0

Why can cdc7(ts) mutants not mate?

cdc7 functions after START

Why is cdc7(ts) useful?

Distinguishes pre- and post-START control

What is a chemical synchronisation method?

Hydroxyurea to deplete dNTPs and block S phase

How does hydroxyurea arrest cells?

Prevents DNA synthesis by inhibiting ribonucleotide reductase

What is a drawback of using hydroxyurea?

It stresses cells and alters metabolism

What is α-factor arrest?

Use of pheromone to arrest haploid cells in G1 before START

What mating type responds to α-factor?

MATa cells

Why does α-factor arrest only haploids?

Diploids express both mating alleles and are insensitive to pheromone

What happens when α-factor is removed?

Cells re-enter the cycle synchronously

Why is synchronisation never perfect?

Cells vary in age

Why is synchrony transient?

Cells immediately begin drifting into different phases once block is removed

Why are synchrony windows short?

Cyclins and checkpoints rapidly reintroduce variability

What should be avoided when studying synchronised cells?

Long-duration assays that blur population synchrony

What is the goal of blocking and release experiments?

Follow ordered events from a defined moment in the cycle

What property of yeast supports easy synchronisation?

Asymmetric budding — daughter cells differ from mothers

How do researchers monitor synchrony?

Microscopy of bud morphology and markers of DNA content

What tool quantifies synchronisation?

Flow cytometry measuring DNA per cell

Why are ts mutants better than drugs?

They give cleaner arrest

What protein feature makes ts alleles temperature-sensitive?

Mutations destabilise tertiary structure

How is restrictive temperature chosen?

Just high enough to destabilise mutants without killing wild-type cells

Why should ts strains be grown carefully?

Slow growth at permissive temps can alter physiology

Can ts mutants be used in mammals?

No — ts studies are primarily used in microbes and yeast

What happens if a ts mutant is kept too long at restrictive temperature?

Secondary stresses trigger alternative pathways and stress responses

How do synchronisation methods complement each other?

Elutriation enriches G1 cells; α-factor arrests new daughters; ts mutants allow precise halts

What happens if all START cyclins are defective?

Population accumulates before START similar to cdc28(ts)

Why is synchronisation essential for mapping phosphorylation?

CDKs fire sequentially

What type of proteins are most informative in synchronised experiments?

Cyclins

How does synchronisation support discovery of MPF?

Early oocyte studies relied on uniform timing of mitotic entry

Why is synchronisation more precise in yeast than mammals?

Yeast cycle is faster

How do synchronised cultures contribute to drug screening?

Allow testing effects on specific checkpoints

What experiment validates synchronisation quality?

Measuring two independent markers — e.g.

What happens when synchronised diploids starve?

They divert from mitosis to meiosis + sporulation

How does synchronisation reveal branching fates?

Cells blocked at different points choose G0

What general lesson comes from ts mutants?

Checkpoint position determines available fate options