Module 9: Cell Cycle Regulation

What are the main phases of the cell cycle?

• G1: Cell grows, gene expression, protein synthesis

• S: DNA replication → sister chromatids formed

• G2: Prepares for mitosis

• M: Mitosis → divides chromosomes into daughter cells

• G0: Quiescent state (non-dividing)

Why is cell cycle regulation important?

• Ensures DNA replication and division alternate properly

• Prevents cell death or over-proliferation (e.g., cancer)

• Most cells are in G0, not actively dividing

What can go wrong with poor regulation?

• Cell death if replication/division fail

• Over-proliferation → cancer

• Tissue cannot be repaired if division stops

What are the possible fates of a stem cell entering the cell cycle?

• Self-renewal → 2 identical stem cells

• Enter G0 temporarily or permanently

• Begin differentiation → specialized cell (e.g., neuron, RBC)

Why is balance between division and differentiation important?

• Too much division → tumors

• Too much differentiation → loss of regeneration

What are the phases of mitosis?

• Interphase (G1, S, G2): Prepares for mitosis

• Prophase: Chromosomes condense, centrosomes separate. mitotic spindle assembles

• Prometaphase: Chromosomes attach to spindle via kinetochores

• Metaphase: Bipolar attachment, chromosomes align at equator

• Anaphase: Sister chromatids pulled apart

• Telophase: Chromosomes decondense, nuclear structures reform, mitotic spindles disassemble

• Cytokinesis: Cell membrane pinches → two daughter cells

What labels are used in imaging mitosis?

• DAPI = DNA (blue)

• Anti-beta-tubulin antibody = spindle microtubules (green)

What are the key events during mitosis shown in the animation?

• Prophase: Chromosomes condense → 4n; centrosomes move to poles

• Prometaphase: Nuclear envelope breaks down; kinetochores attach to microtubules

• Metaphase: Chromosomes align at equator via kinetochore microtubules

• Anaphase: Sister chromatids pulled to opposite poles

• Telophase: Nuclear membrane reforms; chromosomes decondense

• Cytokinesis: Contractile ring (actin + myosin II) → 2 identical daughter cells (2n)

What two classes of proteins regulate the sequence of mitosis?

• Cyclin-dependent kinases (CDKs):

  • Heterodimers (CDK + cyclin)

  • Phosphorylate target proteins → trigger cell cycle events

• E3 ubiquitin ligases:

  • Target proteins for degradation via proteasome

  • Remove cyclins or inhibitors at key checkpoints

What are the four major Cyclin-CDK complexes in the cell cycle?

• G1 Cyclin-CDK: Active in G1 → prepares for S phase

• G1/S Cyclin-CDK: Transitions G1 to S phase

• S-phase Cyclin-CDK: Initiates DNA replication

• Mitotic Cyclin-CDK: Regulates prophase & mitotic changes

What is common across all Cyclin-CDK complexes?

• Same structure

• Same kinase activity

• Differ in targets and timing

What are the 3 major E3 ligase complexes and their functions?

• SCF complex:

  • Promotes G1 to S phase transition

• APC-Cdc20:

  • Regulates metaphase to anaphase transition

• APC-Cdh1:

  • Mediates exit from mitosis

APC (anaphase promoting complex)

What do Cdc20 and Cdh1 do?

• Accessory proteins that determine APC’s target specificity

What are the 3 main targets of G1 Cyclin-CDK?

1. APC-Cdh1:

   • Phosphorylation signals mitosis completion

2. Transcription factors:

   • Activated to express S-phase genes (e.g., DNA polymerase)

3. S-phase inhibitors:

   • Phosphorylation → target for SCF ligase → degraded → S-CDK activated → initiates DNA replication

What are the functions of G1/S Cyclin-CDK?

1. Activating mitosis related genes

   • Activates transcription of mitotic genes (e.g., M-phase cyclins)

2. Preparing centrosomes

   • Phosphorylates proteins involved in centrosome replication, an important part of forming the mitotic spindle

What are the functions of S-phase Cyclin-CDK?

1. Triggering DNA replication

   • Activates pre-replication complex at origins of replication on the DNA

2. Preventing over-replication

   • Phosphorylated proteins to ensure that each origin fires only once per cycle

3. Delaying mitosis if DNA isn’t ready

   • Phosphorylates M-phase CDK to inhibit it until DNA replication is complete

What does M-phase Cyclin-CDK phosphorylate during prophase?

• Chromosomal proteins → condensation

• Nuclear lamins → envelope breakdown

• MAPs → spindle assembly

• Kinetochore proteins → chromosome-spindle binding

• APC complex → mitotic progression

When does protein ubiquitination/degradation occur in mitosis?

1. Anaphase onset: Anaphase inhibitors degraded

2. Mitotic exit: Mitotic cyclins degraded via MEN (mitotic exist network)

What is MPF and how was it discovered?

• MPF (Maturation/Mitosis Promoting Factor) discovered in frog eggs (Xenopus)

  • Induces meiosis completion & 11 mitotic divisions → forms blastocyst → further cell division + differentiation into tadpole

  • Identified by Masui & Markert, 1971

• Later shown to be M-phase Cyclin-CDK

Why use synchronized embryos in cell cycle research?

• High synchrony makes it easier to isolate and study cell cycle regulators biochemically

What did Tim Hunt and Joan Ruderman discover using sea urchin embryos?

• Identified cyclins: proteins with cyclic synthesis/degradation

• Used radiolabeled proteins and gel electrophoresis

• Observed Cyclin B levels oscillate with mitotic activity

Graph:

• Pink line = how much Cyclin B was present over time.

• Blue line = how many cells were in mitosis at that time.

Correlation between cyclin levels and mitosis:

• ↑ Cyclin B → ↑ cells in mitosis

• ↓ Cyclin B → ↓ mitotic cells

• Showed Cyclin B regulates M-phase Cyclin-CDK activity and directly correlates with whether or not a cell is dividing.

What did live imaging of Cyclin B in HeLa (human) cells show?

• Cyclin B present during interphase & early mitosis

• Rapid drop in Cyclin B during anaphase

• This drop in Cyclin B is a key signal for the cell to exit mitosis and finish division

Why was it surprising that cyclin regulates the cell cycle?

Cyclin has no enzymatic activity, unlike MPF (a kinase)

• Big Question: How could a non-enzymatic protein like Cyclin B control something as important as the cell cycle?

What did Andrew Murray’s in vitro experiment involve?

• Used egg extracts (mRNA + proteins for cell division)

• Measured:

  1. MPF activity - via histone H1 phosphorylation

  2. Cyclin B levels - via gel

  3. Mitotic behaviors - observed by adding sperm nuclei into the extract to see if they behaved like they do in real cells (e.g., chromosome condensation, nuclear envelope breakdown)

What happened when sperm nuclei were added to egg extracts?

• Cyclic mitotic behaviors occurred:

  • Condensation of chromosomes and breakdown of the nuclear envelope (typical of early mitosis).

  • Decondensation and reformation of the envelope (typical of late mitosis).

• Synchronized with ↑/↓ Cyclin B and MPF activity:

  • ↑ Cyclin B levels = ↑ MPF activity increased → mitosis started.

  • ↓ Cyclin B levels dropped = ↓ MPF activity decreased → cells exited mitosis.

What did RNase-treated extract experiments show?

• No mRNA (but tRNA and sRNA for protein synthesis intact) → No new Cyclin B made → No MPF activity

• Conclusion: Cyclin B is necessary for MPF activation and mitosis

What happens when Cyclin B mRNA is added to RNase-treated extract?

• Restores synchronized cycling behaviors

• Only Cyclin B is synthesized

• Cycling CDK activity and mitosis behaviors return

• Conclusion: Cyclin B is sufficient to restart the cycle, even if it’s the only new protein made.

What happens when nondegradable Cyclin B mRNA is added?

• Cyclin B levels stay high

• CDK activity remains high

• Mitotic arrest occurs

  • The chromosomes condensed but never decondensed, and mitosis didn’t finish

What do these experiments show?

• Cyclin B is necessary for CDK activation

• Its degradation is needed to complete mitosis

What did microscopy results show for degradable vs. nondegradable Cyclin B mRNA?

Degradable:

• Chromosomes: anaphase → telophase

• Spindle: assembles/disassembles

Non-degradable:

• Chromosomes: undergo anaphase but fail to decondense

• Spindle: fails to disassemble

  Conclusion: Cyclin B degradation is required to exit mitosis

What complexes degrade Cyclin B and the outcome?

1. APC-Cdc20: Activated at anaphase, it begins Cyclin B degradation.

2. APC-Cdh1: Takes over after anaphase to finish degrading Cyclin B, allowing the cell to exit mitosis and reset.

Outcome:

• CDK inactivation

• Cell exits mitosis

What sequence allows APC-Cdc20 to recognize Cyclin B and its significance?

Destruction box (D-box) near N-terminus: RxxLxxxxN/Q

• Position 1: Arginine

• Position 4: Leucine

• Position 9: Asparagine/Glutamine

Significance:

• Necessary: mutations prevent degradation

• Sufficient: adding it to another protein (GFP) causes cyclical degradation

What happens when D-box peptide is added to in-vitro extracts?

• Low amounts of D-box peptide caused delays in mitosis.

• High amounts completely blocked cells in metaphase—they couldn’t move into anaphase.

Why?

• Because APC was so busy binding the excess D-box peptides, it couldn't bind and degrade a real protein needed to progress.

• This suggested APC has a second critical target: an anaphase inhibitor.

What is the anaphase inhibitor?

Securin

• Sister chromatids are held together by a cohesin complex (Smc1, Smc3, Scc1).

• A protein called separase can cut Scc1 to start chromatid separation—but it’s kept inactive by securin.

• When APC-Cdc20 is activated, it degrades securin.

• This frees separase, which cuts Scc1, a key component of the cohesin ring.

• Once cut, the chromatids separate and anaphase begins.

So, APC-Cdc20 is essential to initiate anaphase by removing the block (securin) on chromosome separation.

How is securin targeted for degradation?

• By APC-Cdc20

• Cdc20 acts as specificity factor for APC

Additional note

• APC subunit is phosphorylated by CyclinB-CDK, prepping for anaphase

How is Cyclin B degraded at mitotic end?

1. APC switches specificity:

• After anaphase, APC changes its regulatory subunit from Cdc20 → Cdh1.

2. APC-Cdh1 targets Cyclin B:

• During telophase, APC-Cdh1 ubiquitinates Cyclin B, marking it for proteasomal degradation.

3. Inactivation of CDK:

• Cyclin B degradation → inactivates CDK (M-phase kinase).

• This allows the cell to exit mitosis and enter G1 phase.

4. APC deactivation in G1:

• Without Cyclin B-CDK activity, phosphatases dephosphorylate APC → APC is inactivated in G1.

5. Cyclin B initiates its own end:

• Interestingly, Cyclin B-CDK is required to activate APC at mitotic entry.

• So, Cyclin B helps trigger the system that eventually leads to its own destruction.

What is SCF and its function?

• E3 Ligase SCF = Skp, Cullin, F-box protein complex

• Active in mid-G1 phase

• Targets S-phase inhibitor Sic1

• Sic1 inhibits S-phase CDK until the cell is ready

• G1-CDK phosphorylates Sic1 → SCF recognizes and ubiquitinates Sic1 → Sic1 degraded by proteasome

• S-phase CDK is activated → cell enters S-phase

• Ensures irreversible, one-way progression through cell cycle

What regulates progression through the cell cycle?

• CDK-cyclin complexes → phosphorylation

• E3 ligases (APC, SCF) → degradation of regulators

Animation Summary

• Early G1:

  • DNA prereplication complexes dephosphorylated

  • Assemble at replication origins

• Late G1:

  • G1-CDKs synthesized → activate transcription factors

  • Induce expression of S-phase CDK components

  • S-phase CDK blocked by inhibitor (e.g., Sic1)

• Start of S phase:

  • G1-CDK phosphorylates inhibitor → degradation

  • S-phase CDK activated

  • Triggers DNA replication (1 round only)

  • Cohesins hold sister chromatids together

• S phase & G2:

  • Mitotic CDKs produced, but kept inactive

• M phase:

  • Mitotic CDKs activated → initiate mitosis

  • APC activated → degrades cohesin regulators

  • Allows chromatid separation (anaphase)

• End of M phase:

  • APC degrades mitotic CDKs

  • Cytokinesis completes → new cell cycle starts

How are novel cell cycle regulators identified?

• Use of genetic screens (unbiased approach)

• Random mutations created across genome

• Screen for cell cycle phenotypes:

  • Inhibited division

  • Excessive division

What is a temperature-sensitive (TS) mutation and how is it used?

• TS mutation = protein functional at low temp (24°C), misfolds at high temp (37°C)

• Enables on/off control of protein function

• Mutant cells: grow at 24°C, but not at 37°C

• Wild-type cells: grow at both temperatures

What is Schizosaccharomyces pombe and why is it used?

• A model organism: fission yeast (S. pombe)

• Elongated, rod-shaped cells

• Used to study cell cycle and division mechanisms

What does the movie of fission yeast show?

• Fluorescent DNA labeling

• Shows nuclear division → followed by cytokinesis

What are the two main phenotypes of cdc mutants?

• Elongated phenotype: G2 delay, continued growth

• Wee phenotype: Early mitosis entry, smaller cells

What do different cdc2 mutations cause?

• Wild-type (cdc2⁺) → normal division

• Loss-of-function (cdc2⁻) → elongated phenotype

• Gain-of-function/dominant (cdc2ᴰ) → wee phenotype

What is the role of Cdc2 protein in the cell cycle?

• Cdc2 promotes mitotic entry and cell division

• Loss → no division (elongated)

• Gain → early/frequent division (wee)

• Cdc2 = CDK of MPF

• Is a 34 kDa protein with kinase activity, forms heterodimer with Cdc13 cyclin

What is the role of Cdc13 in S. pombe?

• Regulates MPF activity

• Loss → elongated; Gain → wee

• Oscillating concentration during cell cycle

• Cyclin B homolog (Xenopus)

• Forms Cdc2–Cdc13 complex = MPF

• Only one CDK (Cdc2) and one cyclin (Cdc13) in S. pombe

  • Functions as all CDKs (M-phase, S-phase, G-phase)

What does Cdc25 regulate, and how?

• Loss (cdc25⁻) → elongated

• Gain (cdc25ᴰ) → wee

• Cdc25 = activator of MPF

• Promotes entry into M-phase

What does Wee1 regulate, and how?

• Loss (wee⁻) → wee phenotype

• Gain (weeᴰ) → elongated

• Wee1 = inhibitor of MPF

• Delays M-phase entry

• Opposes Cdc25 function

What are the roles of Wee1 and Cdc25 in MPF regulation?

• Wee1: Tyrosine kinase → adds inhibitory phosphate (Tyr 15 on Cdc2)

• Cdc25: Phosphatase → removes Tyr 15 phosphate → activates MPF

• Balance between Wee1 and Cdc25 controls mitotic entry

How is MPF activity regulated by phosphorylation?

• Cyclin binding to CDK (Cdc2) → required for activity

• Wee1 kinase → phosphorylates Y15 (inhibitory) → MPF off

• CAK kinase → phosphorylates T161 (activating) → not enough alone

• Cdc25 phosphatase → removes phosphate from Y15 → MPF fully activated

• Double mutant (no Wee1 & Cdc25) → slow division shows CAK alone works, but less efficient and synchronized

• Multiple regulators = tight, efficient cell cycle control

What is a key difference in budding yeast (S. cerevisiae) compared to fission yeast?

• Forms daughter bud in G1, before S-phase

• Same cell cycle regulators in S. cerevisiae as S. pombe

What does the Nomarski microscope movie show?

• Budding yeast cells dividing

• Visualizes cell morphology changes during division

What phenotype is caused by cell cycle mutations in S. cerevisiae?

• Arrest in G1

• Daughter bud forms, but no S-phase

• Cdc28 = homolog of Cdc2 in fission yeast

• Single CDK controls cell cycle in both yeasts

• Phenotypes differ slightly despite functional homology

What is functional complementation and how is it used?

• Technique to identify gene that rescues mutant phenotype

• Wild-type gene introduced into mutant cells

Step 1: Start with a temperature-sensitive (TS) mutant

• At 25°C (permissive): cells divide normally

• At 35°C (restrictive): cells arrest in G1 due to a mutation in an unknown gene

What is the purpose of using a cDNA library in functional complementation?

Step 2: Add genes from a cDNA library

• A cDNA library contains DNA versions of expressed genes (no introns), made from mRNA using reverse transcriptase

• This library is introduced into the mutant cells, one gene at a time, to see if any restore normal function

• Screen each cDNA at restrictive temperature:

  • Gene X/Y → no rescue

  • Gene Z → rescues cell division = likely wild-type of mutated gene

What does isolating gene Z reveal?

Step 3: Identify Gene Z

• Gene Z is carried in a plasmid in bacterial cells

• Extract plasmid → sequence the cDNA

• Result: Gene Z = Cdc28 in budding yeast

  • Cdc28 codes for CDK1 protein = same as Cdc2 in fission yeast

  • Functional complementation shows conserved cell cycle genes across species

What does Cdc28 do in budding yeast?

• Cdc28 = single CDK in S. cerevisiae

• Binds to cyclins to regulate cell cycle

• G1/S cyclins: Cln1, Cln2, Cln3 → form SPF (S-phase promoting factor)

• M-phase cyclins: Clb1, Clb2 → form MPF (mitosis promoting factor)

• CDK + Cyclin = active heterodimer → phosphorylates targets

Are cell cycle regulation mechanisms conserved across species?

• Yes, conserved in all eukaryotes

• Cyclin-CDK complexes perform similar roles in each phase

• Regulatory enzymes (kinases, phosphatases) also conserved

• Vertebrates: multiple CDKs + multiple cyclins, but homologous functions

Applied Lecture

Mitotic Spindle Dynamics

What are the key components of the mitotic spindle and their functions?

• Metaphase

• Green = Microtubules

• Blue = DNA

• Red = Kinetochores (anchor centromeres to microtubules)

• Cells use microtubule dynamics + motor proteins to build spindle

How are microtubules dynamic and polar?

• Alpha-tubulin: always bound to GTP (-) end

• Beta-tubulin: can hydrolyze GTP → GDP (+) end

• GTP-β-tubulin → promotes growth

• GDP-β-tubulin → promotes shrinkage

• Subunits added at (+) end (β-tubulin exposed)

• Growth/shrinkage critical during cell division

What is the basic structure and behavior of microtubules?

• Made of 13 protofilaments

• Undergo assembly/disassembly

• Dynamic behavior varies with cell cycle phase

• Both growth & shrinkage occur during mitosis

What is the direction of movement for motor proteins in cell division?

• Motor proteins = essential for mitosis

• Dynein: moves toward (–) end to cell interior

• Kinesin: moves toward (+) end to cell membrane

• Both have head domain, coiled coin, and tail domain for cargo

What are the key types and functions of kinesins?

• Kinesin-1/2: Organelle transport (2 ATP heads + cargo tail)

• Kinesin-5: Bipolar, slides MTs apart (4 ATP heads)

• Kinesin-13: No motor activity, promotes end disassembly

  • Usually seen in (-) end, sometimes seen in (+) end

What do microtubules do during mitosis?

1. Prophase: form bipolar spindle

2. Prometaphase/Metaphase: attach chromosomes

3. Anaphase: separate chromatids

• Motor proteins assist in all stages

What are MTOCs and their role in spindle formation?

• Centrosome = main MTOC (near nucleus in interphase)

• MTs grow from + end, anchored at – end in MTOC

• Centrosomes duplicate before mitosis → form spindle poles

How do centrosomes help assemble the spindle?

• Each centrosome has 2 centrioles (mother and daughter perpendicular)

• Around the centrioles is a cloudy area called the PCM (pericentriolar material) — this is where microtubules (MTs) grow from.

• The PCM contains γ-TuRC (gamma-tubulin ring complex).
  → This acts like a "baseplate" that helps start microtubule growth.

• Microtubules start growing from their minus (–) ends in the PCM, and extend outward from their plus (+) ends, forming the spindle.

When and how do centrosomes duplicate?

• Happens during G1/S phase, alongside DNA replication

• Triggered by CDKs + Plk4 kinase

• Centrioles separate → new daughter centrioles bud

• G2 phase: daughter centriole growth completes

How do centrosomes separate during mitosis?

• Triggered by M phase CDKs

• Each centrosome nucleates MTs → becomes a spindle pole

• Centrioles move to opposite sides

• Occurs in prophase, before nuclear envelope breakdown

How does kinesin-5 help separate centrosomes?

• Microtubules nucleate at centrosomes

• Kinesin-5 binds 2 antiparallel MTs

• Uses ATP to "walk" toward + ends

• Sliding action pushes centrosomes apart

What is the model for centrosome separation?

• Kinesin-5 walks toward + ends of MTs

• Slides overlapping MTs apart

• Centrosomes are pushed to opposite poles

What are the 3 types of spindle MTs and their roles?

• Astral MTs – link spindle to cell cortex

• Kinetochore MTs – attach to chromosomes

• Polar MTs – overlap in center; push poles apart (anaphase)

Different locations, same structure.

Cell cortex: thin layer of actin filaments and proteins that underlie the cell membrane

How do kinetochore MTs capture chromosomes?

• Prometaphase: MTs attach to kinetochores at centromeres

• First: monopolar attachment

• Then: bipolar attachment → metaphase plate alignment

What is the role of kinetochore MTs in mitosis?

• Attach to kinetochore protein complexes at centromeric regions of each sister chromatid

• Undergo dynamic growth/shrinkage

• Kinetochores contain proteins to stabilize MT-chromosome connection

  • Dynein motor proteins connected to kinetochore complex + sister chromatid, walk towards (-) end spindle poles

  • Kinesin 13 depolymerizing

How is bipolar attachment achieved?

2 Stages:

1. Kinetochore MT binds one sister chromatid

2. Dynein moves sister chromatid pair to allow opposite MT to bind second chromatid

• Spindle assembly checkpoint monitors attachment + tension

What happens during Anaphase A?

• Kinetochore MTs shorten

  • Kinesin 13 depolymerizing

• Forces at kinetochores pull chromosomes to poles

• Daughter chromosomes move poleward

What happens during Anaphase B?

• Spindle poles move apart

• 2 forces involved:

  1. Kinesin-5 slides overlapping polar MTs apart

  2. Dynein pulls on astral MTs at cortex

• Polar MTs grow at + ends

What key events occur during anaphase?

• Anaphase A:

  • Cohesins cleaved

  • Chromosomes pulled to poles (MT depolymerization at kinetochores by Kinesin-13)

• Anaphase B:

  • Kinesin-5 slides polar MTs

  • Dynein pulls astral MTs

    • Helps move the two spindle poles farther apart stretching the spindle and separating the chromosomes.