Nucleus/Cell Division (18)

Mammalian Red Blood Cells

Has no nuclei (Anucleate), one of three

• Allows for biconcave shape, due to losing nucleus

• Avian and amphibian blood cells are not (circle)

Skin Epidermis

Has no nuclei (Anucleate), one of three

• Regenerative layer (related to EB) cells differentiate

• This causes them to lose their nucleus and flatten

• This forms stratum corneum

• Cytoplasm lost in process and these cells become membrane

• This membrane serves as the body's primary protective barriers.

Lens of the Eye

Has no nuclei (Anucleate), one of three

• Lens epithelial cells at equator of eye differentiate into lens fibers (comprise bulk of lens, the germanative zone)

Nuclear Envelope (tied together)

• 2 membranes

• Connected to the ER, due to how nucleus is connected to mitosis

What is the benefit of a lens without nuclei in the fibers?

• No nuclei in the center of lens

• Light will bend as it can’t scatter off nuclei, allowing for clearer vision.

Nuclear Pores

A complex protein complex

• Covers 50% of envelope

• Pass proteins by diffusion as long as < 60,000 Daltons (ex. histones)

• Play a role in DNA packaging and regulation

Non-Histones

• Proteins that are not histones (can’t pass through nuclear pores)> 60,000 Daltons

• They still assist in various functions related to chromatin structure and gene expression.

Nucleoplasmin

• Found in X.Laevis (African Clawed Toad)

• About 1-% of nuclear proteins

• “First” chaperone protein discovered

Function:

• Gene stability

• Transcriptional Regulation

• It’s a pentamer (really big), 160 Daltons

What does nucleoplasmin require to function, how was this discovered

Goes to nuclear pore

• requires a receptor + ATP to enter

This was figured out by:

• Conjugate nucleoplasmin with gold

• inject into an egg cell

• Cool egg cell down to 4 degrees C

• ATP doesn’t work now

• Can determine if something is ATP dependent

• You can see binding of nucleoplasmin at the nucleopore (confirming it is receptor-mediated)

Lamins (intermediate filaments)

Come in 3 types (A,B,C), are about 60000 Daltons

Lamins make up the karyoskeleton

• Provides structural support to the nucleus, involved in nuclear organization and shape maintenance.

• Connect chromosomes

Nucleus in mitosis

• Nuclear envelope undergoes dissolution “dissolving” and disappears

  • This happens due to MPF

  • This triggers phosphorylation of Lamins

  • Envelope dissembled

Hutchinson-Gilford Progeria Syndrome

Farnesyl - anchors Lamin A onto the nuclear envelope

• Lamin A gene is mutated in this disease and cannot be separated from farnesyl

• Lamin A piles up at the nuclear envelope

Lonafarnib

This drug is a farnesyltransferase inhibitor used to treat Progeria

• However, not an easy drug for kids to take

Howard and Pelc

Came up with cell cycle in 1953

• Came up with evidence of this by looking at a broad bean (plant)

Cell Cycle

Defines cancer, important in embryogenesis (embryo development)

• NO MISTAKES ALLOWED, cells have to divide properly or fixed until they can

• Requires equilibrium with cell death rate

Arthur Pardee

Used 3T3 cells to show that there are growth factors required for cells to move from G1 to S phase

• PDGF

• EGF

• Insulin

  • GF’s are presented in this order

Also discovered that G1 is characterized by:

• Early response genes

  • High mRNA after an hour

• Late response genes

  • High mRNA after early response gene’s activity comes down

• if early response gene mRNA levels are kept high, then there is no late response gene mRNA activity

Also discovered:

• Checkpoint + restriction point regulate the start of cycle (G0 to G1, aka Pardee checkpoint)

• And another restriction point at (G1 to S), this one however is “go or no go point”

Cell Synchrony

Arthur Pardee couldn’t work with cells since they were all at different stages of the cell cycle.

• All cells go through the cell cycle in the same phase

• Done by:

  • Amino acid deprivation – all cells stall in G1

  • Serum deprivation – all cells stall in G1

  • Protein synthesis inhibitors – all cells stall in G1

  • Microtubule inhibitors – all cells stall in M

  • DNA synthesis inhibitors – all cells stall in Sthe longest phase of the cell cycle, making precise timing challenging.

Limited number of transits since G1 phase is most variable in time leading to later cells not synchronizing

Gloucester Marine Genomics Institute (GMGI)

Discovered that Sea Urchins can live long and avoid cancer

• Sea urchins are a cancer resistant model studied by MGMI

• Comparing local sea urchins to red ones that have more tumor suppressors

What controls the cell cycle phases? (Ruderman and Hunt)

Cyclins,

Sea Urchin embryos are an example of a natural cell synchrony system (done by Ruderman and Hunt)

• SDS gel electrophoresis were used to look at protein abundance

• Correlated cyclin A/B abundance with the phases of cell cycle in sea urchins

• Later found that it’s the same in human cell cycle

Cyclin Dependent Kinases

• bind to cyclins to influence the progression through different phases of the cell cycle.

Is cyclin D/CDK required for cell cycle transit?

• Take a G0 cell, and add growth factors to make it G1

  • Add ³H-thymadine (radioactive precursor) and do autoradiography

  • OR, use BrdU, a fluorescent dye that is similar

Experiment:

• G₀ cells given growth factors to induce cell cycle re-entry

• Microninject anti-cyclin D antibody into one group

• Add BrdU to detect DNA synthesis

• Measure % of BrdU-positive (S-phase) cells after 16 hrs

Conclusion:

Cyclin D is required early (10–14 hrs) for cells to enter S phase after growth stimulation.

• Without cyclin D (antibody group), cells fail to enter S phase (low BrdU at 10–14h).

• By 16h, BrdU uptake restored → cyclin D function complete by then.

Are cell cycle transit times the same in normal cells versus cancer cells? (Cell cycle kinetics)

G1 Transit time - Synchronize cells so all are in G0 and then
add 3H-thymidine. Look for first appearance of radioactive
cells.

S Transit time – Use randomly cycling cells and add 3H-
thymidine. Count percent in S and multiply by total cell cycle
time

G2 Transit time – Use randomly cycling cells. Add 3H-
thymidine for 30 mins and then look for radioactive M cells

M Transit time – Use randomly cycling cells. Count percent
in M and multiply by total cell cycle time

Conclusion - No difference between cancer vs normal cells

G0 phase

• quiescent (quiet) period

• Differentiated cell without the intention of dividing

G1 phase

9± hours of a 24 hour cell cycle period

Growth factors required for cells to move from G1 to S phase

• PDGF

• EGF

• Insulin

  • GF’s are presented in this order

G1 is characterized by:

• Early response genes

  • High mRNA after an hour

• Late response genes

  • High mRNA after early response gene’s activity comes down

• if early response gene mRNA levels are kept high, then there is no late response gene mRNA activity

Also discovered:

• Checkpoint + restriction point regulate the start of cycle (G0 to G1, aka Pardee checkpoint)

• And another restriction point at (G1 to S), this one however is “go or no go point”

S phase

DNA synthesis

• DNA copies itself before cell division.

• Each new DNA has 1 old strand + 1 new strand (semi-conservative).

• DNA polymerase adds bases in the 5′ → 3′ direction.

• Replication starts at origins and moves in both directions — this is called bidirectional replication.

• This creates bubbles that grow as forks move outward.

• Scientists proved this with labeled nucleotides in autoradiography — both sides of the origin lit up (DNA synthesis is bi-directional)

G2 phase

Cell verifies that all of the DNA has been
correctly duplicated and all DNA errors
have been corrected

Chromosome condensation is initiated

Early organization of the cell cytoskeleton

Mitotic cyclin dependent kinases initiate
activity

M phase

• Shortest (30 mins)

• Prophase

• Metaphase

• Anaphase

• Telophase/Cytokinesis

Maturation Promoting Factor/Mitosis Phase Factor

Maturating promoting factor:

• Using African clawed toad oocytes (eggs).

  • Injection of MPF triggers G₂-arrested oocyte to enter Meiosis I.

  • Leads to progression through meiosis and early embryonic divisions.

  • Shows cytoplasm has a factor that starts cell cycle progression.

Mitosis Phase Factor (MPF):

• Found by fusing mitotic cells with G₁ cells.

• G₁ nucleus starts condensing chromosomes → prematurely enters mitosis.

• Shows mitotic cells contain a factor that induces mitosis in other cells.

What is MPF (Cyclin B/CDK)

• Cyclins + CDKs = MPF, the engine for cell cycle progression.

• Cyclin B binds to CDK → activates MPF to trigger mitosis.

• MPF peaks in metaphase, then cyclin B is destroyed in anaphase.

• APC/C targets cyclin B for destruction via ubiquitination, ending mitosis.

• Other cyclins:

  • Cyclin D: G₁ phase

  • Cyclin E: G₁ to S

  • Cyclin A: S to G₂

  • Cyclin B: G₂ to Mitosis

Ruth Sager (Checkpoint Controls)

Fused normal and cancer cells and created a heterokaryon

• Fused a normal cell with a cancer cell, creating a heterokaryon (cell with two nuclei).

• Over time, the heterokaryon formed a single nucleus, becoming a hybridoma.

• Let the hybrid cells divide for multiple generations.

• Initially, the hybridoma showed normal (non-cancerous) behavior.

• But later, cancer traits returned, showing uncontrolled growth.

• Led to the idea that normal cells may contain tumor suppressor genes that are gradually lost or inactivated.

p53

• "guardian of the genome" — a major tumor suppressor that stops the cell cycle when DNA is damaged.

• Normally unstable, but becomes stable when phosphorylated by ATM/ATR after DNA damage.

• Activates p21, which blocks G1 CDKs → halts the cell cycle.

• If p53 is lost or mutated, cells bypass checkpoints, increasing cancer risk — like in Sager’s hybridomas.

If p53 is triggered due to an error in DNA

• Can be fixed (mild)

• OR, tell the cell to die (apoptosis)

  • Ex. radiation works against cancer because it triggers p53

Yeast

Used as a model for cell cycle

• Budding Yeast (S. cerevisiae):

  • Asymmetric division → new bud forms.

  • Start checkpoint before bud emerges.

  • Visible bud size marks cell cycle stage.

  • Used to study G1-S transition.

• Fission Yeast (S. pombe):

  • Symmetric division → cell elongates, then splits.

  • G2 is the longest phase (unlike in budding yeast).

  • No visible bud, so rely on length and nuclei to tell stage.

  • Used to study G2-M transition.