cell lecture 12

The cell cycle is how cells grow and divide. It includes:

1. Interphase (growth + DNA replication)

• G1 phase: Cell grows and functions normally.

• S phase: DNA is duplicated.

• G2 phase: Cell prepares for division.

2. M phase (cell division)

• Mitosis: Nuclear division.

• Cytokinesis: Division of the cell's cytoplasm.

In single-celled organisms, this cycle depends on environmental cues. In multicellular organisms, it's tightly regulated.

DNA Replication

• Bidirectional: DNA replication happens in two directions starting from a single origin of replication.

• Origin: This is the specific spot on DNA where replication starts.

• Replication Forks: At each origin, the DNA opens up like a zipper into "bubbles" and forms forks.

  • Eukaryotes (like humans): have many origins.

  • Prokaryotes (like bacteria): usually have one.

• The parent DNA strands unwind, and each strand is used as a template to build a new strand.

Overview

• Leading Strand:

  • Made continuously in the 5’ → 3’ direction (going toward the replication fork).

• Lagging Strand:

  • Made in small pieces (called Okazaki fragments) also in the 5’ → 3’ direction, but away from the fork.

• Key Players:

  1. Helicase – Unzips DNA.

  2. Single-strand binding proteins (SSBs) – Keep DNA strands apart.

  3. Primase – Lays down RNA primer.

  4. DNA Polymerase III – Adds nucleotides to make new DNA strands.

  5. DNA Polymerase I – Replaces RNA primers with DNA.

  6. DNA Ligase – Seals gaps between Okazaki fragments.

Replication Machinery – Eukaryotes & Prokaryotes

• Different DNA Polymerases for eukaryotes and prokaryotes.

  • In bacteria:

    • DNA Pol III does most of the DNA synthesis.

    • DNA Pol I removes RNA primers and fills in with DNA.

  • Eukaryotic polymerases are more complex (like Pol α, δ, ε).

• Other shared enzymes:

  • Helicase, Primase, Ligase, Topoisomerase, SSBs.

  • These help unwind DNA, synthesize RNA primers, and prevent tangling.

Discontinuous Replication

• Leading Strand:

  • Made in one smooth continuous strand (5’ → 3’).

• Lagging Strand:

  • Made in short fragments (Okazaki fragments) since DNA polymerase can only add in the 5’ → 3’ direction.

• Fragments are stitched together by DNA ligase.

• This is why it’s called discontinuous replication.

• Both strands are made at the same time using a machine called a replisome.

• The leading strand is continuous.

• The lagging strand is made in fragments.

• Tsuneko and Reiji Okazaki discovered these fragments in 1968 (~100 base pairs long).

Replisome

• A replisome is a group of enzymes working together at the replication fork.

  • Includes: helicase, primase, polymerase, ligase, SSB, topoisomerase, and more.

• Ensures coordination between leading and lagging strand synthesis.

• The sliding clamp helps keep the polymerase attached to the DNA.

• On the lagging strand, clamps are reloaded for each fragment.

• The trombone model shows how lagging strand loops so both polymerases move in the same direction.

Key Definitions

Here’s what these important terms mean:

• Chromosome: A condensed structure of DNA.

• Chromatid: One half of a replicated chromosome.

• Centromere: Region where sister chromatids are attached.

• Centrosome: Organelle that organizes microtubules; contains centrioles.

• Metaphase Plate: Middle zone where chromosomes line up before being split.

• Mitotic Spindle: Microtubules that pull chromatids apart.

• Centriole: Microtubule-organizing structure inside the centrosome.

The Cell Cycle

The cell cycle includes phases a cell goes through to grow and divide:

• G1 phase: Cell grows.

• S phase: DNA is replicated (synthesized).

• G2 phase: Cell prepares for mitosis.

• M phase (Mitosis + Cytokinesis): Cell divides.

• G0 phase: Resting phase (cells not dividing).

Most cells spend the majority of their time in interphase (G1, S, G2).

Mitotic Index

• Mitotic Index = % of cells in mitosis at a given time.

• It tells how actively a tissue is dividing.

• Formula:

  Mitotic Index = (Number of cells in mitosis / Total number of cells)×100

G1 Phase & Cell Fate

• G1 is a checkpoint: should the cell divide or not?

• If the cell exits the cycle → enters G0.

  • In G0, the cell may:

    • Stay dormant

    • Or undergo terminal differentiation (e.g., becoming a neuron)

Once terminally differentiated, most cells don’t divide anymore.

Mitosis + Cytokinesis (M Phase)

• M phase = mitosis (nucleus divides) + cytokinesis (cell splits).

• It’s short but critical!

Microtubule Reorganization Drives Mitosis

• This image shows microtubules (green) organizing around chromosomes (red).

• Microtubules constantly reorganize to:

  • Form the mitotic spindle,

  • Attach to chromosomes, and

  • Pull chromatids apart.

• This reorganization is essential for mitosis to occur correctly.

Centrosomes

• Centrosomes = MTOCs (Microtubule-Organizing Centers).

• In dividing cells:

  • Centrosomes replicate.

  • They migrate to opposite poles of the cell.

• During mitosis:

  • Centrosomes increase MT (microtubule) nucleating activity.

• MTs grow out from centrosomes and form spindle fibers that help pull chromosomes apart.

• Without centrosomes, cells still divide but have less organized spindles.

Centrosomes – Centrioles

• A centrosome contains a pair of centrioles, surrounded by pericentriolar material.

• Each centriole is made of 9 triplets of microtubules (like a hollow cylinder).

• Function:

  • Help organize mitotic spindle fibers.

  • Play a role in spatial orientation of the cell during division.

γ-Tubulin Ring Complexes (γ-TuRCs)

• These structures are found in the pericentriolar material of centrosomes.

• γ-TuRCs are essential for:

  • Nucleating (starting) microtubule growth.

  • Anchoring the minus (-) ends of microtubules.

• MTs grow outward from the MTOC with fixed polarity:

  • Minus end at centrosome.

  • Plus end extends outward toward chromosomes.

If a cell lacks γ-TuRC, it can’t build new microtubules, which blocks mitotic spindle formation.

Overview of Mitosis Phases

Mitosis is divided into 5 phases, based on the appearance and behavior of chromosomes:

1. Prophase – Chromosomes condense, nucleolus disappears.

2. Prometaphase – Nuclear envelope breaks down, spindle fibers attach to kinetochores.

3. Metaphase – Chromosomes line up at the metaphase plate.

4. Anaphase – Sister chromatids are pulled apart.

5. Telophase – Chromosomes decondense, nuclei reform.

6. Cytokinesis – Division of cytoplasm (often overlaps with telophase).

Prophase

• Happens after G2 phase of interphase.

• Chromosomes condense and become visible.

• Sister chromatids are held together at the centromere.

• Nucleoli disappear, centrosomes start forming the mitotic spindle and move toward opposite poles.

Prometaphase

• Begins with nuclear envelope breakdown.

• Spindle microtubules attach to chromosomes at their kinetochores.

• 3 types of microtubules form:

  • Kinetochore microtubules (pull chromosomes),

  • Astral microtubules (anchor spindle to cell cortex),

  • Polar microtubules (push spindle poles apart).

Metaphase

• Chromosomes are fully condensed and aligned at the metaphase plate.

• Microtubules exert tension on sister chromatids.

• Drugs like colchicine can stop mitosis at this stage by blocking microtubule polymerization (used in labs and plant breeding).

Anaphase

• The shortest phase.

• Sister chromatids separate and are pulled to opposite poles.

• Kinetochore microtubules shorten, polar microtubules lengthen to stretch the cell.

Telophase

• Chromatids arrive at spindle poles.

• Chromosomes decondense.

• Nuclear envelope and nucleolus reform.

• Occurs at the same time as cytokinesis begins.

Cytokinesis

• This is not part of mitosis itself but follows it.

• It splits the cytoplasm into two cells using a contractile ring of actin filaments.

• A cleavage furrow forms and deepens until the cell pinches in two.

Cytokinesis in Plants

• Plant cells can’t form a cleavage furrow due to the rigid cell wall.

• Instead, vesicles from the Golgi align at the spindle equator and fuse to form the cell plate, which becomes the new cell wall.

Cell Cycle Control:

Regulation

• Why control the cell cycle?

  • To ensure proper timing and accuracy of division.

  • To make sure cells only divide when external conditions (like nutrients and growth signals) are right.

• Different cells grow at different rates:

  • Some (like stem cells, blood cells) divide constantly.

  • Others (like neurons, muscle cells) rarely divide once mature.

Checkpoints (G1/S, G2/M, Metaphase/Anaphase)

1. G1-S checkpoint ("Restriction Point"):

   • Is the cell ready to copy its DNA?

   • Cells that don’t pass this checkpoint go to G0 (resting phase).

2. G2-M checkpoint:

   • Has DNA been copied correctly?

   • Any damage? If yes, the cell activates repair or apoptosis.

3. Metaphase-Anaphase checkpoint:

   • Are chromosomes attached to spindle fibers?

   • If not, the cell will pause mitosis to avoid chromosome mis-segregation.

Cyclins and CDKs (Key Cell Cycle Regulators)

• Cyclins = regulatory proteins.

• CDKs (cyclin-dependent kinases) = enzymes that need cyclins to function.

• When cyclins bind to CDKs, they activate kinase activity → triggers progression through the cell cycle.

Cyclin Levels Fluctuate

• CDKs are always present, but cyclin levels rise and fall depending on the phase.

• The graphs show how:

  • Cyclin B peaks during G2/M (mitosis).

  • Different cyclins (D, E, A, B) are active in specific phases to guide the cycle forward.

MPF = Maturation Promoting Factor

• MPF = Cyclin B + Mitotic Cdk.

• When Cyclin B reaches a threshold in G2, it activates Mitotic Cdk → MPF is formed.

• MPF activity starts mitosis by:

  1. Breaking down the nuclear envelope.

  2. Condensing chromosomes.

  3. Forming the spindle.

MPF Activation (More Detailed Mechanism)

• Binding Cyclin B to Cdk isn’t enough.

• The complex is still inactive due to inhibitory phosphate groups.

• To activate it:

  • An activating kinase adds a helpful phosphate.

  • A phosphatase removes the 2 inhibitory phosphates → active MPF!

What MPF Does

• MPF (Cdk-cyclin complex) phosphorylates targets like:

  • Lamins → break nuclear envelope.

  • Proteins → condense chromosomes.

  • Spindle components → build mitotic spindle.

• MPF also activates APC (Anaphase-Promoting Complex) → starts chromatid separation.

APC (Anaphase-Promoting Complex)

• APC targets securin, which normally blocks separase.

• When securin is destroyed, separase becomes active → cleaves cohesin (holds sister chromatids together).

• APC also triggers breakdown of cyclin B to stop mitosis.

Without APC, sister chromatids would stay stuck together → anaphase couldn’t happen.

Growth Factor Regulation

• Growth factors (a.k.a. mitogens) are signaling proteins that tell cells when to divide.

• Different tissues use different growth factors:

  • PDGF – for wound healing and blood vessel growth

  • EGF – for skin and epithelial tissues

  • VEGF – promotes blood vessel growth (vascular system)

  • NGF – supports nerve cell survival/growth

  • BDNF – for neural stem cells and brain development

• Most growth factors signal through tyrosine-kinase receptors, which activate pathways like HER2 (remember the earlier slide).

G1-S Restriction Point

• This is a major decision point: the cell commits to divide.

• Controlled by Cyclin D1, D2, and Cyclin E.

• If conditions aren't right (not enough growth factors, DNA damage, etc.), the cell enters G0 instead of continuing the cycle.

p53 – DNA Damage Checkpoint

• p53 is a tumor suppressor protein—the “guardian of the genome.”

• It checks for DNA damage at:

  • G1-S

  • G2-M

• If damage is found, p53 pauses the cell cycle and triggers repair.

How p53 Works

• ATM and ATR detect DNA breaks.

• These activate checkpoint kinases (Chk1, Chk2).

• These kinases phosphorylate p53 → activating it.

• p53 turns on p21, which inhibits:

  • MPF (stopping G2→M)

  • G1-S Cdk-cyclin (blocking DNA replication)

Result: cell cycle arrest to give the cell time to fix itself.

Normal vs. DNA Damage Conditions

• Under normal conditions, p53 is constantly degraded by MDM2 (a regulatory protein).

  • MDM2 adds ubiquitin to p53 → marks it for destruction.

• When DNA damage occurs:

  • ATM/ATR phosphorylate p53 → p53 can’t bind MDM2 anymore.

  • Now p53 is stabilized and enters the nucleus to act as a transcription factor.

DNA Damage Triggers Response

• Damage ➝ ATM/ATR ➝ Chk1/Chk2 ➝ phosphorylate p53 ➝ p53 breaks free from MDM2.

• Phosphorylated p53:

  • Stays in the nucleus

  • Activates repair genes and p21

  • Prevents cell division until fixed

If Repair Fails → Cell Death (Apoptosis)

• If damage is too severe:

  • p53 activates Puma, which removes inhibition of apoptosis.

  • This shuts down Bcl-2, a protein that normally prevents apoptosis.

  • Result: the cell undergoes programmed death to protect the organism.

• More than 50% of cancers have p53 mutations, which allows damaged cells to divide uncontrollably.

G1-S Restriction Point and Role of Rb (Retinoblastoma protein)

• Goal: Decide if the cell should proceed to the S phase (DNA synthesis).

• Key players:

  • Rb (Retinoblastoma protein): A tumor suppressor.

  • E2F: A transcription factor needed for S phase genes.

  • Cdk-cyclin (G1 Cdk-cyclin): Enzyme that modifies Rb.

How it works:

1. When Rb is unphosphorylated (inactive):

   • Rb binds to E2F and blocks it.

   • This prevents transcription of genes needed for DNA replication.

   • Result: Cell cannot proceed to S phase.

2. When growth factors are present:

   • They activate the Ras pathway, which leads to Cdk-cyclin activation.

   • Cdk-cyclin phosphorylates Rb (adds phosphate groups).

   • Phosphorylated Rb releases E2F.

   • E2F is now active → turns on genes for the S phase.

3. Cyclins make this process cyclical:

   • Cyclin levels rise and fall throughout the cycle, controlling timing.

   • G1 cyclins (Cyclin D, Cyclin E) trigger Rb phosphorylation.

How Growth Factors Signal Through Ras to E2F

This shows the upstream pathway that kicks off Rb inactivation:

1. Growth factor binds receptor → activates Ras.

2. Ras activates Raf → MAPK cascade.

3. MAPK turns on early genes like Jun, Fos, Myc (transcription factors).

4. These early genes activate delayed genes, like:

   • E2F, G1-cyclins, and Cdks.

Mutation in Ras (making it always on) = uncontrolled cell division → cancer.

Growth Factor Suppression

• Not all growth factors promote division.

• TGFβ (Transforming Growth Factor Beta):

  • Acts as a brake.

  • Inhibits cell cycle → promotes cell differentiation and tissue maintenance.

  • Works via Smad proteins to turn on genes that stop the cell cycle.

Summary of Cell Checkpoints

• The cell cycle has a rhythm controlled by cyclins and Cdks.

• At each stage (G1, S, G2, M), different cyclin-Cdk combos act like "timers" to move forward.

• The restriction point in G1 is a key decision-making step:

  • If growth factors are present → G1 Cdk-cyclin activates → Rb is phosphorylated → cell commits to divide.

  • If no growth factors or if TGFβ is active → cell stays in G1 or exits cycle.

• Understand that cell death is essential for:

  • Normal development (e.g. digit formation)

  • Pathologies (e.g. tissue damage, stroke)

• Two main types of cell death:

  1. Necrosis → uncontrolled, due to injury

  2. Apoptosis → programmed, regulated

Homeostasis and Cell Death

• Healthy cells are in homeostasis (balance).

• When a cell is injured:

  • It tries to adapt or activate repair.

  • If damage is reversible, the cell recovers.

  • If irreversible, the cell undergoes cell death:

    • Either necrosis or apoptosis

• There's a "point of no return" — past this, damage = death.

Causes of Cell Death

1. Injurious agents (external/internal) → necrosis

   • External: trauma, lack of oxygen, toxins

   • Internal: immune attacks, abnormal proteins, poor nutrition

2. Programmed death → apoptosis

   • Self-destruct mechanism

   • Can be normal (development) or due to disease

Necrosis Overview

• Uncontrolled, messy cell death

• Rapid and dramatic → whole cell disintegrates

• Caused by ion imbalance (especially Ca²⁺) leading to:

  • Swelling

  • Mitochondrial & ER damage

  • Vacuole formation

  • Activation of lysosomal proteases (degrade the cell)

Ion Homeostasis & Ca²⁺

• Normally, cells regulate Ca²⁺ tightly.

• In necrosis:

  • Energy failure stops Ca²⁺-ATPase, so Ca²⁺ floods the cell.

  • ER becomes leaky, releases more Ca²⁺.

• This Ca²⁺ overload:

  • Activates calpain (Ca²⁺-sensitive protease)

  • Calpain ruptures lysosomes

  • Lysosomes release cathepsins → chew up the cell

Necrotic Injury: Stroke Example

• Ischemic stroke = blocked blood flow → oxygen/energy loss

  • Energy failure = Ca²⁺ influx

  • Ca²⁺ damages membranes, activates calpain

  • Glutamate (released in stress) also causes Ca²⁺ influx

• Leads to tissue damage & liquefaction

  • Seen as a dark necrotic area in the brain image

What is Programmed Cell Death (PCD)?

Apoptosis is a controlled, tidy process where cells “self-destruct” for the benefit of the organism.

Why is it important?

• Normal development

  • Shapes body parts (e.g., fingers/toes — removing webbing).

  • Eliminates extra neurons during brain development.

• Defense mechanism

  • Kills infected cells (e.g. by viruses).

  • Removes cells with DNA damage or irreparable harm to prevent diseases like cancer.

How does Apoptosis Happen?

1. Cell shrinks, nucleus condenses.

2. DNA is chopped into fragments (“laddering”).

3. Cell breaks into bubbles → apoptotic bodies.

4. These are cleared by phagocytes → no inflammation!

This is in contrast to necrosis, which causes messy cell rupture and inflammation.

Two Main Pathways of Apoptosis

The extrinsic pathway is triggered by external signals, like when a death receptor on the cell surface is activated. This leads to activation of caspase-8, which then activates caspase-3, resulting in apoptosis.

The intrinsic pathway is triggered by internal stress, such as DNA damage or lack of survival factors. This causes the mitochondria to release cytochrome c, which forms the apoptosome and activates caspase-9. Caspase-9 then activates caspase-3, leading to apoptosis.

Caspase Family

Caspases are proteases (enzymes that cut proteins) with:

• Initiators (e.g. caspase-8, caspase-9)

• Executioners (e.g. caspase-3) → break down the cell

They’re called Cysteine-aspartate proteases (cut after aspartate residues).