Lecture 6Cell Cycle Regulation, DNA Replication, and Cell Death

Cell and Molecular Biology: Cell Division, Differentiation, and Apoptosis

Cell division is a fundamental biological process crucial for the continuity of life, growth, development, and tissue repair. It ensures that organisms can develop from a single cell, replace old or damaged cells, and maintain cellular homeostasis. The process involves precise replication of genetic information and its accurate distribution to daughter cells.

The Cell Cycle

The cell cycle describes the life of a cell from its formation through division until it divides again. It is an integral part of cellular life, ensuring that cells produce genetically identical offspring.

Phases of the Cell Cycle

The classical cell cycle "clock" consists of four main phases: G₁, S, G₂, and M.

• Interphase (G₁, S, and G₂ phases): Accounts for approximately 90% of the cell cycle.
  • G₁ phase (“first gap”):
    • Cell grows and performs basic functions.
    • Typically the longest and most variable phase.
    • A critical restriction point decision is made: to enter S phase or enter a resting G₀ stage.
    • Signals (e.g., mitogen factors, nutrient availability, DNA repair status) determine this decision.
  • S phase (“synthesis”):
    • DNA replication occurs, where the cell makes an identical copy of each chromosome.
    • Histone synthesis also takes place.
    • Centrioles duplicate in the cytoplasm.
  • G₂ phase (“second gap”):
    • Cell growth continues, with synthesis of proteins and organelles.
    • Mitochondria divide.
    • Tubulin is synthesized, and microtubules are formed in preparation for mitosis.
    • Another checkpoint ensures DNA integrity and replication completeness before entering M phase.
• Mitotic (M) phase:
  • Includes mitosis (or meiosis in germ cells) and cytokinesis (cytoplasm division).
  • Results in the distribution of DNA and non-nuclear organelles into two daughter cells.

Cell-Cycle Times

The duration of the cell cycle varies greatly among different cell types and depends on external conditions (e.g., nutrients, oxygen).

A typical human cell proliferating in culture has a total cycle time of approximately 24 hours.

G₀ Phase

The G₀ phase is a specialized resting or quiescent state where cells are not dividing but are performing their specific functions. Most cells of an adult organism spend significant time in G₀. Cells can enter G₀ due to:

• Nutrient deprivation.
• Lack of growth factors or density-dependent inhibition.
• Genetic programs (e.g., differentiation).

Cells can re-enter G₁ upon receiving appropriate signals, such as growth factors or other extracellular stimuli, often induced by injury or normal cell turnover.

DNA Replication

DNA replication is the process of copying the nucleotide sequence of one DNA strand into a complementary DNA sequence, ensuring the accurate transmission of genetic information to daughter cells.

Mechanism of DNA Replication

• The two strands of the DNA double helix unwind.
• Each strand serves as a template for the synthesis of a new complementary strand.
• This “semiconservative” process generates two double-stranded daughter DNA molecules, each composed of one original (conserved) strand and one newly synthesized strand.
• DNA polymerases are enzymes that catalyze the addition of new nucleotides.

Key Features of DNA Replication

• Accuracy: DNA must be replicated with extraordinary accuracy to prevent mutations. The human genome, with its 3 billion base pairs, must be copied faithfully.
• Semiconservative: Each new DNA helix contains one original strand from the parent molecule and one newly synthesized strand.
• Origins of Replication: Replication begins at specific sequences rich in A-T base pairs, recognized by initiator proteins that unwind the DNA.
• Replication Bubble and Forks: As DNA opens, a replication bubble forms with two Y-shaped replication forks. These forks are sites where a “replication machine” (a multienzyme complex) moves along the DNA to synthesize new strands.
• Multiple Origins in Eukaryotes: Eukaryotic chromosomes have many replication origins (e.g., ~220 per human chromosome), allowing faster replication of large genomes.

DNA Polymerases

These enzymes are central to DNA synthesis:

• They catalyze the addition of nucleotides to the 3' end of a growing DNA strand.
• The polymerization energy comes from the hydrolysis of dNTPs.
• They always require a template and a primer (a short pre-existing sequence).
• They can only add nucleotides in the 5' to 3' direction.
• They possess proofreading activity (3' to 5' exonuclease activity) to correct errors.
• Eukaryotic cells contain five main types: α, β, γ, δ, and ε (polymerase γ is mitochondrial).

Primase and Primers

• Primase, an RNA polymerase, synthesizes a short RNA segment (about 10 nucleotides long) called a primer, using the DNA strand as a template.
• The primer provides the necessary 3' end for DNA polymerase to begin synthesis.

Leading and Lagging Strands

Due to DNA polymerase's 5' to 3' synthesis direction and the antiparallel nature of DNA strands, replication proceeds differently on the two template strands:

• Leading strand: Synthesized continuously in the 5' to 3' direction towards the replication fork with only one primer.
• Lagging strand: Synthesized discontinuously in short segments called Okazaki fragments (about 200 nucleotides long each), moving away from the replication fork. Each fragment requires a new primer.

Processing Okazaki Fragments

1. RNA primers are removed by nucleases.
2. Repair polymerase (a DNA polymerase) fills the gaps with DNA.
3. DNA ligase joins the fragments by forming a phosphodiester bond, requiring ATP.

Accessory Proteins in DNA Replication

A “replication machine” involves several proteins:

• DNA helicase: Unwinds the DNA double helix at the replication fork.
• Single-strand DNA-binding proteins: Prevent re-annealing of separated strands and keep them elongated.
• Sliding clamp: A ring-shaped protein that holds DNA polymerase to the template, increasing processivity.
• Topoisomerase: Works ahead of the replication fork to relieve torsional stress (supercoiling) by making temporary nicks in the DNA.

Accuracy and Repair Mechanisms

DNA polymerase has an error rate of about 1 in $10^7$ bases. To prevent mutations, cells have multiple repair mechanisms:

1. Proofreading: DNA polymerase's 3' to 5' exonuclease activity removes incorrectly added nucleotides during synthesis.
2. Mismatch repair: A post-replication mechanism that corrects mispaired bases not caught by proofreading. A protein complex identifies the mismatch, removes the incorrect section, and DNA polymerase refills the gap, sealed by DNA ligase.
3. DNA damage repair pathways: Detect and correct damage throughout the cell cycle.
   • Direct reversal: Enzymes directly “undo” certain chemical damages.
   • Excision repair:
     • Base excision repair: Removes only the damaged base.
     • Nucleotide excision repair: Removes a patch of nucleotides around the damage.
   • Double-stranded break repair: Fixes chromosome breaks.
     • Non-homologous end joining: Joins broken ends, often with some loss or addition of nucleotides.
     • Homologous recombination: Uses an undamaged homologous chromosome as a template for repair, resulting in a cleaner repair with fewer mutations.

Mutations in these repair genes can lead to hereditary cancers (e.g., Lynch syndrome from mismatch repair defects, Xeroderma pigmentosum from nucleotide excision repair defects).

DNA Replication Fidelity & Genetic Stability

The remarkably high fidelity of DNA replication and maintenance is critical for two types of cells:

• Germ cells: Must be protected against high mutation rates to preserve the species.
• Somatic cells: Must be protected from genetic changes to maintain organized body structure. Accumulation of DNA changes in somatic cells can lead to cancer.

Telomeres and Telomerase

Eukaryotic chromosomes are linear, posing a challenge for lagging strand replication at the ends. This causes chromosomes to shorten with each division. Specialized DNA “caps” called telomeres protect the chromosome ends.

• Telomeres consist of repeated non-coding sequences (e.g., 5'-TTAGGG-3' in humans).
• The repeats are slowly eroded over many cell divisions, preventing loss of essential genetic information.
• Telomerase, a ribonucleoprotein, maintains telomere length.
  • It contains an RNA template and a reverse transcriptase enzyme.
  • It adds multiple copies of the telomeric repeat sequence to the template strand, extending it.
  • This extended template allows conventional DNA replication machinery to complete the lagging strand synthesis.
• Clinical Relevance: Telomerase activity is usually low in somatic cells but high in germ cells, some adult stem cells, and many cancer cells. Inhibiting telomerase in cancer cells is a potential therapeutic strategy.
• Telomere shortening is linked to cell aging and limits the number of cell divisions.

Polymerase Chain Reaction (PCR)

PCR is an in vitro technique to amplify a specific DNA region, mimicking DNA replication.

• Components: DNA template, DNA primers (flanking the target region), dNTPs, and a thermostable DNA polymerase (e.g., Taq polymerase).
• Steps per cycle:
  1. Denaturation: Heat DNA to separate strands.
  2. Annealing: Cool to allow primers to bind to complementary sequences.
  3. Extension: Incubate with DNA polymerase and dNTPs to synthesize new DNA strands.
• Repeated cycling amplifies DNA exponentially.
• Uses: Research, diagnostics, forensics.

Cell Cycle Control System

The cell cycle is tightly regulated by a sophisticated control system that acts like a timer, triggering events in sequence and preventing out-of-control division that could lead to cancer.

Cell Cycle Checkpoints

Checkpoints are stages where the cell pauses to examine internal and external cues before proceeding with division.

• G₁ Checkpoint (G₁/S transition):
  • Main decision point (restriction point).
  • Checks for: cell size, nutrients, growth factors, DNA damage.
  • If “go-ahead cues” are absent, the cell may enter G₀.
  • Once passed, the cell commits to the cell cycle.
• G₂ Checkpoint (G₂/M transition):
  • Ensures DNA is in perfect condition before mitosis.
  • Checks for: DNA damage, DNA replication completeness.
  • Pauses for repair; if damage is irreparable, the cell may undergo apoptosis.
• Spindle Checkpoint (Metaphase to Anaphase transition):
  • Checks if sister chromatids are correctly attached to spindle microtubules.
  • Pauses mitosis if chromosomes are misplaced, allowing time for correction.

Cell Cycle Regulators: Cyclins & Cyclin-Dependent Kinases (Cdks)

These are central components of the control system, highly conserved across eukaryotes.

• Cyclins:
  • Undergo cycles of synthesis and degradation throughout the cell cycle.
  • Four basic types in humans: G₁ cyclins, G₁/S cyclins, S cyclins, and M cyclins.
  • Their fluctuating levels lead to cyclic assembly and activation of cyclin-Cdk complexes.
• Cyclin-Dependent Kinases (Cdks):
  • Levels remain constant, but their activity changes based on cyclin binding.
  • When bound to cyclin, Cdk becomes active, phosphorylating specific intracellular proteins that initiate or regulate cell cycle events.
  • Cyclin binding also directs Cdk to specific target proteins.
  • Activation involves cyclin binding and phosphorylation at specific sites, while negative regulation can involve phosphorylation at other sites by proteins like Wee1 or dephosphorylation by Cdc25.

Cyclin-Cdk Complex Activity

• G₁/S-cyclin: Forms G₁/S-Cdk complexes, triggering progression through the Start transition.
• S-cyclin: Forms S-Cdk complexes at the start of S phase, triggering DNA replication and early mitotic events.
• M-cyclin: Forms M-Cdk complexes (Maturation Promoting Factor - MPF) during G₂, triggering entry into mitosis.

Regulation of Cyclin-Cdk Activity

• Cdk Inhibitor Proteins (CKIs): (e.g., p27, p16, p21) inactivate cyclin-Cdk complexes, primarily for G₁/S and S-Cdks to control early cell cycle progression.
• Anaphase-Promoting Complex/Cyclosome (APC/C): A ubiquitin ligase that initiates the metaphase-to-anaphase transition by targeting specific proteins for destruction in proteasomes.
  • Destroys M cyclins, pushing the cell out of mitosis and into G₁.
  • Destroys Securin, which releases Separase. Separase then cleaves cohesin, allowing sister chromatid separation.

External Signals Governing Cell Division

• Growth Factors (Mitogens): Proteins that stimulate other cells to divide. They activate signaling pathways that synthesize G₁ and G₁/S cyclins, thereby promoting cell cycle entry from G₀. E.g., Platelet-Derived Growth Factor (PDGF).
• Anchorage Dependence: Normal cells require attachment to a substratum to divide.
• Density-Dependent Inhibition (Contact Inhibition): Cells stop dividing when they make contact with neighboring cells. This triggers the production of CKIs (p16 and p27), inhibiting G₁ Cdks.
• Retinoblastoma (Rb) protein: A tumor suppressor that acts as a negative regulator, blocking progression from G₁ to S phase by binding transcription regulators. Phosphorylation by G₁-Cdks and G₁/S-Cdks releases these regulators, allowing cell proliferation. Defective Rb protein is associated with cancer.

Internal Signals Governing Cell Division

• p53 (“the guardian of the genome”): A tumor suppressor protein.
  • If DNA damage is detected in G₁, p53 concentration and activity increase.
  • p53 activates the transcription of the gene encoding p21 (a Cdk inhibitor protein).
  • p21 binds to G₁/S-Cdk and S-Cdk, arresting the cell cycle in G₁ to allow DNA repair.
  • If damage is too severe, p53 can induce apoptosis.
• Mutations in p53 are found in about half of all human cancers, leading to unrestrained replication of damaged DNA and high mutation rates.

Cancer Cells and Cell Cycle Control

Cancer cells exhibit abnormal cell cycle control:

• They do not respond to normal cell cycle regulatory signals.
• They may not require growth factors, or may produce their own, or bypass dependence on external growth factor signals.
• They often lack density-dependent inhibition and anchorage dependence.

Mitosis

Mitosis is a type of cell division where one mother cell divides to produce two genetically identical daughter cells. It is essential for growth, development, and replacement of old/worn-out cells.

Phases of Mitosis

1. Interphase (Late G₂ phase):
   • DNA has already been copied, so each chromosome consists of two sister chromatids.
   • Chromosome number (2n) is maintained, but DNA content is doubled (2C).
   • Chromosomes are decondensed.
   • Centrosomes have duplicated and will orchestrate mitosis.
2. Prophase:
   • Chromosomes condense, appearing as long, thin threads. Each contains two sister chromatids joined at the centromere by cohesin proteins.
   • Mitotic spindle begins to assemble between the two centrosomes, which move apart.
   • Nucleolus disappears.
   • Nuclear membrane is still visible.
3. Prometaphase:
   • Starts with the breakdown of the nuclear envelope (due to phosphorylation of nuclear lamins by M-Cdk).
   • Mitotic spindle grows and accesses chromosomes.
   • Chromosomes attach to spindle microtubules via their kinetochores and move towards the equator.
4. Metaphase:
   • Chromosomes align at the metaphase plate (equator of the cell).
   • Kinetochore microtubules attach sister chromatids to opposite poles of the spindle.
   • Spindle checkpoint verifies correct attachment and alignment; if not, division halts.
5. Anaphase:
   • Sister chromatids synchronously separate (cohesins break down by Separase activity, which is activated by APC/C after Securin degradation).
   • Chromatids (now individual chromosomes) are pulled towards opposite spindle poles.
   • Kinetochore microtubules shorten (anaphase A), and spindle poles move apart (anaphase B), contributing to chromosome segregation.
6. Telophase:
   • Two sets of chromosomes arrive at opposite poles.
   • Mitotic spindle disassembles.
   • New nuclear envelopes reassemble around each set of chromosomes (due to dephosphorylation of nuclear pore proteins and lamins).
   • Chromosomes decondense, and nucleoli reappear.
   • Cytokinesis begins with the assembly of the contractile ring.
7. Cytokinesis:
   • Division of the cytoplasm to form two new cells.
   • Overlaps with late anaphase and telophase.
   • A cleavage furrow appears on the cell surface, formed by the contractile ring (actin and myosin II filaments).
   • The ring contracts, deepening the furrow until the cell divides.

Mitotic Spindle

The mitotic spindle is a microtubule-based machine responsible for chromosome separation.

• It is a bipolar array of microtubules assembled by M-Cdk.
• Types of microtubules:
  • Interpolar microtubules: Overlap in the spindle mid-zone.
  • Kinetochore microtubules: Attach to sister chromatid pairs at the kinetochores.
  • Astral microtubules: Radiate outward from poles, contacting the cell cortex to position the spindle.
• Microtubule Motor Proteins (kinesins and dyneins) contribute to spindle assembly and function by moving along microtubules.

Cohesin and Condensin

• Cohesin: Forms ring structures around chromosomal DNA, holding sister chromatids together from S phase until anaphase. Mutations in cohesin genes are linked to developmental disorders and cancer (e.g., Roberts syndrome).
• Condensin: Forms ring structures that promote chromosome condensation in prophase, compacting chromatids into rod-like structures.
• Sister-chromatid resolution: Involves decatenation of sister DNAs and partial removal of cohesin from chromosome arms, making sister chromatids distinct.

Kinetochores

Kinetochores are giant, multilayered protein complexes built at the centromeric region of each chromatid, mediating attachment to spindle microtubules. Each sister chromatid has its own kinetochore.

Meiosis

Meiosis is a specialized type of cell division that reduces the chromosome number by half, producing haploid gametes (sperm and eggs) from diploid cells. This is essential for sexual reproduction.

Overview of Meiosis

• Starts with a diploid cell (2n).
• Undergoes one round of DNA replication, followed by two successive rounds of chromosome division: Meiosis I and Meiosis II.
• Results in four haploid daughter cells (1n), each with a single set of chromosomes.

Meiosis I (Reductional Division)

Preceded by an interphase almost identical to mitosis (G₁, S, G₂ phases), where DNA is replicated.

Prophase I: A long and complex phase with 5 substages:

1. Leptotene: Chromosomes appear threadlike; sister chromatids linked by cohesin.
2. Zygotene: Homologous chromosomes pair up (synapsis) to form bivalents (or tetrads). A synaptonemal complex forms between homologs.
3. Pachytene: Chromosomes thicken; synapsis is complete. Crossing over (genetic recombination) occurs between non-sister chromatids at recombination nodules, fostering genetic diversity.
4. Diplotene: Synaptonemal complex disassembles. Homologous chromosomes begin to separate but remain linked by chiasmata (sites of crossing over).
5. Diakinesis: Chromosomes detach from the nuclear envelope, shorten, and thicken. Microtubule spindle begins to develop.

Metaphase I:

• Homologous pairs (bivalents) align at the metaphase plate, with random orientation of maternal and paternal homologs.
• Each homologous chromosome attaches to spindle fibers originating from opposite poles.

Anaphase I:

• Homologous chromosomes move to opposite poles.
• Cohesins holding the sister chromatid arms are degraded, but cohesins in the centromere region remain, keeping sister chromatids together.

Telophase I:

• Separated chromosomes arrive at opposite poles.
• Nuclear membranes may reform, and chromosomes decondense.

Cytokinesis I: Usually occurs simultaneously with Telophase I, forming two haploid daughter cells (1n, 2C), each with chromosomes still consisting of two sister chromatids.

Meiosis II (Equational Division)

Cells move directly from Meiosis I to Meiosis II without DNA replication. This division is similar to mitosis, but for haploid cells (often called “mitosis for haploid cells”):

• Prophase II: Chromosomes re-condense, if necessary. Nuclear envelope breaks down, and spindle forms.
• Metaphase II: Sister chromatids align at the metaphase plate.
• Anaphase II: Sister chromatids separate (cohesins in centromere are degraded) and move to opposite poles.
• Telophase II: Nuclear membranes reform, chromosomes decondense.

Cytokinesis II: Forms four haploid cells (1n, 1C), each with chromosomes having only one chromatid. These become specialized as sperm or egg cells.

Sources of Genetic Variation in Meiosis

Meiosis generates genetically distinct gametes primarily through:

• Crossing over: Random exchange of genetic material between homologous chromosomes during Prophase I.
• Random orientation of homologue pairs: Independent assortment of homologous chromosomes at the metaphase plate in Metaphase I. In humans, this alone allows for over 8 million different gamete combinations.

Gamete Formation (Gametogenesis)

Gamete formation is a complex process where germ cells undergo meiosis and differentiation to become mature gametes.

Oogenesis (Female Gamete Formation)

Occurs in the ovary within ovarian follicles. Characterized by asymmetric division and arrested development.

• Oogonia (46 chromosomes) multiply by mitosis in the embryo.
• They then grow and enter meiosis, becoming primary oocytes (46 chromosomes).
• Primary oocytes arrest in Prophase I (diplotene stage) from birth, for decades.
• Number of primary oocytes: ~7 million in 5-month embryo, ~700,000-2 million at birth, ~400,000 at adolescence, and only ~400 mature by menopause.
• During cyclic growth, a single primary oocyte completes Meiosis I, yielding a large secondary oocyte and a small polar body (due to asymmetric cytokinesis).
• The secondary oocyte arrests in Metaphase II.
• Meiosis II is only completed upon fertilization, producing the ovum and another small polar body. If no fertilization, the ovum dies via apoptosis.

Ovum (Oocyte) Characteristics:

• Largest human cell (~30-200 μm).
• Eccentric nucleus, no centrioles.
• Cytoplasm rich in nutrient reserves (deutoplasm) and RNA reserves.
• Cortical granules: Release proteases upon sperm penetration to prevent polyspermy.
• Zona pellucida: An outer layer of glycoproteins and glycosaminoglycans providing mechanical protection, binding for sperm, and preventing polyspermy.
• Corona radiata: Follicular cells surrounding the zona pellucida, providing nutrition via gap junctions.

Spermatogenesis (Male Gamete Formation)

Occurs in the testes, continuously from puberty.

• Spermatogonia (stem cells) appear at ~3 weeks embryonic age, migrate to testes, and divide by mitosis and meiosis at puberty.
• Spermatogonia → Primary spermatocytes (46 chromosomes) (Prophase I, ~2 weeks).
• Primary spermatocytes complete Meiosis I → Secondary spermatocytes (Meiosis I completion, ~24 days).
• Secondary spermatocytes complete Meiosis II → Spermatids (haploid, cannot move, ~1 day).
• Spermatids undergo spermiogenesis (maturation) → Spermatozoon (motile).

Spermatozoon Characteristics:

• Smallest human cell (~60 μm long), motile due to flagellum.
• Head: Contains the nucleus (DNA is heterochromatic) and an acrosome (secretory vesicle with hydrolytic enzymes for fertilization).
• Neck: Contains mitochondria for ATP production.
• Lacks ER and Golgi apparatus.
• Tail (flagellum): 9x2 + 2 microtubule arrangement.

Cell Differentiation and Stem Cells

Multicellular organisms contain diverse cell types, all originating from a single zygote. This diversity arises through differentiation.

Cell Differentiation

• The process by which cells gain a final cell type identity, becoming more restricted in their developmental potential.
• Controlled by specific gene expression patterns, leading to specialized proteins and functional RNAs.
• Differentiation is influenced by:
  • Intrinsic (lineage) information: Inherited from mother cells (e.g., specific molecules directing lineage).
  • Extrinsic (positional) information: Received from the cell’s surroundings (e.g., chemical signals from neighbors).

Stem Cells

Stem cells are undifferentiated cells capable of self-renewal and differentiation into various cell types.

• They can divide limitlessly via symmetric or asymmetric cell divisions.
• Self-renewal: One daughter cell remains a stem cell, renewing the stem cell pool.
• The other daughter cell differentiates, either directly or through precursor (progenitor) cells.

Types of Potency

Stem cell potency defines the range of differentiated cells they can produce:

• Totipotent: Can produce all cell types of a developing organism, including embryonic and extraembryonic tissues (can form a complete organism). E.g., zygote.
• Pluripotent: Can make cells of the embryo proper, including germ cells and cells from all three primary germ layers (ectoderm, endoderm, mesoderm). E.g., embryonic stem cells.
• Multipotent: Can make cells only within a given germ layer or tissue. E.g., hematopoietic stem cells (blood cells).
• Unipotent: Can make cells of a single cell type but retain self-renewal capacity. E.g., gamete-forming stem cells.

Categories of Stem Cells

• Embryonic Stem (ES) Cells:
  • Pluripotent, derived from the inner cell mass of a blastocyst.
  • Can differentiate into any of the ~220 cell types in the adult body.
• Adult Stem Cells (Somatic Stem Cells):
  • Undifferentiated cells found in differentiated tissues/organs.
  • Multipotent (or sometimes unipotent); play roles in tissue repair and regeneration.
  • E.g., hematopoietic stem cells (bone marrow), neural stem cells (brain), epithelial stem cells (digestive tract), skin stem cells.
• Induced Pluripotent Stem (iPS) Cells:
  • Adult somatic cells genetically reprogrammed (de-differentiated) to an embryonic stem cell-like pluripotent state.
  • Discovered by Yamanaka and Gurdon (Nobel Prize 2012).
  • Advantage: avoids ethical issues of ES cells and reduces immune rejection (can be made from patient’s own cells).
  • Potential for studying development, disease modeling, drug testing, and regenerative medicine (e.g., treating Parkinson’s or Alzheimer’s).

The ability of stem cells to differentiate is largely under genetic control, with varied gene expression distinguishing cell types (e.g. liver cells expressing alcohol dehydrogenase, neurons expressing neurotransmitters).

Cell Death: Necrosis and Apoptosis

Multicellular organisms require mechanisms for cell death to balance cell proliferation, sculpt tissues during development, and eliminate abnormal or harmful cells.

Necrosis (Accidental Cell Death)

• Result of external factors like mechanical trauma, lack of blood supply (hypoxia), or toxic agents.
• Characterized by cell swelling, rupture of the plasma membrane, and leakage of cytoplasmic contents (including lysosomal enzymes).
• Triggers an inflammatory immune response in surrounding tissue.
• Typically affects groups of cells.

Apoptosis (Programmed Cell Death)

Derived from the Greek word “falling off,” apoptosis is a highly regulated, deliberate cellular suicide process fundamental to normal physiological conditions. It is neatly executed without causing inflammation.

Characteristics of Apoptosis

• Cell shrinks and condenses.
• Cytoskeleton collapses, organelles fragment.
• Nuclear chromatin condenses and fragments.
  • DNA fragmentation occurs due to activation of nuclear endonucleases.
  • This produces DNA fragments that show a “ladder pattern” on gel electrophoresis and can be detected by TUNEL technique.
• Cell surface develops bubble-like protrusions (“blebs”).
• Cell breaks into membrane-enclosed apoptotic bodies.
• Apoptotic bodies display phosphatidylserine on their surface, signaling phagocytic cells (macrophages or neighboring cells) to engulf them.
• This process is tidy, preventing leakage of cellular contents and subsequent inflammation.

Physiological Roles of Apoptosis

• Sculpting body parts during embryonic development (e.g., separation of fingers and toes).
• Removal of unneeded neurons during neural circuit “wiring.”
• Quality control: eliminating abnormal, misplaced, nonfunctional, or potentially dangerous cells (e.g., self-reactive immune cells).
• Maintaining tissue homeostasis by removing old cells to make space for new ones.
• Eliminating virus-infected or cancerous cells.
• Clearing pathogen-specific immune cells after infection resolution.

Mechanism of Apoptosis: Caspases

Apoptosis is executed by a family of specialized intracellular proteases called caspases.

• Caspases have a cysteine at their active site and cleave target proteins at specific aspartic acid residues (hence C-asp-ase).
• They are synthesized as inactive precursors and activated during apoptosis via dimerization and proteolytic cleavage.
• Once activated, they trigger a destructive, self-amplifying, and irreversible proteolytic cascade.
• Caspases cleave over a thousand proteins, including:
  • Nuclear lamins: Leads to irreversible breakdown of the nuclear lamina.
  • DNA degrading endonucleases: Frees CAD (Caspase-Activated DNase) from its inhibitor (iCAD), allowing it to cut chromosomal DNA.
  • Cytoskeletal and cell-cell adhesion proteins: Helps the apoptotic cell round up and detach for engulfment.

Two Major Apoptotic Pathways

1. Extrinsic Pathway (Death Receptor Pathway):
   • Triggered by extracellular signal proteins (e.g., Fas ligand, TNF-alpha) binding to cell-surface death receptors (e.g., Fas death receptor, TNF receptor) which belong to the TNF receptor family.
   • Binding of ligand causes aggregation of death receptors, whose intracellular death domains recruit adaptor proteins (e.g., FADD - Fas-associated death domain).
   • FADD then recruits initiator caspases (primarily caspase-8), forming a death-inducing signaling complex (DISC).
   • Activated initiator caspases activate downstream executioner caspases, leading to apoptosis.
2. Intrinsic Pathway (Mitochondrial Pathway):
   • Activated by intracellular stresses such as DNA damage, developmental signals, or absence of growth factors.
   • Involves the release of mitochondrial proteins (e.g., cytochrome c) from the intermembrane space into the cytosol.
   • Cytosolic cytochrome c binds to the adaptor protein Apaf1 (apoptotic protease activating factor-1).
   • Apaf1 oligomerizes into a wheel-like heptamer called an apoptosome.
   • The apoptosome recruits initiator caspase-9, which then activates downstream executioner caspases.

Bcl2 Family Proteins in Intrinsic Pathway

These proteins regulate the release of mitochondrial proteins and have conserved roles in evolution.

• Pro-apoptotic effector Bcl2 family proteins (e.g., Bax, Bak): Become activated in response to apoptotic stimuli, aggregate in the outer mitochondrial membrane, and induce the release of cytochrome c.
• Anti-apoptotic Bcl2 family proteins: Located on the cytosolic surface of the outer mitochondrial membrane, they inhibit apoptosis by binding to and inhibiting pro-apoptotic Bcl2 proteins.
• BH3-only proteins: The largest subclass, produced or activated in response to apoptotic stimuli. They promote apoptosis by inhibiting anti-apoptotic Bcl2 proteins.

IAPs and Anti-IAPs

• Inhibitors of Apoptosis Proteins (IAPs): Negative regulators of caspases and cell death, containing BIR (Baculovirus IAP Repeat) domains. They bind to activated caspases and some polyubiquitylate caspases for destruction.
• Anti-IAPs: Neutralize the inhibitory effects of IAPs, promoting apoptosis. They are produced in response to various apoptotic stimuli.

The ability of cells to escape apoptosis, often due to mutations in these regulatory pathways, is a major cause of cancer development.

Key Takeaways

• Cell division is fundamental for life, involving precise replication and segregation of genetic material.
• The cell cycle is highly regulated by checkpoints and molecular switches like cyclins and Cdks.
• DNA replication is semiconservative, uses origins of replication, and involves DNA polymerases, primase, and numerous accessory proteins.
• Telomeres protect chromosome ends, with telomerase maintaining their length, which is relevant in aging and cancer.
• DNA repair mechanisms are crucial for maintaining genetic stability and preventing mutations that can lead to disease.
• Mitosis ensures genetically identical daughter cells for growth and repair.
• Meiosis produces genetically diverse haploid gametes for sexual reproduction through crossing over and random assortment.
• Cell differentiation, guided by intrinsic and extrinsic signals, creates specialized cell types from stem cells.
• Stem cells differentiate with varying potencies (totipotent, pluripotent, multipotent, unipotent) and have therapeutic potential.
• Apoptosis is a vital programmed cell death process that sculpts tissues, removes damaged cells, and maintains homeostasis, primarily executed by caspases.
• Dysregulation of cell cycle control and apoptosis contribute significantly to cancer pathology.

Cell Division and Related Processes: A Cheatsheet

Cell division is fundamental to life, enabling growth, repair, and reproduction. It involves the precise duplication and distribution of genetic material and cellular components. This cheatsheet summarizes the key aspects of the cell cycle, DNA replication, mitosis, meiosis, differentiation, stem cells, and cell death (apoptosis).

I. The Cell Cycle

The life of a cell from its creation to its own division.

• Rudolf Virchow (1858): "Where a cell arises, there must be a previous cell."

• Continuity of Life: Entirely based on cell division.

A. Purpose of Cell Division

• Unicellular Organisms: Reproduces the entire organism, increasing population.

• Multicellular Organisms:

  • Development: From a single fertilized egg to a complex organism.

  • Maintenance: Renews, repairs, or replaces cells (e.g., wound healing, tissue homeostasis).

B. Fundamental Tasks of a Dividing Cell

1. Genetic Information Transfer: Pass genetic information to the next generation.

2. DNA Replication: Faithfully replicate DNA in each chromosome.

3. Chromosome Segregation: Accurately distribute replicated chromosomes to daughter cells.

4. Organelle Duplication: Duplicate other organelles and macromolecules to prevent cell shrinking.

C. Phases of the Cell Cycle

The classic cell cycle is made of four phases: G1, S, G2, and M.

• Interphase (approx. 90% of the cycle, deceptively uneventful under microscope):

  • G₁ Phase ("first gap"):

    • Cell grows and performs basic functions.

    • Restriction Point: A crucial decision point at the end of G1.

      • Enter S phase (e.g., with mitogen factors).

      • Enter a resting G₀ stage (majority of adult organism cells).

    • Signals dictate division, DNA repair, apoptosis, or G₀ entry.

    • Longest and most variable phase.

  • S Phase ("synthesis"):

    • DNA replication: Identical copy of each chromosome.

    • Histone synthesis.

    • Centrioles duplicate in the cytoplasm.

  • G₂ Phase ("second gap"):

    • Cell growth continues.

    • Protein and organelle synthesis (e.g., mitochondria divide).

    • Tubulin synthesized and microtubules formed (prepares for mitosis).

    • G2 Checkpoint: Ensures DNA is perfect before mitosis (checks for damage, replication completeness). Errors lead to repair pause or apoptosis.

• Mitotic (M) Phase (approx. 1 hour for human cells):

  • Mitosis (nuclear division) or Meiosis.

  • Cytokinesis (cytoplasm division).

D. Cell Cycle Variability

• Cell Type Dependent: Varies greatly (e.g., frog embryo cells: 30 min, human liver cells: ~1 year).

• External Conditions: Influenced by nutrients, oxygen, etc.

• Normal Cells: Have a finite number of divisions; then enter replicative senescence (permanent growth arrest) due to telomere shortening.

II. DNA Replication

The process of copying the nucleotide sequence of one DNA strand into a complementary DNA sequence.

A. Accuracy and Importance

• Extraordinary Accuracy: Crucial for passing unaltered genetic information.

• 3 Billion Base Pairs: Must be copied accurately in human genome.

• Mutation: Errors are rare but can lead to permanent DNA changes, potentially destroying an organism or leading to cancer.

B. Cell Types & Genetic Stability

• Germ Cells: Transmit genetic information; must be protected from mutation (germ-cell stability) to maintain the species.

• Somatic Cells: Form body; must be protected from genetic change (somatic-cell stability) to maintain body structure. Accumulation of changes leads to cancer.

• High Fidelity: DNA replication and maintenance ensure genomic integrity.

C. Basic Mechanism (Semiconservative)

• Unwinding: Two strands of DNA double helix unwind.

• Template Function: Each original strand acts as a template for a new complementary strand.

• Daughter Molecules: Two double-stranded DNA molecules, each identical to the parent.

• Semiconservative: Each new double helix has one conserved (original) strand and one newly synthesized strand. Original strands remain intact through many generations.

D. Key Players in DNA Replication

1. Origins of Replication:

   • Specific sequences, rich in A-T base pairs.

   • Recognized by initiator proteins that break hydrogen bonds.

   • Forms a replication bubble with two Y-shaped replication forks.

   • Bacteria have single origin, eukaryotes have many origins (e.g., ~220/human chromosome) to shorten replication time.

2. Replication Machine: Multienzyme complex at replication forks.

3. DNA Polymerases (The Heart of Replication):

   • Catalyze addition of nucleotides to the 3' end of a growing DNA strand, using a template.

   • Polymerization forms a phosphodiester bond.

   • Substrates are deoxyribonucleoside triphosphates (dNTPs). Energy from hydrolysis of high-energy phosphate bonds.

   • Eukaryotic varieties: α, β, γ, δ, ε (γ in mitochondria).

   • Requirements:

     • Always need a template.

     • Can only add to 3' end.

     • Cannot start from scratch, need a primer.

   • Proofreading: Monitors base-pairing, corrects errors with 3' to 5' exonuclease activity (removes incorrect nucleotide).

4. Primase:

   • An RNA polymerase.

   • Makes a short RNA primer (~10 nucleotides) using DNA as template.

   • Provides the necessary base-paired 3' end for DNA polymerase to start synthesis.

E. Leading and Lagging Strands

• DNA Polymerase Direction: Can only make DNA in the 5' to 3' direction.

• Antiparallel Strands: DNA template strands run in opposite directions, creating a replication problem.

• Solution:

  • Leading Strand: Synthesized continuously in the 5' to 3' direction, towards the replication fork. Requires one primer.

  • Lagging Strand: Synthesized discontinuously in short fragments (Okazaki fragments, discovered by Reiji Okasaki in 1968) away from the replication fork (a "backstitching" maneuver). Requires a new primer for each fragment.

F. Lagging Strand Processing

• RNA primers (approx. 10 nucleotides) are made every ~200 nucleotides.

• Nucleases: Degrade RNA primers, leaving gaps.

• Repair Polymerase (DNA Polymerase): Replaces RNA with DNA.

• DNA Ligase: Joins fragments by forming a phosphodiester bond (nick sealing, requires ATP).

G. Additional Proteins at Replication Fork

• DNA Helicase: Opens DNA helix.

• Single-strand DNA-binding proteins: Prevent re-pairing of separated strands.

• Sliding Clamp: Holds DNA polymerase in place.

• Topoisomerase: Prevents supercoiling ahead of the fork by making temporary nicks.

H. Telomeres & Chromosome Ends

• Linear Chromosome Problem: The lagging strand cannot fully replicate at the very end, leading to progressive shortening with each division.

• Telomeres: Specialized DNA "caps" at eukaryotic chromosome tips (e.g., 5'-TTAGGG-3' in humans).

  • Non-coding repeats: Slowly eroded over divisions.

  • Protection: Safeguard internal gene-bearing regions.

• Telomerase: A ribonucleoprotein (RNP).

  • Telomerase RNA: Provides template for telomeric repeat synthesis.

  • Telomerase reverse transcriptase: (RNA-dependent DNA polymerase) extends the template strand.

  • Activity: Not usually active in somatic cells, but active in germ cells (resets telomeric clock) and some adult stem cells.

  • Clinical Relevance: Active in many cancer cells; telomerase inhibition is a potential cancer therapy target.

  • Telomere Shortening: Linked to cell aging and limited division capacity.

I. DNA Repair Mechanisms

Cells have mechanisms to correct errors and repair damage.

• Proofreading: Corrects errors during DNA replication (by DNA polymerase).

• Mismatch Repair: Corrects mispaired bases right after replication.

  • Recognition and binding by a protein complex.

  • Cutting near mismatch.

  • Excision of incorrect patch by enzymes.

  • Replacement by DNA polymerase.

  • Sealing by DNA ligase.

• DNA Damage Repair Pathways: Detect and correct damage throughout cell cycle (e.g., from UV, chemicals, X-rays, spontaneous reactions).

  • Direct Reversal: Enzymes directly undo some chemical reactions.

  • Excision Repair: Removes damaged bases.

    • Base Excision Repair: Removes single damaged base.

    • Nucleotide Excision Repair: Removes a patch of nucleotides.

  • Double-stranded Break Repair: For full chromosome breaks.

    • Non-homologous End Joining: Joins broken ends, often with nucleotide loss/addition ("messy").

    • Homologous Recombination: Uses homologous chromosome as template ("cleaner," fewer mutations).

• Clinical Relevance (Hereditary Cancers): Mutations in repair genes are linked to cancers (e.g., defects in mismatch repair leading to colon cancer, defects in nucleotide excision repair leading to extreme UV sensitivity/skin cancer).

J. PCR (Polymerase Chain Reaction)

"DNA replication in a tube."

• Technique: Makes billions of copies of a specific DNA region in vitro.

• Components:

  • DNA primers: Two specific primers flank the target region.

  • Thermostable DNA Polymerase: E.g., Taq polymerase from Thermus aquaticus.

  • dNTPs (deoxyribonucleoside triphosphates).

• Three Steps per Cycle:

  1. Denaturation: Heat to separate DNA strands.

  2. Annealing: Cool to allow primers to hybridize to complementary sequences.

  3. Extension: Incubate with DNA polymerase and dNTPs for DNA synthesis.

• Uses: Research, diagnostics, forensics.

III. Cell Cycle Control

Sophisticated control processes ensure accurate cell cycle progression and prevent uncontrolled division (cancer).

A. The Cell-Cycle Control System

• Timer Analogy: Triggers events in a set sequence via molecular switches.

• Conserved: Similar machinery across eukaryotes.

• Adaptable: Modifiable for cell types and signals.

B. Cell Division Frequency

• Varies with Cell Type: Crucial for growth, development, maintenance.

  • Frequent: Skin cells, gut lining, bone marrow.

  • Reserve Capacity: Liver cells, endothelial cells, fibroblasts.

  • Terminally Differentiated: Mature nerve and muscle cells (do not divide).

• Cancer Cells: Escape normal cell cycle controls.

C. Cell Cycle Checkpoints

Stages where cells examine internal/external cues to decide on division.

1. G₁ Checkpoint (G₁/S transition): Main decision point (restriction point).

   • Checks: Cell size, nutrients, growth factors, DNA damage.

   • No "go-ahead" cues enters G₀.

   • Once passed, cell is committed to cycle.

2. G₂ Checkpoint (G₂/M transition):

   • Checks: DNA damage, DNA replication completeness.

   • Errors pause for repair; irreparable damage apoptosis.

3. Spindle Checkpoint (Metaphase to Anaphase transition):

   • Checks: Sister chromatids correctly attached to spindle microtubules.

   • Misplaced chromosomes pause mitosis until fixed.

D. Cell Cycle Regulators

Internal and external signals activate/inactivate core proteins.

• Cyclins:

  • Undergo cycles of synthesis and degradation; levels vary.

  • Four types: G1, G1/S, S, M cyclins.

  • Form cyclin-Cdk complexes at specific stages.

  • Not only activate Cdks but also direct them to specific target proteins.

• Cyclin-Dependent Kinases (Cdks):

  • Enzymes whose levels are constant.

  • Activity rises and falls, controlled by cyclin levels.

  • Phosphorylate intracellular proteins to initiate/regulate cell cycle events.

  • Inactive without cyclin (active site obscured).

  • Require phosphorylation at a specific site for full activity.

  • Can be negatively regulated by phosphorylation at other sites (e.g., by Wee1 kinase).

• Key Cyclin-Cdk Complexes: (Concentrations of cyclins oscillate, Cdk concentration is constant)

  • G₁/S-Cdk: Triggers progression through Start transition.

  • S-Cdk: Triggers DNA replication, early mitotic events.

  • M-Cdk (MPF - Maturation-Promoting Factor): Triggers entry into mitosis (G2/M transition).

    • Accumulates during G2, activated by Cdc25 phosphatase (removes inhibitory phosphates).

    • Phosphorylates proteins for nuclear envelope breakdown, chromosome condensation, cytoskeleton rearrangement, Golgi apparatus rearrangement.

    • Deactivated by destruction of M-cyclin (cyclin B) in proteasome after mitosis.

• Cdk Inhibitor Proteins (CKIs): (e.g., p27, p16, p21)

  • Inactivate cyclin-Cdk complexes, primarily G1/S- and S-Cdks in early cell cycle.

  • Binding causes structural rearrangement in Cdk active site.

• APC/C (Anaphase-Promoting Complex/Cyclosome):

  • A ubiquitin ligase that triggers metaphase-to-anaphase transition by protein destruction (polyubiquitylates target proteins for proteasomal degradation).

  • Targets:

    • M cyclins: Destroyed in anaphase, pushes cell out of mitosis into G1.

    • Securin: Destruction allows separase (which securin inhibits) to cleave cohesin, separating sister chromatids.

E. External Signals

• Growth Factors (Mitogens):

  • Proteins released by cells to stimulate division.

  • Bind to cell surface receptors, activating signaling pathways.

  • Stimulate synthesis of G1 and G1/S cyclins, and proteins for DNA/chromosome duplication.

  • Induce exit from G₀ arrest (e.g., PDGF for fibroblasts).

• Retinoblastoma (Rb) Protein:

  • A tumor suppressor protein.

  • Negative regulator: Blocks progression from G1 to S phase.

  • Binds to transcription regulators, preventing gene activation for cell proliferation.

  • Mitogens activate G1-Cdks/G1/S-Cdks, which phosphorylate Rb. This alters Rb, releasing transcription regulators to activate proliferation genes.

• Anchorage Dependence & Density-Dependent Inhibition:

  • Normal cells: Require attachment to a substratum to divide (anchorage dependence).

  • Density-dependent inhibition: Stop dividing when they contact neighboring cells (due to production of CKIs like p16 and p27).

  • Cancer cells: Lack both anchorage dependence and density-dependent inhibition.

F. Internal Signals

• p53 ("the guardian of the genome"):

  • A tumor suppressor protein.

  • Activated by DNA damage in G1 (increases p53 concentration and activity).

  • Acts as a transcription regulator, activating transcription of p21 (a Cdk inhibitor).

  • p21 binds to G1/S-Cdk and S-Cdk, arresting the cell cycle in G1 (allows time for repair).

  • If damage is too severe, p53 induces apoptosis.

  • Clinical Relevance: Mutations in p53 are found in about half of all human cancers, leading to high mutation rates and cancerous cells.

IV. Mitosis (M Phase)

A type of cell division producing two genetically identical daughter cells from a single mother cell.

• Karyokinesis: Division of the nucleus.

• Cytokinesis: Division of the cytoplasm.

• Purpose: Development, growth, and replacement of old/worn-out cells.

• Walther Flemming (1882): First detailed chromosomal movements during mitosis.

A. Stages of Mitosis

1. Pre-Mitosis (Late G₂ Interphase):

   • DNA copied, chromosomes have two sister chromatids (2n, 2C).

   • Chromosomes are decondensed.

   • Centrosomes duplicated, preparing for spindle formation.

   • M-Cdk activated at G2/M transition via Cdc25.

2. Prophase:

   • Cell breaks down structures and builds new ones.

   • Replicated chromosomes condense (appear as long threads).

   • Each chromosome has two sister chromatids joined at the centromere by cohesins.

   • Mitotic spindle begins to assemble between separating centrosomes.

   • Nucleolus disappears.

   • Nuclear membrane still visible.

3. Prometaphase:

   • Nuclear envelope breaks down (M-Cdk phosphorylation of nuclear lamins).

   • Mitotic spindle grows and accesses chromosomes.

   • Chromosomes attach to spindle microtubules via kinetochores.

   • Active movement of chromosomes towards the equator.

   • Nucleus no longer visible.

4. Metaphase:

   • Spindle captures all chromosomes.

   • Chromosomes align at the metaphase plate (middle of the cell).

   • Kinetochore microtubules attach sister chromatids to opposite poles.

   • Spindle Checkpoint: Ensures all chromosomes are correctly aligned and attached. Halts division if not.

5. Anaphase:

   • Sister chromatids synchronously separate (cohesins break down by separase).

   • Each former chromatid is now an individual chromosome (1C).

   • Chromosomes are pulled towards opposite spindle poles.

   • Anaphase A: Kinetochore microtubules shorten, pulling chromosomes poleward.

   • Anaphase B: Spindle poles move apart, further segregating chromosomes (involving motor proteins and interpolar/astral microtubules).

   • APC/C activity initiated (destruction of securin and M-cyclins).

6. Telophase:

   • Two sets of chromosomes arrive at spindle poles.

   • Mitotic spindle disassembles.

   • New nuclear envelope reassembles around each set of chromosomes (due to dephosphorylation of nuclear pore proteins/lamins).

   • Chromosomes begin to decondense.

   • Nucleoli reappear.

   • Cytoplasm division begins with contractile ring assembly.

7. Cytokinesis:

   • Division of the cytoplasm, overlaps with late telophase.

   • Cleavage furrow appears on the cell surface.

   • Formed by the contractile ring (actin filaments, myosin II, structural/regulatory proteins).

   • Ring contracts, inserting new membrane, fully dividing the cell into two.

B. Mitotic Spindle

• Microtubule-based machine, bipolar array.

• Minus ends: Focused at spindle poles.

• Plus ends: Radiate outward.

• M-Cdk: Triggers assembly.

• Types of Microtubules:

  • Interpolar MTs: Overlap in spindle mid-zone.

  • Kinetochore MTs: Attach to sister-chromatid pairs at kinetochores.

  • Astral MTs: Radiate from poles, contact cell cortex, position spindle.

• Motor Proteins: (Kinesins, Dyneins) Contribute to spindle assembly and function, moving along MTs.

C. Cohesin & Condensin

• Structurally Related: Both form ring structures around chromosomal DNA.

• Cohesin: Holds sister chromatids together from S phase until anaphase. Disruption causes developmental disorders (e.g., Roberts Syndrome).

• Condensin: Promotes chromosome condensation (compaction) and sister-chromatid resolution for separation.

D. Kinetochores

• Giant, multilayered protein complex built at the centromeric region of each chromatid.

• Connect sister chromatids to spindle microtubules.

• Each sister chromatid has its own kinetochore.

• Can bind 10-40 MTs in animal cells.

V. Meiosis

A specialized cell division process that converts a diploid cell (2n) into haploid cells (1n), essential for sexual reproduction.

A. Overview

• Sexual Reproduction: Mixes parental genomes to create genetically distinct offspring.

• Diploid Organisms (2n): Contain two copies of each chromosome (homologs).

• Haploid Cells (n): Gametes (sperm and eggs) produced by meiosis, containing a single copy of each chromosome.

• Fertilization: Fusion of sperm and egg forms a diploid zygote.

B. Key Events

• One Round of Chromosome Replication.

• Two Successive Rounds of Division:

  • Meiosis I: Homologue pairs separate (reductional division).

  • Meiosis II: Sister chromatids separate (equational division, similar to mitosis).

• Each division round includes Prophase, Metaphase, Anaphase, Telophase (I and II).

C. Meiosis I Stages

1. Interphase (G₁, S, G₂): Similar to mitosis.

   • S phase: DNA replicated; each chromosome has two sister chromatids (2c).

2. Prophase I: Longest and most complex phase.

   • Homologous chromosomes pair (synapsis) to form bivalents (tetrads).

   • Crossing Over/Recombination: Exchange of genetic material between non-sister chromatids.

     • Increases genetic diversity.

     • Occurs at sites called recombination nodules along the synaptonemal complex.

   • Synaptonemal Complex: Protein structure that facilitates synapsis.

   • Chiasmata: Inter-homolog connections post-crossing over.

   • Substages: Leptotene, Zygotene, Pachytene, Diplotene, Diakinesis.

   • X and Y Chromosomes: Pair in a small homologous region.

3. Metaphase I:

   • Homologous pairs line up at the metaphase plate.

   • Random Orientation: Orientation of each homologous pair is random, further increasing genetic diversity.

4. Anaphase I:

   • Homologous chromosomes separate, pulled to opposite poles.

   • Sister chromatids remain together (cohesins at centromere persist).

5. Telophase I & Cytokinesis:

   • Separated chromosomes arrive at poles.

   • Nuclear membrane reforms, chromosomes decondense.

   • Cytokinesis usually occurs, forming two haploid daughter cells (1n, 2C). Each chromosome still has two sister chromatids.

D. Meiosis II Stages

• No DNA Replication between Meiosis I and II.

• Cells are haploid (1 chromosome from each homologous pair), but chromosomes still have two sister chromatids.

• Similar to mitosis ("mitosis for haploid cells").

• Anaphase II: Sister chromatids separate and are pulled to opposite poles.

• Telophase II & Cytokinesis: Nuclear membranes form, chromosomes decondense.

  • Forms four haploid cells (1n, 1C), each with one chromatid.

  • In humans, these are sperm or egg cells.

E. Genetic Diversity Sources

1. Crossing Over: Random exchange of segments between homologous chromosomes.

2. Random Orientation of Homologue Pairs: During Metaphase I. (e.g., >8 million possible gametes in humans based on this alone).

F. Gamete Formation (Humans)

• Oogenesis (Female):

  • Ovum/Oocyte: Largest cell, eccentric nucleus, no centrioles. Has nutrient reserves, RNA, cortical granules.

  • Surrounding Layers: Zona Pellucida (mechanical protection, sperm binding, polyspermy barrier) and Corona Radiata (nutritional follicular cells).

  • Timing: Primary oocytes enter Prophase I in embryo and arrest for decades. Secondary oocyte forms after Meiosis I, arrests in Metaphase II.

  • Meiosis II only completed upon fertilization. If no fertilization, ovum dies by apoptosis.

  • Asymmetric division: Results in one large ovum and small polar bodies.

• Spermatogenesis (Male):

  • Spermatogonia: Divide by mitosis and meiosis from puberty.

  • Primary Spermatocytes: Undergo Meiosis I.

  • Secondary Spermatocytes: Undergo Meiosis II.

  • Spermatids: Haploid, non-motile.

  • Spermatozoon: Mature, motile (spermiogenesis maturation).

  • Spermatozoon Structure: Head (nucleus, acrosome with hydrolytic enzymes), Neck (mitochondria), Tail (flagellum).

  • Symmetric division results in four functional sperm.

VI. Cell Differentiation & Stem Cells

The process by which cells become specialized, guided by intrinsic and extrinsic information.

A. Cell Differentiation

• Process: Cells gain a final cell type identity.

• Developmental Potential: Becomes more restricted with differentiation.

• Gene Control: Largely controlled by genes (specific gene expression patterns for each cell type).

• Information Guiding Behavior:

  • Intrinsic (Lineage) Information: Inherited from mother cell (e.g., neural lineage molecules).

  • Extrinsic (Positional) Information: Received from surroundings (e.g., chemical signals from neighbors).

B. Stem Cells

• Characteristics:

  • Undifferentiated: Can differentiate into various cell types.

  • Unlimited Division: By symmetric and asymmetric divisions.

  • Self-Renewal Capability: Produce functionally identical daughter cells.

• Asymmetric Cell Division: Produces two different daughter cells: one remains a stem cell (self-renewal), the other differentiates or becomes a precursor.

• Precursor/Progenitor Cells: Unspecialized, can divide to yield specialized cells, but not capable of self-renewal.

C. Stem Cell Potency

• Totipotent: Can produce all cell types of the developing organism, including embryonic and extraembryonic tissues (can form a complete organism).

• Pluripotent: Can make any cell of the body (embryo proper, germ cells, all three germ layers).

• Multipotent: Can make cells within a given germ layer (e.g., hematopoietic stem cells make blood cells, but not neural cells).

• Unipotent: Makes cells of a single cell type, but can self-renew (e.g., gamete-forming stem cells).

D. Types of Stem Cells

• Embryonic Stem Cells (ESCs):

  • Pluripotent: Differentiate into all three germ layers (>220 cell types).

  • Derived from the inner cell mass of a blastocyst.

  • Ethical issues associated with their use.

• Adult Stem Cells (Somatic Stem Cells):

  • Undifferentiated, found in differentiated tissues/organs.

  • Perform tissue repair and regeneration.

  • Typically multipotent (limited range of cell types).

  • Examples: Hematopoietic, neural, epithelial, skin stem cells.

• Induced Pluripotent Stem Cells (iPSCs):

  • Adult cells genetically reprogrammed (de-differentiated) to an ESC-like state.

  • Express genes and factors important for ES cell properties.

  • Pluripotent: Can generate cells of all three germ layers.

  • Nobel Prize 2012 (Yamanaka and Gurdon).

  • Advantage: No need for embryos, cells from patient avoid immune rejection.

  • Applications: Study development, model diseases, drug testing, potential regenerative therapies (e.g., Parkinson's, Alzheimer's).

VII. Cell Death

Essential for growth, development, and maintenance of multicellular organisms.

A. Balance of Life & Death

• Proliferation balanced by cell death to limit organism size.

• Controlled Process: Cell death is not random but highly regulated.

B. Types of Cell Death

1. Necrosis:

   • Accidental: Caused by trauma, lack of blood, toxins.

   • Only type seen in unicellular organisms.

   • Messy: Cell swells and bursts, spilling contents and causing inflammation.

   • Typically affects cell groups.

2. Apoptosis (Programmed Cell Death):

   • Deliberate: Built-in suicide mechanism, triggered by specific regulatory signals.

   • Physiologic conditions: Normal removal of cells.

   • Tidy: Cell shrinks, condenses, forms "blebs" and apoptotic bodies (membrane-enclosed fragments).

   • Phagocytosis: Apoptotic bodies display phosphatidylserine (usually inner membrane) as an "eat me" signal for macrophages, preventing inflammation.

C. Functions of Apoptosis

• Sculpting Tissues: E.g., forming digits during embryonic development.

• Neuronal Pruning: Removing unneeded neurons.

• Quality Control: Eliminating abnormal, misplaced, nonfunctional, or dangerous cells (e.g., self-reactive lymphocytes).

• Homeostasis: Removing old cells for new ones.

• Eliminating Threats: Infected cells, cancerous cells, pathogen-specific immune cells after infection.

• DNA Damage: When defects are too severe to repair.

D. Mechanisms of Apoptosis: Caspases

• Caspases: Family of specialized intracellular proteases.

  • Cysteine at active site, cleave target proteins at aspartic acids.

  • Synthesized as inactive precursors, activated only during apoptosis.

  • Form an amplifying proteolytic cascade (destructive, self-amplifying, irreversible).

• Initiator Caspases: (e.g., Caspase-8, Caspase-9)

  • Begin the apoptotic process.

  • Exist as inactive monomers; an apoptotic signal leads to formation of large protein platforms (e.g., DISC, apoptosome).

  • Activate executioner caspases.

• Executioner Caspases:

  • Exist as inactive dimers; cleaved by initiator caspases to become active.

  • Catalyze widespread protein cleavage events that kill the cell.

• Target Proteins of Caspases:

  • Nuclear lamins: Cleavage breaks down nuclear lamina.

  • DNA degrading endonuclease inhibitor: Cleavage (of iCAD) frees CAD endonuclease to cut chromosomal DNA into fragments (ladder pattern on gel electrophoresis; detectable by TUNEL technique).

  • Cytoskeleton and cell-cell adhesion proteins: Cleavage helps cell round up and detach.

E. Apoptotic Pathways

1. Extrinsic Pathway:

   • Triggered by extracellular signal proteins binding to cell-surface death receptors.

   • Death Receptors: Transmembrane proteins (e.g., Fas death receptor, TNF receptor) with an intracellular death domain. Activated by death ligands (e.g., Fas ligand).

   • FasL/FasR Model: Activation of Fas death receptor by Fas ligand leads to adaptor protein (FADD) binding, then initiator caspase-8 binding to form DISC (death-inducing signaling complex).

   • Activated initiator caspases then activate executioner caspases.

2. Intrinsic (Mitochondrial) Pathway:

   • Triggered by intracellular factors (DNA damage, developmental signals, absence of growth factors).

   • Involves mitochondria: release of mitochondrial proteins into the cytosol.

   • Cytochrome c: Key protein released from mitochondria into cytosol.

   • Released cytochrome c binds to Apaf1 (adaptor protein), forming a wheel-like apoptosome.

   • Apoptosome recruits and activates initiator caspase-9, which then activates executioner caspases.

F. Regulators of Intrinsic Pathway (Bcl2 Family)

• Bcl2 Family Proteins: Control release of cytochrome c and other intermembrane mitochondrial proteins.

  • Pro-apoptotic effectors (Bax, Bak): Activated by apoptotic stimuli, form oligomers in mitochondrial outer membrane, induce protein release.

  • Anti-apoptotic (Bcl2, BclXL): Prevent protein release, inhibit pro-apoptotic family members.

  • BH3-only proteins (Puma, Noxa, Bad): Largest subclass; produced/activated by apoptotic stimuli; promote apoptosis by inhibiting anti-apoptotic Bcl2 proteins.

• IAPs (Inhibitors of Apoptosis Proteins):

  • Negative regulators of caspases and cell death.

  • Bind with activated caspases (via BIR domain) and mark them for destruction by proteasomes (polyubiquitylation).

• Anti-IAPs: Neutralize the inhibitory effect of IAPs, produced in response to apoptotic stimuli.

• Clinical Relevance: The ability of cells to escape apoptosis is a major cause of cancer.