Chapter 1: Stem Cells and Their Differentiation

Stem Cell Fate Decisions

Stem cells are unique cells capable of both self-renewal and differentiation. Their fate is tightly regulated by intrinsic and extrinsic factors, and disruptions in these controls can lead to disease or be harnessed for clinical applications.

• Self-renewal: The process by which stem cells divide to produce more stem cells, maintaining the stem cell pool.

• Differentiation: The process by which stem cells become specialized cell types.

Potency Hierarchy

Stem cells are classified by their potency, or the range of cell types they can produce.

Example: Hematopoietic stem cells (HSCs) are multipotent, giving rise to all blood cell types but not other tissues.

Stem Cell Source Comparison

The Niche

• Definition: The microenvironment that maintains stem cell properties through active signaling (e.g., Wnt, Notch, BMP antagonists).

• Example: Lgr5+ intestinal stem cells require signals from adjacent Paneth cells.

Clinical Correlates

• Metaplasia: Reversible replacement of one differentiated cell type by another, driven by stem cell reprogramming (e.g., Barrett esophagus: squamous → columnar).

• Cancer stem cell hypothesis: Tumor regrowth is driven by a small, self-renewing subpopulation.

• OSKM (Yamanaka) factors: Oct4, Sox2, Klf4, c-Myc reprogram somatic cells to iPSCs; c-Myc increases oncogenic risk.

Additional Testable Details

• MAPK signaling: Promotes differentiation, opposes pluripotency.

• STAT signaling: Supports pluripotency.

• Wnt signaling: Promotes stemness in niche contexts.

• Chromatin: Undifferentiated cells have open (euchromatin-rich) chromatin for broad transcriptional access.

• Adherens junctions: E-cadherin/β-catenin anchor stem cells in the niche; loss leads to premature differentiation or cell loss.

• microRNAs: Regulate both stemness and differentiation post-transcriptionally.

Chapter 2: Extracellular Matrix and Cell Adhesion

Core ECM Components

The extracellular matrix (ECM) is a dynamic structure providing support and signaling cues to cells. Defects in ECM components lead to characteristic diseases.

Genetic ECM Disorders

Adhesion Structures

Collagen Synthesis and Related Deficiencies

• Vitamin C: Required for prolyl/lysyl hydroxylase; deficiency causes scurvy.

• Lysyl oxidase: Cross-links collagen/elastin; copper-dependent; defective in Menkes disease.

• GAG sulfation: Most GAGs are sulfated except hyaluronic acid.

Adhesion Receptor Specifics

• Integrins: Heterodimers; β2 subunit is leukocyte-specific.

• Selectins: Mediate initial leukocyte rolling on endothelium.

• Basement membrane assembly: Laminin assembles first, then recruits type IV collagen.

Chapter 3: Biological Membranes

Membrane Lipids

Membrane Asymmetry and Apoptosis

• Flippases and floppases maintain phospholipid asymmetry.

• During apoptosis, phosphatidylserine (PS) is externalized, signaling phagocytosis.

Membrane Fluidity

• Increased unsaturated fatty acids: ↑ fluidity

• Increased chain length: ↓ fluidity

• Cholesterol: Buffers fluidity (↓ at high temp, ↑ at low temp)

• Increased temperature: ↑ fluidity

Membrane Transport Types

Lipid Rafts and Caveolae

• Lipid rafts: Cholesterol/sphingolipid-rich microdomains, concentrate signaling proteins.

• Caveolae: Flask-shaped rafts formed by caveolin, involved in endocytosis and signaling.

• Disruption impairs signal transduction, not passive diffusion.

Glycocalyx and Osmotic Balance

• Glycocalyx: Carbohydrate coat for recognition, protection, signaling.

• Body water: 50–60% of weight; extracellular fluid rich in Na⁺, Cl⁻.

• Osmolality governs water distribution; osmotic shock can cause cell lysis.

Membrane Protein Mobility

• Some proteins are anchored to the cytoskeleton, restricting movement.

• FRAP measures protein mobility; slow recovery = restricted movement.

• CFTR in cystic fibrosis: Folding/trafficking defect, not a lipid problem.

Chapter 4: The Cytoskeleton

The Three Filament Systems

Actin Dynamics

• Polymerizes by adding ATP-G-actin; ATP hydrolysis destabilizes subunits, favoring depolymerization at the minus end.

• Treadmilling: Plus end grows, minus end shrinks at equal rates.

Motor Proteins

Intermediate Filament Types

Microtubule Dynamic Instability

• Microtubules grow/shrink via GTP cap; loss of cap triggers rapid depolymerization (catastrophe).

• Drugs: Colchicine/vinca alkaloids block polymerization; Taxol stabilizes microtubules, blocking disassembly.

RBC Membrane Skeleton

• Spectrin network cross-linked to actin, anchored via ankyrin and protein 4.1.

• Defects cause hereditary spherocytosis (fragile, spherical RBCs).

Ciliary Structure

• Motile cilia: 9+2 axoneme, powered by dynein.

• Primary cilia: 9+0, sensory, lack central pair.

• Defects: Primary ciliary dyskinesia (motile), PKD (primary cilia, mechanosensing).

Cytoskeletal Disease Beyond Filament Structure

• Duchenne muscular dystrophy: Dystrophin links actin to ECM; loss destabilizes muscle membrane.

• All cytoskeletal systems are integrated, not independent.

Chapter 5: The Organelles

The Nucleolus

• Site of rRNA synthesis and ribosome assembly; organized around NORs on acrocentric chromosomes.

Protein Targeting to the ER

• SRP recognizes N-terminal signal, halts translation, docks ribosome at ER.

• Defects in SRP impair ER targeting, not glycosylation.

The Protein-Processing Chain

Lysosomal Storage Diseases

Mitochondrial Genetics

• Maternal inheritance; heteroplasmy explains variable expressivity.

• High-energy tissues most affected (e.g., muscle, CNS).

• Examples: Leber hereditary optic neuropathy, Pearson syndrome.

Peroxisomal Disease

• X-linked adrenoleukodystrophy: Impaired very-long-chain fatty acid breakdown.

• Zellweger syndrome: Defective peroxisomal protein import.

Lysosomal Acidification and Pharmacology

• v-ATPases maintain acidic pH (~5); chloroquine raises pH, disrupting function.

ER Stress and the Unfolded Protein Response

• UPR reduces global protein synthesis to allow chaperones to catch up.

Autophagy and Apoptosis Signals

• Autophagosomes deliver damaged organelles to lysosomes for degradation.

• Cytochrome c release from mitochondria triggers intrinsic apoptosis pathway.

Chapters 6 & 7: Eukaryotic Genomes and DNA Replication

Chromatin Packaging Hierarchy

• DNA (2 nm) → nucleosome (~10 nm) → 30 nm fiber → loop domains → chromosome.

• Euchromatin: Dispersed, active, early S phase replication.

• Heterochromatin: Condensed, silent, late S phase replication.

• Constitutive heterochromatin: Always silent (centromeres, telomeres).

• Facultative heterochromatin: Reversibly silent (e.g., Barr body).

Epigenetic Regulation

DNA Replication: Enzymes and Steps

• PCNA: Sliding clamp for processivity.

• Topoisomerase: Relieves supercoiling.

• SSBs: Prevent premature reannealing.

Genome Composition and Repetitive DNA

• ~1.5% protein-coding; rest is regulatory and repetitive DNA.

• LINEs/SINEs: Largest fraction of repeats.

• Satellite DNA: Centromeres/telomeres; alpha satellite DNA for centromere function.

• Trinucleotide repeat expansions: Cause anticipation in diseases like Huntington's.

The Telomere Problem and Solution

• End-replication problem: Lagging strand can't fully replicate 3′ end.

• Telomerase: RNA-dependent DNA polymerase extends 3′ overhang.

• Active in stem cells, germ cells, most cancers.

DNA Repair Overview

• HR: High-fidelity, uses sister chromatid.

• NHEJ: Fast, error-prone, no template.

• PARP inhibitors: Exploit synthetic lethality in BRCA-deficient cells.

Chapter 8: Transcription

The Three RNA Polymerases

Prokaryotic vs. Eukaryotic Transcription

Promoter Recognition and Initiation

• TATA box (~25–30 bp upstream) is bound by TFIID (TBP subunit).

• TFIIH unwinds DNA and phosphorylates Pol II CTD to initiate elongation.

• CTD phosphorylation status coordinates transcription and processing.

mRNA Processing Steps

1. 5′ capping: 7-methylguanosine cap added co-transcriptionally; required for translation initiation and stability.

2. Splicing: Spliceosome removes introns (GU...AG boundaries); alternative splicing allows multiple isoforms.

3. Cleavage and polyadenylation: ~200 adenine poly(A) tail added at 3′ end; protects from degradation, regulates translation.

Post-Translational Modification Location Map

Transcriptional Regulation

• Transcription factors have DNA-binding and transactivation domains.

• Chromatin accessibility (nucleosome positioning, histone marks, DNA methylation) regulates promoter access.