Cell Biology: Experimental Models, Biomolecules, Membranes, and the Cytoskeleton

Experimental Models in Cell Biology

Why Use Non-Human Cells to Study Human Biology?

All present-day cells share a common evolutionary ancestor, resulting in the conservation of fundamental molecular mechanisms across species. This unity allows scientists to extrapolate findings from non-human cells to human biology.

• Shared Properties: All cells use DNA as genetic material, are surrounded by plasma membranes, and utilize similar energy metabolism mechanisms.

• Experimental Generalization: Discoveries in one cell type can often be applied to others, including human cells.

Characteristics of a Good Experimental Model

• Simplicity: Simpler than humans, making molecular studies more manageable.

• Rapid Reproduction: E.g., E. coli divides every 20 minutes; yeast every 2 hours.

• Short Life Cycles: E.g., Drosophila completes a generation in 2 weeks.

• Manageable Genomes: Smaller genomes facilitate genetic analysis.

• Genetic Amenability: Susceptible to genetic manipulation (mutations, recombinant DNA).

Choosing an Experimental Model

• Research Question: The model is chosen based on the biological property under investigation.

• Complexity: Use the simplest organism that exhibits the property of interest.

• Practical Considerations: Cost, speed, and genome availability influence choice.

Common Experimental Models and Their Uses

• Escherichia coli (E. coli): Fundamental for molecular biology and biochemistry.

• Yeasts (S. cerevisiae): Simplest eukaryotic model; studies of cell division, protein sorting, transcription.

• Caenorhabditis elegans: Multicellular development and cell differentiation.

• Drosophila melanogaster: Genetics and animal development, body plan formation.

• Arabidopsis thaliana: Plant molecular biology.

• Zebrafish: Vertebrate development; transparent embryos for observation.

• Mouse (Mus musculus): Closest to human biology; disease models.

• Animal Cell Culture: Study of isolated cells (e.g., HeLa cells) for signaling and cell behavior.

• Viruses: Simple systems to study gene expression and cancer mechanisms.

Origins of Life and Biomolecules

Stanley Miller’s Experiment

Stanley Miller demonstrated that organic molecules could form spontaneously under prebiotic Earth conditions by discharging electric sparks into a mixture of H2, CH4, and NH3 with water, resulting in amino acid formation. This experiment supported the plausibility of abiotic synthesis of life's building blocks.

RNA World Hypothesis

• Proposes that early life was based on self-replicating RNA molecules.

• RNA can serve as both genetic material and a catalyst (ribozyme activity).

• Suggests RNA preceded DNA and proteins in evolution.

Evolution of Metabolism

1. Glycolysis: Anaerobic breakdown of glucose to pyruvate (net 2 ATP).

2. Photosynthesis: Harnessing sunlight, producing O2 as a byproduct.

3. Oxidative Metabolism: Efficient ATP production (36–38 ATP per glucose) using O2.

Endosymbiosis: Origin of Mitochondria and Chloroplasts

• Mitochondria evolved from aerobic bacteria internalized by an archaeal ancestor.

• Chloroplasts evolved from photosynthetic bacteria (cyanobacteria) inside plant ancestors.

Prokaryotes vs. Eukaryotes

Feature	Prokaryotes	Eukaryotes
Nucleus	Absent	Present
Organelles	Absent	Present (e.g., mitochondria, Golgi)
DNA	Single, circular	Multiple, linear
Size	~1 μm	10–100 μm
Ribosomes	Present	Present

Chemical Bonds in Biology

• Covalent Bonds: Sharing of electron pairs; strongest bonds.

• Ionic Bonds: Transfer of electrons, forming charged ions held by electrostatic attraction.

• Hydrogen Bonds: Weak, noncovalent interactions between a hydrogen atom (partial positive) and an electronegative atom (N or O).

Hydrophilic, Hydrophobic, and Amphipathic Molecules

• Hydrophilic: Polar, water-soluble, form hydrogen bonds with water.

• Hydrophobic: Nonpolar, water-insoluble, cluster together in aqueous environments.

• Amphipathic: Contain both hydrophilic and hydrophobic regions (e.g., phospholipids).

Functions of Major Biomolecules

• Carbohydrates: Energy storage (glycogen, starch), structural roles, cell recognition.

• Lipids: Energy storage, membrane structure, signaling (hormones).

• Nucleic Acids: Information storage (DNA), protein synthesis and catalysis (RNA).

• Proteins: Enzymes, structure, transport, signaling, defense.

Building and Breaking Down Macromolecules

• Dehydration (Condensation) Reactions: Join monomers by removing water.

• Hydrolysis: Break polymers by adding water.

Carbohydrate Structure and Bonds

• General Formula: (CH2O)n

• Monomers: Monosaccharides (e.g., glucose).

• Bond: Glycosidic bond (formed by dehydration).

Complex Sugars and Their Roles

• Glycogen (animals) & Starch (plants): α-1,4 glycosidic bonds; easy to break down for energy.

• Cellulose: β-1,4 glycosidic bonds; forms rigid, unbranched chains for structural support.

Phospholipids and Unique Membrane Lipids

• Common Features: Amphipathic; two hydrophobic fatty acid tails, hydrophilic phosphate head.

• Sphingomyelin: Only non-glycerol phospholipid; backbone from serine, not glycerol.

Triglyceride Structure

• Three fatty acids covalently joined to a single glycerol molecule.

Glycolipids

• Lipids with carbohydrate (e.g., glucose) head groups instead of phosphate.

DNA vs. RNA

Feature	DNA	RNA
Strandedness	Double-stranded	Single-stranded
Sugar	2'-deoxyribose	Ribose
Bases	A, T, G, C	A, U, G, C

Nucleic Acid Terminology

• Nucleoside: Base + sugar.

• Nucleotide: Nucleoside + phosphate(s).

• Nucleic Acid: Polymer of nucleotides (DNA or RNA).

Purines and Pyrimidines

• Purines: Adenine (A), Guanine (G) – two rings.

• Pyrimidines: Cytosine (C), Thymine (T), Uracil (U) – one ring.

• Base Pairing: A–T (DNA), A–U (RNA), G–C (both); via hydrogen bonds.

Amino Acids, Proteins, and Enzymes

Common Features of Amino Acids

• Central α carbon bonded to an amino group (NH3+), carboxyl group (COO–), hydrogen atom, and side chain (R group).

Functions of Proteins

• Enzymes, structural support, transport, signaling (hormones), defense (antibodies).

Hydrophobic vs. Hydrophilic Amino Acids

• Hydrophobic: Nonpolar side chains (e.g., alanine, valine, leucine, phenylalanine).

• Hydrophilic: Polar, uncharged side chains (e.g., serine, threonine, tyrosine).

• Basic: Positively charged (e.g., lysine, arginine).

• Acidic: Negatively charged (e.g., aspartic acid, glutamic acid).

Peptide Bonds

• Formed by dehydration between the α carboxyl group of one amino acid and the α amino group of another.

Levels of Protein Structure

• Primary: Amino acid sequence.

• Secondary: Local folding (α helix, β sheet) via hydrogen bonds.

• Tertiary: 3D folding of a single polypeptide.

• Quaternary: Association of multiple polypeptide chains (not all proteins have this).

Secondary Structure Bonds

• Hydrogen bonds between CO and NH groups of peptide bonds stabilize α helices and β sheets.

Prions

• Misfolded, self-replicating proteins that induce normal proteins to misfold, causing disease.

Intrinsically Disordered Proteins/Regions (IDPs/IDRs)

• Lack stable 3D structure; enable flexible, multivalent interactions and formation of biomolecular condensates.

Enzymes and Catalysis

• Enzymes are biological catalysts that increase reaction rates by lowering activation energy ($E_A$).

• They are not consumed or permanently altered and do not change reaction equilibrium.

Enzyme Models

• Lock and Key: Substrate fits exactly into the enzyme's active site.

• Induced Fit: Enzyme and substrate both change conformation for optimal binding and catalysis.

Cofactors

• Prosthetic Groups: Tightly bound, e.g., heme.

• Coenzymes: Organic molecules (often vitamins), chemically altered during reaction but recycled.

Allosteric Regulation and Feedback Inhibition

• Allosteric Regulation: Noncompetitive inhibition via binding at a site other than the active site, altering enzyme activity.

• Feedback Inhibition: End product inhibits an early enzyme in its biosynthetic pathway (e.g., isoleucine inhibits threonine deaminase).

Enzyme Binding Sites

• Active Site: Substrate and coenzymes bind here.

• Allosteric Site: Noncompetitive inhibitors bind here.

Protein Kinases and Phosphatases

• Kinases: Add phosphate groups (phosphorylation) to serine, threonine, or tyrosine residues.

• Phosphatases: Remove phosphate groups (dephosphorylation).

• Phosphorylation: Can activate or inhibit enzyme activity by altering conformation.

Membrane Structure and Function

Spontaneous Membrane Formation

• Phospholipids are amphipathic and self-assemble into bilayers in water, with hydrophobic tails inward and hydrophilic heads outward.

Roles of Membrane Proteins

• Enzymatic activity, structural support, transport, signaling, and defense.

Lipid Mobility and Membrane Fluidity

• Lipids move laterally and rotate within the bilayer; flip-flop between leaflets is rare and requires flippases.

• Fluidity is affected by temperature, fatty acid chain length, saturation, and cholesterol content.

• Cholesterol acts as a fluidity buffer, stabilizing membranes at high temperatures and preventing freezing at low temperatures.

Lipid Rafts

• Microdomains enriched in sphingomyelin, glycolipids, and cholesterol; concentrate specific proteins for signaling and trafficking.

Membrane Asymmetry

• Outer leaflet: Phosphatidylcholine, sphingomyelin.

• Inner leaflet: Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine (net negative charge).

• Maintained by flippases and other transporters.

Classes of Membrane-Associated Proteins

• Integral: Embedded in the bilayer; require detergents for extraction.

• Peripheral: Associated via ionic or protein–protein interactions; can be removed by polar reagents.

• Lipid-Linked: Covalently attached to lipids anchoring them to the membrane.

Transmembrane Protein Structure

• Hydrophobic α helices anchor proteins; amphipathic helices can form channels.

• GPCRs have seven transmembrane domains and function as receptors, not channels.

Glycocalyx

• Carbohydrate-rich coat (glycolipids and glycoproteins) on the cell surface; protects cells and mediates interactions.

Membrane Transport

Resting Membrane Potential

• Inside of the cell is negative (~−70 mV) due to ion gradients and selective permeability.

• K+ leak channels and the Na+/K+ pump maintain gradients.

Types of Membrane Transport

• Passive Diffusion: Direct movement through the bilayer; no energy required.

• Facilitated Diffusion: Via channels or carriers; no energy required.

• Active Transport: Against gradient; requires energy (usually ATP).

Barriers to Diffusion

• Hydrophobic core blocks ions and large polar molecules; small nonpolar and some small polar molecules can pass.

Channels vs. Carriers

• Channels: Open pores for rapid diffusion; no energy required.

• Carriers: Bind and transport specific molecules; slower, can mediate active or passive transport.

Channel Gating Mechanisms

• Ligand-Gated: Open in response to molecule binding (e.g., neurotransmitters).

• Voltage-Gated: Open in response to membrane potential changes.

• Second Messenger-Gated: Open in response to intracellular signals (e.g., cAMP).

Selectivity Filter and Voltage Response

• Selectivity Filter: Determines which ions pass (e.g., K+ channels lined with carbonyl oxygens).

• Voltage Response: Gating occurs when membrane potential reaches threshold, causing conformational change.

Transporter Examples

• Glucose Transporter: 12 α-helical segments; alternates between conformations to move glucose.

• Aquaporin: 6 α helices; forms water-selective channels.

• Na+/K+ Pump: Uses ATP to export 3 Na+ and import 2 K+.

• ABC Transporter: Two transmembrane and two ATP-binding domains; uses ATP hydrolysis for transport.

Importance of Na+/K+ Pump

• Maintains osmotic balance, cell volume, and ion gradients for signaling and nutrient uptake.

The Cytoskeleton: Actin, Intermediate Filaments, and Microtubules

Three Main Cytoskeletal Filaments

Filament	Diameter	Main Functions
Actin (Microfilaments)	~7 nm	Cell shape, movement
Microtubules	~25 nm	Organelle transport, mitosis
Intermediate Filaments	10–12 nm	Mechanical strength

Actin Filament Dynamics

• G Actin: Monomeric, binds ATP.

• F Actin: Filamentous, helical polymer.

• Polymerization: Favored by ATP-actin; profilin promotes ATP exchange.

• Dissociation: Favored by ADP-actin; cofilin severs filaments.

• Nucleation: Initial formation of actin trimer "seed."

• Treadmilling: Addition at plus end, loss at minus end; maintains length with turnover.

Actin-Binding Proteins

• Formins: Nucleate long, unbranched filaments.

• Arp2/3 Complex: Initiates branched filaments.

• Profilin: Promotes ATP exchange on actin.

• Cofilin: Severs filaments for remodeling.

• Tropomyosin: Stabilizes filaments.

• Spectrin: Cross-links actin in the cell cortex.

• α-Actinin: Cross-links filaments in contractile bundles.

Microvilli and Stereocilia

• Microvilli: Increase surface area for absorption (e.g., intestine).

• Stereocilia: Specialized for sound detection in auditory hair cells.

Rho Proteins

• Small GTPases that regulate actin dynamics in response to signals, activating formins and Arp2/3.

Intermediate Filaments

• Provide mechanical strength; not involved in movement.

• Composed of coiled-coil dimers, tetramers, protofilaments, and final ropelike filaments.

• Found in nuclear lamina, epithelial cells (keratins), muscle (desmin), and neurons (neurofilaments).

• Desmosomes (cell–cell) and hemidesmosomes (cell–matrix) anchor intermediate filaments for tissue stability.

Microtubules

Functions of Microtubules

• Determine cell shape, enable movement (cilia, flagella), transport organelles, and separate chromosomes during mitosis.

Microtubule Structure and Dynamics

• Composed of α- and β-tubulin dimers; 13 protofilaments form a hollow tube.

• Grow by adding GTP-tubulin to the plus end; GTP hydrolysis to GDP favors disassembly.

• Exhibit dynamic instability: alternating growth (rescue) and shrinkage (catastrophe).

Centrosome and γ-Tubulin

• Centrosome is the microtubule-organizing center; anchors minus ends and nucleates growth via γ-tubulin ring complexes.

Microtubule Motor Proteins

• Kinesins: Move toward plus end (cell periphery).

• Dyneins: Move toward minus end (centrosome); larger and more complex.

Cilia and Flagella

• Axoneme with "9 + 2" microtubule arrangement; movement driven by axonemal dyneins causing sliding and bending.

Microtubule-Targeting Drugs

• Drugs like vincristine, vinblastine, and taxol disrupt microtubule dynamics, blocking mitosis and selectively affecting rapidly dividing cancer cells.

Spindle Microtubules in Mitosis

• Kinetochore Microtubules: Attach to chromosomes.

• Interpolar Microtubules: Overlap at spindle center for structure.

• Astral Microtubules: Extend to cell cortex, help position spindle.

Mechanisms of Chromosome Separation

• Astral Microtubules: Dynein motors pull spindle poles outward.

• Interpolar Microtubules: Kinesins slide microtubules apart, pushing poles away.

• Kinetochore Microtubules: Kinesins depolymerize microtubules at kinetochores, pulling chromatids toward poles.