Cellular Biology — Study Guide

U1 · Cells, model organisms & microscopy

Cell theory

All organisms are made of cells; cells are the basic units of life; cells arise from preexisting cells (Schleiden, Schwann, Virchow).

Prokaryotes vs eukaryotes

Prokaryotes lack a nucleus and membrane organelles; eukaryotes have both. Eukaryotic cells originated by endosymbiosis (mitochondria from α-proteobacteria, chloroplasts from cyanobacteria).

Model organisms

E. coli (bacteria), S. cerevisiae (yeast), C. elegans (worm — Karen Kim Guisbert uses this), Drosophila, zebrafish, mouse, human cell culture lines (HeLa, HEK293).

Light microscopy

Resolution limit ~200 nm (Abbe). Bright-field, phase contrast, DIC, fluorescence (matters for Johnson's lab).

Confocal microscopy

Pinhole excludes out-of-focus light → optical sections of thick samples.

Super-resolution microscopy

STED, STORM, PALM beat the diffraction limit (~20-50 nm).

Electron microscopy

TEM (transmission, ~0.1 nm) and SEM (surface, ~1 nm). Requires fixation, sections.

Immunofluorescence (IF)

Primary antibody binds antigen; fluorescent secondary antibody binds primary. Used to localize proteins in cells.

U2 · Cell chemistry & biosynthesis

Water properties

Polar; H-bonds give high specific heat, surface tension, cohesion. Hydrophobic effect drives folding + membrane assembly.

Macromolecules

Proteins (AA), nucleic acids (nucleotides), polysaccharides (sugars), lipids (FAs + glycerol/sterol).

ΔG (free energy change)

ΔG < 0 = spontaneous (exergonic); ΔG > 0 = nonspontaneous (endergonic). Cells couple endergonic to exergonic via ATP hydrolysis.

ATP

Energy currency. Hydrolysis of γ-phosphate releases ~−7.3 kcal/mol under standard conditions; much more in cells.

Enzyme catalysis

Lower activation energy. Michaelis-Menten: v = V_max[S] / (K_m + [S]). K_m = [S] at half V_max.

Allosteric regulation

Effector binds non-active site, changes conformation + activity. Foundation of cell-signaling switches.

U3 · Proteins

Primary structure

Amino acid sequence (peptide bonds).

Secondary structure

Local folds — α-helix, β-sheet — stabilized by backbone H-bonds.

Tertiary structure

3D fold of one polypeptide; stabilized by H-bonds, ionic, hydrophobic interactions, disulfides.

Quaternary structure

Multi-subunit assembly (hemoglobin = 2α + 2β).

Chaperone

Hsp70, Hsp90, GroEL/ES — assist folding; rescue misfolded proteins.

Ubiquitin-proteasome

Tags damaged/regulated proteins with poly-Ub chain → 26S proteasome degrades.

Motor proteins

Myosin (actin), kinesin + dynein (microtubules); ATP-driven directional movement.

Phosphorylation

Kinase adds phosphate (Ser/Thr/Tyr), phosphatase removes. Most common reversible regulatory PTM.

U4 · DNA, chromosomes & replication

DNA structure

Antiparallel double helix; AT (2 H-bonds), GC (3). 10.5 bp/turn, major + minor grooves.

DNA replication

Semiconservative. Helicase unwinds, primase lays RNA primer, DNA pol III extends 5'→3'. Leading vs lagging strand (Okazaki fragments). Telomerase extends telomeres.

DNA repair

Mismatch repair (MMR), base excision (BER), nucleotide excision (NER), homologous recombination (HR), non-homologous end joining (NHEJ).

Chromosome structure

DNA + histones = nucleosome (~146 bp around 8 histones). 30-nm fiber → loops → chromatid in mitosis.

Centromere / kinetochore

Heterochromatic region where spindle attaches via kinetochore protein complex.

Telomere

(TTAGGG)n caps; shortens with each replication. Telomerase active in germ + stem + many cancer cells.

U5 · Gene expression

Transcription

RNA pol II makes mRNA from DNA template. Promoter (TATA, etc.) + general TFs + enhancers + activators.

RNA processing

5' cap (m7G), poly-A tail, splicing (spliceosome removes introns; alternative splicing diversifies proteome).

Translation

Ribosome reads mRNA codons via tRNA anticodons. Initiation (Met-tRNA, eIFs) → elongation → termination (release factors at stop codon).

Genetic code

Triplet, redundant (degenerate), nearly universal. Wobble at 3rd codon position.

Transcription factor (TF)

DNA-binding protein that activates/represses transcription (e.g., p53, Myc, NF-κB).

Epigenetic marks

DNA methylation (CpG), histone modifications (H3K4me3 active, H3K27me3 repressive, H3K9ac active).

miRNA

~22 nt; binds 3'UTR → translational repression or mRNA decay (RISC complex).

U6 · Membranes & transport

Lipid bilayer

Amphipathic phospholipids; fluid mosaic. Cholesterol modulates fluidity; sphingolipids cluster in lipid rafts.

Membrane proteins

Integral (transmembrane), peripheral (cytosolic-facing), lipid-anchored (GPI-anchored, prenylated).

Passive transport

Diffusion (down gradient, no ATP); facilitated diffusion through channels or carriers.

Active transport

Against gradient, requires ATP. Primary: Na⁺/K⁺-ATPase, Ca²⁺-ATPase, H⁺-ATPase. Secondary: symport/antiport coupled to ion gradient.

Na⁺/K⁺-ATPase

3 Na⁺ out, 2 K⁺ in per ATP. Sets resting potential, drives secondary transport.

Aquaporin

Water-selective channel; key in kidney, RBCs.

Action potential prep

Voltage-gated channels open in response to depolarization. (Cross-reference NEUR 1520 U4!)

U7 · Internal compartments & sorting

ER (rough)

Studded with ribosomes; site of secretory + membrane protein synthesis. Co-translational import via SRP + signal sequence.

ER (smooth)

Lipid synthesis, Ca²⁺ storage, detox (P450 enzymes in liver).

Golgi apparatus

Cis → medial → trans cisternae. Modifies glycoproteins, sorts to lysosomes / plasma / secretion.

Lysosome

Acidic (pH ~4.5) hydrolytic compartment; hydrolases tagged with mannose-6-phosphate.

Peroxisome

β-oxidation of very-long-chain fatty acids; H₂O₂ neutralized by catalase.

Mitochondrion

Double membrane; matrix has TCA cycle + mtDNA; inner membrane houses ETC + ATP synthase. Imports nuclear-encoded proteins post-translationally via TOM/TIM.

Nuclear localization signal (NLS)

Lys/Arg-rich sequence recognized by importin α/β; transport through nuclear pore.

U8 · Vesicle traffic

COPII

ER → Golgi anterograde traffic; Sar1 GTPase coordinates assembly.

COPI

Golgi → ER (retrograde) and intra-Golgi; Arf1 GTPase.

Clathrin

Plasma membrane endocytosis + Golgi → endosome. Adaptor proteins (AP1/2) link cargo to coat.

SNARE

v-SNARE (vesicle) + t-SNARE (target) zip into 4-helix bundle → membrane fusion. Specificity for compartment pairing.

Endocytosis types

Phagocytosis (large particles, immune cells), pinocytosis (fluid uptake), receptor-mediated endocytosis (LDL, transferrin).

Exocytosis

Constitutive (continuous) or regulated (Ca²⁺-triggered, e.g., insulin secretion, neurotransmitters).

U9 · Mitochondrial energy conversion

Glycolysis

Cytosolic, glucose → 2 pyruvate, net 2 ATP + 2 NADH.

TCA / Krebs cycle

Mitochondrial matrix. Acetyl-CoA → CO₂ + 3 NADH + 1 FADH₂ + 1 GTP per acetyl-CoA.

Electron transport chain

Complex I (NADH-DH), II (succinate-DH), III (cyt bc1), IV (cyt c oxidase). Electrons from NADH/FADH₂ → O₂ → H₂O. Pumps H⁺ to intermembrane space.

Chemiosmosis

Proton gradient drives ATP synthase (Complex V): F₀ rotor + F₁ catalytic head. ~32 ATP per glucose total.

Uncoupling protein (UCP1)

Brown adipose; dissipates proton gradient as heat (thermogenesis).

U10 · Cell signaling

Receptor classes

Cell-surface: GPCR, RTK, ion-channel-coupled, integrins. Intracellular: nuclear receptors (steroid, thyroid).

GPCR signaling

Ligand → Gα-GTP → effectors (adenylyl cyclase → cAMP → PKA; PLCβ → IP₃/DAG → Ca²⁺/PKC). Desensitization by GRKs + arrestins.

RTK signaling

Ligand → dimerization → trans-autophosphorylation → SH2/PTB-domain adaptors (Grb2, Shc, PI3K). Examples: EGFR, insulin receptor, VEGFR.

MAPK cascade

Ras → Raf → MEK → ERK → nuclear TFs (Elk1, Fos). Drives proliferation. Mutated in many cancers.

PI3K-AKT-mTOR

Survival + growth. PIP₂ → PIP₃ recruits AKT; PTEN reverses. mTORC1 controls translation/autophagy.

Notch

Juxtacrine; intramembrane proteolysis releases NICD → nucleus → CSL → HES/HEY targets. Lateral inhibition.

Wnt/β-catenin

Wnt → Frizzled/LRP → Dishevelled → inhibits destruction complex (APC, GSK3, axin) → β-catenin to nucleus → TCF/LEF target genes.

TGF-β / BMP

Receptor Ser/Thr kinase phosphorylates SMADs → nucleus.

Second messengers

cAMP, cGMP, IP₃, DAG, Ca²⁺, NO.

U11 · Cytoskeleton

Actin (microfilaments)

~7 nm; G-actin polymerizes (ATP-dependent) to F-actin. Polar (+/-). Regulators: profilin, cofilin, Arp2/3, formins. Cell shape, migration.

Microtubules

~25 nm; α/β-tubulin dimers (GTP-dependent). 13 protofilaments. Polar — minus end at centrosome, plus end outward. Dynamic instability (catastrophe / rescue).

Intermediate filaments

~10 nm; non-polar; coiled-coil dimers → tetramers → filaments. Mechanical support. Examples: keratins (epithelia), vimentin (mesenchymal), nuclear lamins.

Myosin

Actin-based motor. Myosin II = muscle + cytokinesis. Myosin V = vesicle transport.

Kinesin

MT + end-directed motor (anterograde transport, mitotic spindle).

Dynein

MT − end-directed (retrograde), also drives cilia/flagella.

Cilia/flagella

9+2 axoneme. Beat by dynein-driven sliding of doublets.

U12 · Cell cycle & cell death

Cell-cycle phases

G1 → S (DNA replication) → G2 → M (mitosis + cytokinesis). G0 = quiescent.

Cyclin/Cdk

CDKs are kinases; cyclins are regulatory subunits, periodically expressed. G1: cyclin D + CDK4/6; S: cyclin E/A + CDK2; M: cyclin B + CDK1.

Cell-cycle checkpoints

G1/S (Restriction): DNA damage → p53 → p21 → CDK inhibition. G2/M: damage / unreplicated DNA. M (SAC): unattached kinetochores hold APC/C.

Mitosis stages

Prophase → prometaphase → metaphase (alignment) → anaphase (cohesin cleaved by separase) → telophase → cytokinesis.

Apoptosis

Programmed cell death. Intrinsic (mitochondrial cyt c → apoptosome → caspase-9 → effector caspase-3/7); extrinsic (death receptor Fas/TNFR → caspase-8). Bcl-2 family balance.

Necroptosis / pyroptosis

Lytic forms of cell death — RIPK + MLKL; gasdermin pores (matters for Chivero's NEUR 1520 inflammasome content!).

Autophagy

Self-degradation via autophagosome → lysosome. mTORC1 inhibits, ATG genes execute.

U13 · Cell-cell junctions — Johnson focus

Tight junctions (zonula occludens)

Apical seal; claudins + occludin form paracellular barrier. Define apical/basolateral polarity.

Adherens junctions

E-cadherin (Ca²⁺-dependent) homophilic binding. Cytoplasmic tail binds β-catenin → α-catenin → actin. Johnson's specialty.

Desmosomes

Spot-weld between cells; desmocollins/desmogleins → plakoglobin/desmoplakin → intermediate filaments (keratin).

Gap junctions

Connexin hexamer = connexon; two connexons span the gap → channel for small molecules (<1 kDa: ions, cAMP, IP₃). Johnson's specialty.

Hemidesmosomes

Cell-to-ECM (basal lamina) via integrin α6β4 → keratin IFs.

Focal adhesions

Integrins + actin; mechanosensing; signal via FAK + Src.

Cadherin switching in EMT

E-cadherin ↓, N-cadherin ↑ during epithelial-to-mesenchymal transition (development + cancer metastasis).

Catenins as signals

β-catenin doubles as a Wnt-pathway TF when not sequestered at adherens junctions.

U14 · Cancer + tissues + ECM

Hanahan-Weinberg hallmarks

Sustained proliferation, evasion of growth suppressors, resisting cell death, replicative immortality, angiogenesis, invasion + metastasis, deregulated metabolism, immune evasion + inflammation, genome instability.

Oncogenes

Gain-of-function mutations in proto-oncogenes (Ras, Myc, EGFR, BRAF). Dominant.

Tumor suppressors

Loss-of-function (p53, Rb, APC, PTEN, BRCA1/2). Recessive (Knudson 2-hit).

Metastasis

EMT → local invasion → intravasation → circulation → extravasation → colonization.

ECM

Collagen (most abundant), elastin, proteoglycans (heparan/chondroitin sulfate), fibronectin, laminin (basal lamina).

Basal lamina

Sheet of laminin + type IV collagen + perlecan. Separates epithelium from CT. Barrier disrupted in carcinoma invasion.

Stem cells

Self-renewal + multipotency. Niche-dependent. Adult (intestinal crypt, bone marrow, hair follicle).

U15 · Lab techniques (Northam's lab block)

Tissue culture

Maintain mammalian cells in vitro: medium with FBS, sterile technique, passaging. Common lines: HeLa, HEK293, NIH-3T3, MCF-7.

Trypsinization

Protease detaches adherent cells for splitting/passaging.

Transfection

Introduce DNA/RNA: lipofection, electroporation, viral vectors (lentivirus).

Immunofluorescence (IF)

Fix → permeabilize → block → primary Ab → fluorescent secondary Ab → DAPI for nuclei → image. Localize proteins.

Western blot

SDS-PAGE → transfer → primary + secondary HRP Ab → ECL detection. Quantify protein.

RT-qPCR

RNA → cDNA (reverse transcriptase) → SYBR/TaqMan qPCR. Quantify mRNA. ΔΔCt analysis vs housekeeping gene.

Flow cytometry / FACS

Fluorescent markers + cytometer → analyze or sort cells by surface or DNA-content phenotypes.

siRNA / shRNA / CRISPR

Loss-of-function: siRNA transient, shRNA stable, CRISPR-Cas9 genomic edit.

Live-cell imaging

Fluorescent proteins (GFP/mCherry) + confocal/spinning disk; FRAP for diffusion; FRET for protein-protein interaction.

High-throughput assays

Multi-well plate readouts: viability (MTT, ATP), reporter, image-based phenotyping. Used in drug discovery.