21 chapters · NCBI Bookshelf Alberts MBoC 4e (free) + 7e PDF + iBiology video

Cellular Biology — Textbook Reader

Detailed chapter-by-chapter scaffolding from Molecular Biology of the Cell (Alberts). Each chapter gives you a Big Idea, key mechanisms, common exam pitfalls, and direct links to NCBI Bookshelf 4e (fully free).

Ch 1 Cells & Genomes Ch 2 Cell Chemistry Ch 3 Proteins Ch 4 DNA & Chromosomes Ch 5 DNA Replication + Repair Ch 6 DNA → Protein Ch 7 Gene Expression Control Ch 8 Manipulating DNA/RNA/Proteins Ch 9 Visualizing Cells Ch 10 Membrane Structure Ch 11 Membrane Transport Ch 12 Compartments + Sorting Ch 13 Vesicle Traffic Ch 14 Energy Conversion Ch 15 Cell Communication Ch 16 Cytoskeleton Ch 17 Cell Cycle + Death Ch 18 Cell Division Ch 19 Junctions + ECM (JOHNSON) Ch 20 Cancer Ch 21 Lab Methods (NORTHAM)

Ch 1Cells & Genomes

Big idea. All known life is built from cells, descended from a last universal common ancestor (LUCA) ~3.5 billion years ago. Three domains — Bacteria, Archaea, Eukarya — split early. Eukaryotes arose by endosymbiosis: an archaeal host engulfed an α-proteobacterium (mitochondrion) and a cyanobacterium (chloroplast).

Cells are bounded compartments separating "self" from environment. The simplest cells (Mycoplasma, ~0.3 μm) approach the theoretical minimum genome (~500 essential genes) needed for autonomous life. Bacterial cells (~1 μm) lack membrane-bound organelles. Eukaryotic cells (~10 μm) compartmentalize functions into organelles, allowing specialization + larger genomes.

Endosymbiotic theory (Margulis 1967): mitochondria + chloroplasts evolved from free-living bacteria engulfed by ancestral eukaryotes. Evidence: their own circular genomes, 70S bacterial-style ribosomes, double membranes (host + bacterial), bacterial-style protein synthesis machinery, division by binary fission. Mitochondria most closely related to Rickettsiales (α-proteobacteria); chloroplasts to cyanobacteria.

Model organisms: E. coli (genetics workhorse), S. cerevisiae (eukaryotic genetics), C. elegans (development; Karen Kim Guisbert's organism), Drosophila (genetics + development), zebrafish (vertebrate development), mouse (mammalian genetics), human cell lines (HeLa, HEK293, MCF-7).

LUCAthree domainsendosymbiotic theoryα-proteobacteriacyanobacteriaHeLaHEK293C. elegansS. cerevisiaeMycoplasma
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Ch 3Proteins

Big idea. Proteins are the workhorses of cells — enzymes, structural elements, receptors, motors, antibodies. Their function depends entirely on 3D shape, which is determined by amino-acid sequence (Anfinsen 1972 Nobel). Folding is robust but can fail catastrophically (prion diseases, Alzheimer's amyloid).

20 standard amino acids vary in side chain (R-group): hydrophobic (Ala, Val, Leu, Ile, Phe), polar uncharged (Ser, Thr, Tyr, Cys), positive (Lys, Arg, His), negative (Asp, Glu), special (Gly, Pro). Proteins fold to bury hydrophobic residues + expose polar ones to water — the hydrophobic effect drives folding.

Four levels of structure: 1° sequence (peptide bonds, N→C); 2° α-helix + β-sheet (backbone H-bonds); 3° 3D fold of one chain; 4° multi-subunit assembly. Domains (~50–200 AA) are independent folding units, often corresponding to functions.

Misfolding is dangerous. Cells deploy chaperones (Hsp70, Hsp90, GroEL/GroES) to prevent aggregation. Damaged proteins are tagged with polyubiquitin (Lys48-linked) and degraded by the 26S proteasome. Eric Guisbert at UNomaha studies the heat shock response — chaperone induction during stress.

Allostery: effector binding at non-active site changes conformation + activity. Enables on/off + graded regulation. Hemoglobin's cooperative O₂ binding (T ↔ R state transition) is the classic example.

20 amino acidshydrophobic effectα-helixβ-sheetchaperoneHsp70/90GroEL/ESproteasome (26S)polyubiquitin K48allosteryinduced fit
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Ch 5DNA Replication + Repair

Big idea. DNA replication is semiconservative, fast, and astonishingly accurate (1 error/10⁹–10¹⁰ bp due to multi-layer fidelity). Multiple DNA repair pathways correct different damage types. Defects in repair → cancer susceptibility.

Replication starts at origins (oriC in E. coli, multiple in eukaryotes), proceeds bidirectionally. Helicase unwinds DNA; SSB coats single-stranded DNA; primase lays RNA primers; DNA pol III (bacterial) extends 5'→3'; leading strand synthesized continuously, lagging strand in Okazaki fragments later joined by ligase.

Telomerase is an RNP with intrinsic RNA template that extends 3' overhangs of telomeres. Active in germ cells + many cancers; usually OFF in somatic cells → telomere shortening with each division → senescence (Hayflick limit ~50 divisions). ~85% of cancers reactivate telomerase via TERT promoter mutations.

DNA repair pathways:
  1. Mismatch repair (MMR) — corrects misincorporations after replication. MutSα recognizes mismatch; MutLα + Exo1 excise; pol δ resynthesizes. Defects → Lynch syndrome (HNPCC).
  2. Base excision repair (BER) — small base damage (oxidation, deamination). Glycosylase removes damaged base; AP endonuclease cuts; gap filled.
  3. Nucleotide excision repair (NER) — bulky lesions (UV pyrimidine dimers). XP proteins; defects → xeroderma pigmentosum, severe UV sensitivity.
  4. Homologous recombination (HR) — error-free DSB repair using sister chromatid template. BRCA1, BRCA2, Rad51. Defects → breast/ovarian cancer.
  5. NHEJ — error-prone end joining (Ku70/80, DNA-PKcs, Lig4). Default in G1.
oriChelicaseprimaseDNA pol III/δSSBOkazaki fragmentstelomeraseHayflick limitMMRBERNERHR (BRCA1/2)NHEJLynch syndrome
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Ch 11Membrane Transport

Big idea. The lipid bilayer is impermeable to charged + polar molecules — this is a feature, not a bug. Transport proteins selectively move solutes; some are passive (down gradient), others active (against gradient, ATP-driven).

Channels form pores; once open, transport is fast (~10⁸ ions/sec) but always passive (down gradient). Channels can be ungated (water — aquaporin), voltage-gated (Na_v in neurons), ligand-gated (nicotinic ACh receptor), mechanically gated (cochlear hair cells).

Carriers (transporters) bind solute, undergo conformational cycle, release on other side. Slower than channels (~10⁴/sec). Either passive (GLUT family — facilitated diffusion) or active.

Active transport: primary uses ATP directly (Na⁺/K⁺-ATPase pumps 3 Na⁺ out + 2 K⁺ in per ATP — sets up the resting potential and gradients exploited by neurons + secondary transport). Secondary couples downhill ion gradient to uphill solute movement (Na⁺/glucose symport in intestine, Na⁺/H⁺ antiport).

Voltage-gated channels drive action potentials. The Hodgkin-Huxley model (1952 Nobel) describes Na⁺ + K⁺ kinetics. Patch clamp (Sakmann/Neher 1991 Nobel) records single-channel currents — molecular basis of bioelectricity.

channelcarrieraquaporinNa/K-ATPaseprimary active transportsecondary active transportsymportantiportvoltage-gatedligand-gatedpatch clamp
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Ch 12Intracellular Compartments + Protein Sorting

Big idea. Eukaryotic cells solve the routing problem with topogenic signals — short sequences that target proteins to nucleus, ER, mitochondria, peroxisomes, or secretion. The signal hypothesis (Blobel 1999 Nobel) explains how every protein finds its destination.

Co-translational ER import: signal peptide emerges from ribosome; SRP binds; halts translation; SRP-receptor at ER docks ribosome; translocon Sec61 opens; polypeptide threads through; signal peptidase cleaves signal. Then folding + N-glycosylation in ER lumen.

NLS (nuclear localization signal — Lys/Arg-rich) recognized by importin α/β; transit through nuclear pore complex; RanGTP releases cargo in nucleus. Mitochondrial matrix targeting sequence (positively charged amphipathic helix) binds TOM (outer) + TIM23 (inner) translocases — POST-translational. Peroxisomal targeting signal PTS1 (C-terminal SKL).

ERAD (ER-associated degradation): misfolded ER proteins retrotranslocated to cytosol → ubiquitinated → proteasome. UPR (unfolded protein response): IRE1 + PERK + ATF6 sensors → halt translation, upregulate chaperones, expand ER → if unresolved → CHOP-mediated apoptosis.

signal peptideSRPSec61NLSimportinNPCRanGTPmatrix targeting sequenceTOM/TIMPTS1/PTS2ERADUPR
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Ch 15Cell Communication

Big idea. Cells coordinate via signaling pathways that detect, amplify, integrate, and respond to extracellular cues. Pathway dysregulation underlies most cancers + many other diseases.

Receptor classes: cell-surface (GPCR, RTK, ligand-gated channel, integrin) vs intracellular (nuclear receptors for steroids, thyroid hormone — direct DNA binding via zinc-finger or related domains).

GPCR signaling: ~800 GPCRs in humans (~30% of FDA drug targets). Ligand → heterotrimeric G protein → Gα-GTP → effectors (adenylate cyclase makes cAMP → PKA; PLCβ makes IP₃ + DAG → Ca²⁺ + PKC). Desensitization by GRK + β-arrestin (clathrin endocytosis).

RTK signaling: ~60 RTKs in humans (EGFR, insulin R, VEGFR, etc.). Ligand → dimerization → trans-autophosphorylation of cytoplasmic tail Tyr → SH2/PTB-domain adaptor proteins (Grb2/SOS for Ras pathway; PI3K for PI3K-AKT pathway).

Ras-MAPK cascade: ligand → RTK → Grb2/SOS → Ras-GTP → Raf → MEK → ERK → nuclear TFs (Elk1, Fos, Myc) → proliferation. Mutated in ~30% of human cancers (KRAS G12V/D classic). BRAF V600E in melanoma.
PI3K-AKT-mTOR: RTK or GPCR → PI3K → PIP₂ → PIP₃ → recruits PH-domain proteins (AKT, PDK1) → AKT phosphorylates many targets including TSC2 → mTORC1 → translation, growth. PTEN dephosphorylates PIP₃ → tumor suppressor.

Wnt/β-catenin: Wnt → Frizzled → Dishevelled → inhibits APC/GSK3/axin destruction complex → β-catenin to nucleus → TCF/LEF target genes. APC mutated in colon cancer (familial adenomatous polyposis + sporadic).

Notch (juxtacrine): Delta/Jagged on neighbor → ADAM10 + γ-secretase cleave Notch → NICD enters nucleus → CSL → HES/HEY genes. Lateral inhibition in development.

TGF-β/SMAD: receptor Ser/Thr kinase phosphorylates R-SMADs → bind co-SMAD4 → nucleus → target genes.

GPCRRTKnuclear receptorsecond messengercAMP/PKAIP3/DAG/PKCCa²⁺Ras-Raf-MEK-ERKPI3K-AKT-mTORPTENWnt-β-cateninNotchTGF-β-SMADJAK-STAT
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Ch 16Cytoskeleton

Big idea. Three filament systems (actin, microtubules, intermediate filaments) shape cells, drive movement, and segregate chromosomes. Each has different polymerization dynamics, binding proteins, and motors.

Actin (microfilaments, 7 nm): G-actin (ATP-bound) polymerizes to F-actin. Polar (+ end fast-growing, − end slow). Treadmilling at steady state. Nucleators: Arp2/3 (branched networks → lamellipodia), formins (linear unbranched → stress fibers, contractile ring). Motor: myosin family (II = muscle + cytokinesis; V = vesicle transport).

Microtubules (25 nm): αβ-tubulin dimers polymerize with GTP. 13 protofilaments. Polar (− at MTOC/centrosome, + outward). Dynamic instability: GTP cap stabilizes growth; cap loss → catastrophe (rapid shrinkage); rescue restarts growth. Motors: kinesin (+ end, anterograde) and dynein (− end, retrograde). Drugs: colchicine + vincristine bind tubulin → block polymerization (anti-cancer); paclitaxel (Taxol) stabilizes MT → blocks depolymerization (anti-cancer).

Intermediate filaments (10 nm): coiled-coil dimers → tetramers → 8-tetramer staggered units → filaments. Non-polar, no motors, very stable. Pure mechanical scaffolds. Examples: keratins (epithelia), vimentin (mesenchymal), nuclear lamins (nuclear envelope), desmin (muscle), GFAP (astrocytes).

Cilia + flagella: 9+2 axoneme (9 outer doublets + 2 central singlets). Dynein arms drive sliding of doublets → bending. Defects → primary ciliary dyskinesia (Kartagener syndrome). Primary cilium = single immotile cilium found on most vertebrate cells; sensory + signaling (Hedgehog).

actinmicrotubuleintermediate filamenttreadmillingdynamic instabilityArp2/3forminkinesindyneinmyosin9+2 axonemecolchicine/Taxolkeratin/vimentin/lamins
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Ch 17Cell Cycle + Programmed Cell Death

Big idea. The cell cycle is regulated by oscillating CDK activity, phase-specific cyclins, and checkpoints that gate progression. p53 + Rb are master tumor suppressors. Programmed cell death (apoptosis + others) eliminates damaged or unwanted cells.

Phases: G1 (growth, decision) → S (DNA replication) → G2 (pre-mitotic) → M (mitosis + cytokinesis); G0 = quiescent.

Cyclin/CDK pairings: D + CDK4/6 (G1 → R point), E + CDK2 (G1/S), A + CDK2 (S), A + CDK1 (G2), B + CDK1 (M).

Restriction point (R) in late G1: Rb hyperphosphorylated → releases E2F TF → S-phase genes transcribed. p53/p21 can block this. In quiescent cells, Rb hypophosphorylated, E2F sequestered.

p53 ("guardian of genome"): DNA damage → ATM/ATR → p53 stabilized (otherwise MDM2 ubiquitinates it) → induces p21 (CDK inhibitor → G1 arrest) + pro-apoptotic genes (Bax, Puma). p53 mutated in ~50% of human cancers.

SAC (Spindle Assembly Checkpoint): unattached kinetochores generate Mad2-MCC → blocks APC/C-Cdc20 → securin not degraded → separase inactive → cohesin holds sister chromatids. Once all attached, MCC dissolves → APC/C active → securin destroyed → separase cleaves cohesin → anaphase. Cyclin B also degraded → CDK1 inactive → mitotic exit.

Intrinsic apoptosis: stress (DNA damage, growth factor withdrawal) → BH3-only (Bid, Bim, Puma) → BAX/BAK pore in outer mito membrane (MOMP) → cytochrome c release → cyt c + Apaf-1 + ATP form apoptosome → caspase-9 → caspase-3/7 (effector) → DNA fragmentation, cell shrinkage, apoptotic bodies. Bcl-2 + Bcl-xL are anti-apoptotic blockers.
Extrinsic apoptosis: Death ligand (FasL, TNFα) → death receptor → DISC complex → caspase-8 → caspase-3/7. Cross-talk with intrinsic via Bid → tBid → BAX/BAK.

Other PCD modes: pyroptosis (caspase-1/4/5/11 → gasdermin D pore — inflammatory; Chivero focus in NEUR 1520), necroptosis (RIPK1-RIPK3-MLKL), ferroptosis (iron-dependent lipid peroxidation), autophagy-dependent death.

G1/S/G2/Mcyclin/CDKR pointRb-E2Fp53/p21SACAPC/C-Cdc20separasecohesinBAX/BAKcytochrome capoptosomecaspase-9caspase-3/7Bcl-2 familypyroptosisnecroptosis
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Ch 19Cell Junctions, Adhesion, ECM — JOHNSON FOCUS

Big idea. Multicellular life requires cells to stick together, communicate, sense their environment, and maintain tissue architecture. Five junction types do this — and their dysregulation drives cancer metastasis (EMT). Johnson's research focuses here.

Tight junctions (zonula occludens): apical seal between epithelial cells. Strands of claudins + occludin form paracellular barrier. Cytoplasmic adaptor ZO-1 links to actin. Defines apical/basolateral polarity. Different claudins have different permeabilities — Cld-2 leaky in kidney; Cld-5 tight in brain endothelium (BBB).

Adherens junctions (zonula adherens): belt-like, just below tight junctions. E-cadherin (epithelia) makes Ca²⁺-dependent homophilic bonds with E-cadherin on neighbor cells. Cytoplasmic tail binds β-catenin → α-catenin → actin filaments. Critical for tissue integrity. Johnson's specialty.

Desmosomes (macula adherens): spot-welds for mechanical resistance. Cadherin family desmocollin + desmoglein → cytoplasmic plakoglobin + desmoplakin → keratin intermediate filaments. Skin + cardiac muscle. Pemphigus = autoantibodies vs Dsg → skin blistering.

Gap junctions: communicating channels between cells. Connexin hexamer = connexon (hemichannel). Two connexons across the gap form the channel. Permeability ~1 kDa (ions, cAMP, IP₃). Closed by acid pH, high cytoplasmic Ca²⁺, voltage, phosphorylation. Cardiac myocytes coupled via gap junctions for synchronized contraction. Johnson's specialty.

Hemidesmosomes: cell-to-ECM (basal lamina) anchors. Integrin α6β4 binds laminin in basal lamina; cytoplasmic side connects to keratin IFs (NOT actin like other integrin junctions).

Focal adhesions: another cell-to-ECM junction. Integrins (α/β heterodimers) bind ECM proteins → cytoplasmic talin/vinculin/paxillin → actin. Mechanosensing + signaling (FAK, Src). Critical in cell migration.

EMT (epithelial-to-mesenchymal transition):
  1. Epithelial cell receives EMT signal (TGF-β, Wnt, Notch, hypoxia).
  2. EMT transcription factors (Snail, Slug, ZEB1/2, Twist) upregulated.
  3. E-cadherin DOWN, N-cadherin UP (cadherin switch).
  4. Tight junctions disassemble; cell loses apico-basal polarity.
  5. β-catenin re-localizes to nucleus → drives Wnt target genes.
  6. Cytoskeleton remodels (vimentin instead of keratin IFs).
  7. Cell becomes migratory, invasive, mesenchymal-shaped.
  8. Critical in development (gastrulation, neural crest), wound healing, AND cancer metastasis.

ECM: collagen (most abundant; 28 types in humans; type IV in basal lamina), elastin (skin, blood vessels, lungs), proteoglycans (heparan + chondroitin sulfate; massive carbohydrate-protein complexes), fibronectin (ECM scaffold + integrin ligand), laminin (basal lamina). MMPs (matrix metalloproteinases) remodel ECM — normal tissue turnover + cancer invasion.

tight junctionclaudin/occludin/ZO-1adherens junctionE-cadherinα/β-catenindesmosomedesmocollin/gleingap junctionconnexonhemidesmosomeintegrin α6β4focal adhesionEMTcadherin switchcollagen IVlamininbasal laminaMMP
Johnson exam target. Be ready to diagram each junction type with its proteins + cytoskeletal link. Memorize: tight = claudin → actin; adherens = E-cad → β-cat → α-cat → actin; desmosome = Dsc/Dsg → plakoglobin/plakin → keratin; gap = connexin (no cytoskel); hemides = integrin α6β4 → keratin.
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Ch 20Cancer

Big idea. Cancer is a Darwinian process within a body — clonal selection of cells with mutations that enable proliferation + survival in disregard of homeostatic signals. Hanahan + Weinberg's "hallmarks" framework organizes the changes required.

10 Hallmarks of Cancer (Hanahan + Weinberg 2011 update):

  1. Sustained proliferative signaling (oncogenic Ras, EGFR amplification).
  2. Evading growth suppressors (loss of Rb, p53).
  3. Resisting cell death (Bcl-2 overexpression, p53 loss).
  4. Replicative immortality (telomerase reactivation).
  5. Inducing angiogenesis (VEGF).
  6. Activating invasion + metastasis (EMT, MMPs).
  7. Reprogramming energy metabolism (Warburg effect — aerobic glycolysis).
  8. Evading immune destruction (PD-L1).
  9. Tumor-promoting inflammation.
  10. Genome instability + mutation (mismatch repair defects, BRCA loss).

Oncogenes are gain-of-function mutated proto-oncogenes; dominant. Ras (~30% of human cancers) is the most common. Myc amplified or translocated (Burkitt lymphoma t(8;14)). EGFR mutated/amplified in lung adenocarcinoma. BCR-ABL from t(9;22) in CML.

Tumor suppressors are loss-of-function (Knudson 2-hit). p53 (most-mutated, ~50%), Rb (retinoblastoma), APC (FAP/sporadic colon), PTEN (multiple), BRCA1/2 (breast/ovarian), VHL (renal cell carcinoma).

Metastasis cascade: local invasion (EMT, MMP-mediated basal lamina breach) → intravasation → circulation survival → extravasation → colonization at distant site. Most cancer deaths from metastases, not primary tumor.

Modern therapies: targeted (Imatinib for BCR-ABL CML; Trastuzumab for HER2+ breast; PARP inhibitors for BRCA-deficient), immunotherapy (anti-PD-1/PD-L1 checkpoint inhibitors — Pembrolizumab, Nivolumab; CAR-T cells), chemo (DNA-damaging — cisplatin, doxorubicin; antimetabolites — methotrexate; mitotic — Taxol).

10 hallmarksoncogenetumor suppressorKnudson 2-hitRas (KRAS)MycEGFRp53RbAPCBRCA1/2PTENWarburg effectEMTVEGFPD-1/PD-L1checkpoint inhibitorCAR-T
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Ch 21Lab Techniques (Northam's lab block)

Big idea. The catalog description for BIOL 4140 emphasizes "experimental cellular biology" — tissue culture, microscopy, gene expression, high-throughput assays. This chapter scaffolds Northam's lab block.

Mammalian cell culture: keep cells in incubator at 37°C, 5% CO₂ (buffers bicarbonate medium pH), humidified. Sterile work in BSC (biosafety cabinet). Common medium: DMEM or RPMI + 10% FBS (fetal bovine serum) + antibiotics. Trypsinize to detach adherent cells; passage at 70–90% confluence.

Common cell lines: HeLa (cervical cancer, Henrietta Lacks 1951 — first immortal line), HEK293 (embryonic kidney; "easily transfectable"), NIH-3T3 (mouse fibroblast), MCF-7 (breast cancer), U2OS (osteosarcoma).

Transfection: lipofection (cationic lipid vesicles deliver DNA), electroporation (transient electric pulses), viral vectors (lentivirus integrates stably). Selection markers: G418/neomycin, puromycin, hygromycin, blasticidin.

Indirect immunofluorescence (IF):
  1. Fix cells (paraformaldehyde 4% — cross-links proteins).
  2. Permeabilize (Triton X-100 or saponin).
  3. Block (BSA 1–5% or normal serum 10%).
  4. Primary antibody (anti-target, raised in mouse/rabbit/etc.; 1–2 hr).
  5. Wash.
  6. Fluorescent secondary antibody (anti-mouse/anti-rabbit; 1 hr).
  7. Wash.
  8. DAPI (DNA counterstain) + mounting medium with anti-fade.
  9. Image on confocal/widefield.

Western blot: lyse cells → SDS-PAGE → transfer to PVDF/nitrocellulose → block → primary Ab → HRP-secondary Ab → ECL detection. Quantitative protein detection.

RT-qPCR: RNA → cDNA (reverse transcriptase) → qPCR with SYBR Green or TaqMan. ΔΔCt normalizes target Ct to housekeeping (β-actin, GAPDH, 18S) AND to control sample. Fold-change = 2^(−ΔΔCt).

FACS (flow cytometry / sorting): cells in suspension flow past laser; light scatter (size + granularity) + fluorescence (markers) detected. Sort populations of interest. Used for cell-cycle analysis (PI/DAPI), apoptosis (Annexin V/PI), surface marker phenotyping.

CRISPR-Cas9: sgRNA targets Cas9 nuclease to specific genomic site → blunt double-strand break ~3 bp upstream of PAM (NGG). Repair: NHEJ → small indels → frameshift knockout. HDR with donor template → precise edit/knockin. CRISPRi (dCas9-KRAB) represses; CRISPRa (dCas9-VP64) activates.

siRNA: 21-nt dsRNA loaded into RISC; guide strand pairs to target mRNA → cleavage. Transient knockdown (need re-delivery). shRNA: stable expression from plasmid → continuous knockdown.

Microscopy: bright-field (basic), phase contrast (unstained live cells), DIC (3D-shaded), fluorescence (multiplex with antibodies + fluorescent proteins like GFP), confocal (optical sectioning), super-resolution (STED, STORM, PALM — <100 nm), two-photon (deep tissue), light sheet (gentle, fast 3D), TEM (transmission EM, ~0.1 nm), SEM (scanning EM, surface).

FRAP (Fluorescence Recovery After Photobleaching): bleach a region of fluorescent protein → measure recovery → quantify diffusion + turnover.

FRET: donor fluorophore → acceptor energy transfer when 1–10 nm apart. Reports protein-protein interactions or conformational changes.

tissue cultureHeLa/HEK293/MCF-7trypsinizationlipofectionIF protocolWestern blotRT-qPCR ΔΔCtFACSCRISPR-Cas9siRNA/shRNAFRAPFRETconfocalSTED/STORM/PALM
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