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In vitro (Fach) / Zellbio (Lektion)

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• substrate functionalization substrate materials (plastics, glass, hydrogels) can affect cell morphology (cell shape)> can determine molecular state
• 3D culture models standard 3D culture suppors: vessels contain a thin membrane of polymer with a well defined and uniform architecture;                                           environmentally sensitive hydrogel senses changes of pH, temp, concentration of metabolites and release their load as result of such changes; 
• matrix stiffness can be controlled by differential crosslinking of hydrogel networks or collagen concentrations  tumor cells respond to matrix stiffness (more stiff, enhanced growth & disruption of polarity)
• 3D without scaffold spheroid microarray, hanging drop culture z.b. HT29 colon carcinoma 3D nature illustration
• 3D cancer models multicellular tumor spheroids tumorspheres tissue-derived tumor spheres organotypic multicellular spheroids
• 3D vs 2D shape: 3D ellipsoids with dimensions of 10-30um, 2D thickness 3um environment: 3D nealy 100% of their surface area exposed to other cells or matrix, 2D very small percentage behavior: 3D show differences in morphology, differentiation, drug metabolism, proliferation, response to stimuli.. 3D: more similar to cells in living organism
• population cell analysis vs single population averages can obscure heterogeneous cellular response
• experimental strategy of single-cell tech conclusions may be restricted by experimental strategy techs: FACS; single cell RNA-seq; proximity ligation assay (PLA); digital droplet PCR (ddPCR); single cell western (fluor imaging); life cell RNA/protein imaging (dynamics over time); single-cell nanobiopsy (illustration using SOM: each hexagon represents a group of genes whose expression patterns across samples are most similar)
• single-cell nanobiopsy illustration using SOM: each hexagon represents a group of genes whose expression patterns across samples are most similar 2D: spatial proximity corresponds to expression pattern similarity
• perturbation experiment to investigate gene function or resulting phenotype by gene overexpression, deletion and mutation transfection: (non-viral delivery): transposon; episomal non-replicative vectors; modified RNAs; proteins
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• different phenotype obtained by KD vs KO reasons: technical: difference in off-targets(CRISPR vs RNAi)                             diff mechanistics              biological: genetic robustness; network rewiring; genetic redundancy
• genetic compensation changes in RNA or protein levels that can functionally compensate for the loss of function of another gene by KO mutation; transcriptional adaptation; RNA intermediates
• networks transcriptional regulatory network; virus-host network metabolic network protein-protein interaction disease network
• PPI network graphs depiction of signaling pathways: protein interactions are represented as edges between the proteins of the pathway
• network graphs theory terms nodes: connected to each other via edges hubs: nodes with a high proportion of the total edges; removal of hubs and their edges results in a fractured network clustering coefficient: result of nodes and edges forming closed triplets path length between nodes: sum of numbers of edges; shortest as distance random network: probability to place an edge between two nodes > small world network: most nodes are not neighbours, shorter path length, high clustering coeffi >scale-invariance: networks of any size yield similar proportions of edges and nodes
• PPI methods yeast two-hybrid: problems: detection of transient interactions co-immunoprecipitation: only high affinity interactions are detected, most transient interactions are lost; Co-IP-MS(AP-MS) affinity tagging and pull down assays: loss of weakly bound proteins; TAP-MS Co-IP(AP)-MS: cell culture, affinity purification, LC-MS/MS;           variation: adding of in vivo cross-linking can provide info about protein-protein interaction and multiprotein complex structure TAP: bait expression, tandem affinity purification: two seperate tags ensure high signal-to-noise ratio
• design the perfect PPI experiment proper negative control (to identify the backgroud contaminants): Co-IP-MS biological replicates to verify robust interactions batch effect controls closely linked to biological samples minimize overexpression artefeacts consider endogeneous POI
• network perturbation genetic variant-induced perturbations >give rise to altered pheotypes such as disease network per provide explanatory connections between genotype and phenotype
• differential network mutant EGFR complexes in lung cancer e.g. immortalized bronchial epithelial cell lines
• network apply on medicine EGFR network, TAP-MS mutated and drug-sensitive forms of EGFR in lung cancer cell lines EGFR network > functional analysis > drug network > reduced drug network > drug combinations validated
• membrane-bound receptors G protein coupled receptor (GPCR) ion channel coupled receptor tyrosine kinase coupled receptor receptors with intinsic enzymatic activity
• GPCR neutral antagonist, inverse agonist Ga,b,r ligands bind to receptor, Ga releases GDP, binds GTP, subunits seperate (a and br), GaGTP binds to adenylyl cyclase (=effector), makes it active; Ga subunit hydrolyses GTP to GDP and becomes inactive, subunitsrecombine to form an inactive G protein (Ga GDP br) ATP through adenylyl cyclase to cyclic AMP, cyclic AMP through water and phosphodiesterase to AMP
• G proteins stimulating ligand: ACTH (corticotropin) inhibitory ligand: PGE1 (prostaglandin E1) receptors for stimulating/inhibitory hormone & stimulating/inhibi G-protein complex (G protein not ligand)
• GPCR effector isoforms effector: e.g. adenylyl cyclase different isoforms depend on G-protein regulation (GaGTP or Gbr or dual or conditional regulation)
• pleiotropy of GPCR signaling signaling (G-dependent/indepen), endocytosis.. ligand-directed trafficking of receptor signaling
• EGFR epidermal growth factor receptor GPCR ligand: TGF-a: recycling, sustained response             EGF: degradation, transient response response time: 24h-72h
• signal regulation via different pathways of GPCR cAMP pathway initiated by activation of beta-adrenergic receptor (epinephrine), smooth muscle relaxation, vasodilation inositol-phospholipid-Ca pathway by alpha- (epinephrine + norepinephrine), smooth muscle contraction, vasoconstriction
• transcription regulation via GRCR in zellkern CRE: cAMP response element CREB: CRE-binding protein (=TF)
• receptor tyrosine kinase (RTK) EGF and EGF receptor cytosolic tail, extracellular region ligand binds > dimerization of RTK ligand stabilize a specific relationship between 2 individual receptor molecules: without direct contact: nerve growth factor(ligand), TrkA(receptor) ligand-mediated and receptor-mediated dimeri: stem cell factor, KIT receptor-mediated dimeri: EGF, ErbB
• intracellular tyrosine kinase domain (TKD) regulatory mechanism N- and C-lobe inactive TKD structures differ > different regulatory mecha insulin receptor-like = activation loop inhibition, FGFR, activation loop blocks access to protein substrates KIT-like = juxtamembrane inhibition, juxtamembrane region interacts with the active site of kinase (including the activation loop) to stablize the inactive conformation Tie2-like = C terminal tail inhibition, C tail interact EGFR TKD: allosterically activated by direct contacts between C of "Activator" and N of "Receiver" (2 TKDs), no activation loop phosphorylation is required
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• RTK surface abundance and distribution high surface level: homo-/heterodimerization distinct domains within plasma membrane and cortical actin cytoskeleton also affect the receptor activation
• plasma membran/ endosomal localization of RTKs different signaling outputs
• endosomal signaling of RTKs endosomes sustain signals; transmit signals over long distances; as intermediate stations between PM and nucleus; specify signaling scaffolding sequestration catalysis
• EGFR trafficking dynamics (RTK) depend on biased ligands internalization (cytoplasma) recycling (plasma membrane)
• nuclear RTK signaling generation of an intracellular domain fragment, translocated by importin into the nucleus
• signal transduction of RTKs, SH2/3 domain after autophos of tyrosine> binding of cytosolic proteins with SH2 domain. SH2 domain: two major binding pockets on surface + seperated by a bulky tyrosine residue beta D5: pTyr-binding pocket and specificity-determining pocket (pTyr +3), 
• transcription regulation via RTK in zellkern ras pathway, MAPK SRE: serum response element TCF, CRF: ternary complex factor (phosp form bind to SRE)
• sevenless mutant of drosophila wildtype: R8 + Boss, R7 progenitor + Sev > R7 neuron mutant: sev- > conus cell Boss: bridge of sevenless Sev: RTK sevenless
• receptors agonist, antagonist (inverse agonist; neutral anta-competitive/non-comp) affinity, kon and koff: association and dissociation rate constant, kon/koff = affinity constant
• affinity vs intrinsic activity, receptors high affinity: bind maximally even at low concentrations, may produce only weak physiological or pharmacological response (low efficacy) low intrinsic activity: even at maximally effective dose but produce less maximum effect
• solubilization of receptors (membrane protein) critical micelle concentraion (CMC) above CMC: solubilize in micelles (detergent molecules, proteins, lipids) below: no micelles produced ionic detergents (SDS); non-ionic (DM): for integral membrane proteins (short chain may be deactivating) bile acid salts; zwitterionic
• affinity determination (ligand and receptor) surface plasmon resonance sensor (SPRS)
• in vitro vs in vivo Kd in vitro: coprecipitation etc. , analytical ultracentrifugation, SPR, isothermal titration calometry (ITC) in vivo: kd affected by competitive binding (increase kd) and molecular crowding (decrease kd); FRET (energy transfer), single-molecule fluor imaging, FCCS (cross-correlation spectroscopy) FCCS: measure protein mobility, conc, interactions; temporal fluor fluctuation under confocal laser scanning microskope; if two proteins are associated with each other > synchronized pass through, simultaneous fluctuation, increase of amplitude
• identification of receptor cDNA library, FACS
• inhibitor-based strategies for experimental conceptions mutant signaling proteins (for gain- and loss-of-function): small molecule inhibitors protein-protein interaction disruption bivalent inhibitor allosteric regulator
• agonist, antagonist ligand agonist + competitive antagonist: parallel shift of EC50, no maximum change; isoproterenol + propranolol agonist + non-competitive antagonist: no change of EC50, decreased max. response; Norepi + phenoxybenzamine
• RTK activation assay in vivo PathHunter a small peptide epitope is expressed recombinantly on the intra C-terminus of RTK. SH2 are co-expressed with EA (enzyme acceptor). Activation of receptor > dimeri and phospho of RTK with subsequent interaction with SH2. Generates an active beta-gal > chemiluminescent detection
• SH2 domain Plex assay (RTK) SH2 interaction profiling EGF treatment of COS-1 cells Luminex beads conjugated to SH2, only specific SH2 beads will bind to phospho tyrosine proteins, biotin-conjugated anti-phosphotyrosine antibody with addition of streotavidin PE, then Luminex SH2: GRB2, CRK, CRKL..
• Ras mediated kinase cascade (RTK) rasGDP> rasGTP  > GTP hydrolysis > raf activation >MEK binding and phospho > MAP phospho and activation > MAPK phospho
• molecular interaction yeast 2 hybrid: hybrid proteins(bait, prey) > transcriptional activation by hybrid proteins in yeast cells (transfected) > proteins interacting with Ras are "fished" (bait plasmid with Ras and TRP, prey plasmid with cDNA and LEU, transfection of trp-, leu-, his- yeast mutations, selection and plate on his deficient medium) FRET: YFP-RasGDP--RBD-CFP > RBDRasGTP-CFPYFP; when and where Ras is activated in response to cellular stimulation by calculating the ratio of emision at 527 and 475 nm BiFC: nonfluor fragments (YN and YC) fused to putative interaction partners respectively; multicolor BiFC, shared interaction partner (CC155) RBD: ras-binding domain CFP: 433>475 nm (donor fluorophore) YFP: > 527 nm (acceptor)

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