CSB331 Midterm 1 Study Notes

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University of Toronto St. George
Cell and Systems Biology
Katherine Sodek

CSB328 MIDTERM 1 REVIEW OVERVIEW  Epithelial tissue – mechanical stress transmitted from cell to cell across the whole epithelia by cytoskeletal filaments anchored to cell-matrix and cell-cell adhesion sites  Connective tissue –xtracellular matrix directly bears mechanical stress of tension and compression  Occluding junctions o Tight junctions – regulate what can pass through epithelia  Cell-cell anchoring junctions o Adherens junctions – link actin filaments between cells o Desmosomal junctions – link intermediate filaments between cells  Cell-matrix anchoring junctions o Integrins – anchor actin filaments o Hemidesmosomes – anchor intermediate filaments  Light microscopy cannot resolve details smaller than its own wavelength  Cell-cell junctions can be visualized with electron microscopy o Use biochemical and molecular biology methods to study function  Cell-cell junctions are symmetrical ADHERENS JUNCTIONS  Adherens junctions connects actin filament bundle in one cell with that in the next cell  Cadherin (classical cadherin) is the transmembrane adhesion protein that mediates adherens junctions o E-cadherin (many epithelia) o N-cadherin (neurons, heart, skeletal, muscle, fibroblasts) o P-cadherin (placenta, epidermis, breast epithelium) o VE-cadherin (endothelial cells)  E-cadherin expression confers the polarized epithelial phenotype o Loss of E-cadherin = EMT o Gain of E-cadherin = MET  Classical cadherins have five extracellular cadherin repeats o Ca2+ binds between E-cadherin repeats in hinge region o Ectodomain = N-terminal domain o Endodomain = C-terminal domain  Classical cadherins show homophilic binding o Knob and pocket model  EC1 domain at N-terminal  EC1 domain mediates selective binding  Cadherin-mediated adhesion is calcium dependent o Extracellular [Ca2+] > Intracellular [Ca2+] o Extracellular Ca2+ is high enough for Ca2+ to always be bound  Cells cannot regulate the extracellular Ca2+, only intracellular Ca2+ for signalling  Must artificially remove Ca2+ by adding chelator o Ca2+ < 0.05 mM  Hinge regions collapse and fold over  Unable to make interactions  Cadherins susceptible to cleavage o Ca2+ > 1 mM  Rigid rod structure  Cis interactions stabilize cadherin associations for trans interactions  Many cadherins are arranged in parallel to hold cells together  Cadherins use intracellular anchor proteins to link the cytoplasmic tail (endodomain) of the transmembrane protein to cytoskeletal filaments o Intracellular anchor protein = β-catenin and α-catenin o β-catenin binds to the tail of E-cadherin o α-catenin binds to β-catenin and other anchor proteins that connect to actin cytoskeleton  Cadherin-catenin complex o When α-catenin is bound in an adherens complex to β-catenin, α-catenin binds to actin through other anchor proteins o When force is exerted, β-catenin is phosphorylated  dissociates from E-cadherin tail  goes to the nucleus  act as transcriptional co-activator  turn on genes in Wnt signalling pathway o β-catenin is involved in EMT  Adherens junctions in polarized epithelia is maintained through endocytosis and recycling o Cadherin interactions are dynamic o Cells detaching and attaching all the time o Actomyosin contraction is transmitted throughout the epithelial layer through cadherins o Regulated by extracellular and intracellular signalling events  p120 prevents E-cadherin from becoming internalized (clathrin) and degraded (ubiquitination) o p120 is downregulated in invasive cancers via EMT so cancer cells can go off and invade  Cadherins are the main adhesion molecules holding cells in early embryos together o Removing calcium by adding EDTA (chelator) cause embryos to dissociate into single cells o Inactivation of E-cadherin cause embryos to fall apart and die early in development  siRNA knockdown of EP cadherin in xenopus  Genetic knockout of E-cadherin in mice o But there are other adhesion molecules that also require calcium TIGHT JUNCTIONS  PDZ domains (80-90 residues that fold into a β-sandwich of 5-6 β-strands and 2 α-helices) bind to C-terminal PDZ binding domain (4-5 residues) on transmembrane receptor or ion channels o ZO-1,2,3 and MUPP1 (PDZ domains) binds to C-terminal PDZ binding domain of claudins, occludins, JAMs (PDZ binding domains) o ZO family and MUPP1 link claudin to actin cytoskeleton o Scaffolding proteins have PDZ domain which localize them to the C-terminal tail of TM receptor  EC1 regulates pore formation o Claudin 1 forms tight tight junctions via attraction of charges (charge is balanced) o Claudin 2 forms leaky tight junctions via repulsion of charges (surplus of charges)  Claudin 16 and 19 form cation-selective pore in kidney nephron  In thick ascending limb of loop of Henle  enables paracellular Na+ flux from renal interstitium into urinary lumen  creates electropositive gradient  promotes paracellular Mg2+ and Ca2+ flux from urinary lumen into renal interstitium o Na+ from blood to urine through claudin 16 and 19 TJ o Mg2+ and Ca2+ from urine to blood (reabsorbed) by passive diffusion  Mutations in claudins 16 and 19 causes FHHNC o Urinary Mg2+ and Ca2+ wasting (not reabsorbed) causing renal failure because Mg2+ and Ca2+ is low in blood o Will reabsorb Ca2+ from bones instead of urine  Experiment o Immunohistochemical evidence that MDCK cells co-express claudin 1 and 4  Cultures of MDCK cells (epithelial cells) stained with anti-claudin-1,2,3,4  Claudin-1 and claudin-4 localized at polarized epithelial cells  Add CPE toxin which is a polypeptide that interferes with the function of claudins  CPE is selectively binding to or interfering with claudin 4  loss of TJ barrier function  CPE decreased permeability of epithelial cells o Causes diarrhea or death by diarrhea  Claudin 1 is not functional as a barrier on its own without claudin 4 o Transwell electropermeability assay  Check changes in barrier function by measuring electrical current  Ion flow = Increased current = Decreased resistance after CPE treatment  Embryogenesis o TJ  Na+ pump on basal side  Na+ pumped into blastocoel cavity  water pumped into blastocoel cavity  osmotic pressure  hydrostatic pressure  formation of blastocoel cavity in mammals o TJ in trophectoderm  invade into endometrium  Experiment o Effect of CPE on blastocyst formation in mouse embryo  Prevents formation of blastocoel cavity in blastocyst  CPE treatment removes claudins 4 and 6 leading to blastocyst to collapse due to insufficient fluid accumulation in the blastocoel cavity  Claudin 7 localization is not affected but is not functional o PCR to determine expression of claudin 4,6,7 o Immunohistochemistry to confirm localization to edges of blastocyst (trophectoderm) o Transwell electropermeability assay to measure functionality CELL POLARITY  Three protein complexes that define and maintain distinct regions of the cell (apical versus basolateral) o Par complex (Par3, Par6, aPKC)  the polarity complex that gets recruited to TJ and NA  Apical  Par3 and Par6 are scaffolding proteins meaning they have other binding domains  Recruits and activates Rac/Cdc42 (actin cytoskeleton dynamics) o Crumbs complex  Apical o Scribbled complex  Basolateral  Par, Crumbs and Scribbled communicate with cytoskeleton via Rho family of monomeric GTPases RHO FAMILY MONOMERIC GTPases  Certain GAPS and GEFS associated with polarity complex o Each specific to each Rho family monomeric GTPase o Speculated to enable tight temporal and spatial regulation of Rho family activity  Stimulate actin rearrangement o RhoA  stress fibres o Rac  membrane ruffling (lamellipodia) o Cdc42  microspikes (filopodia) o Rac/Cdc42 promotes lamellipodia actin protrusions  interact with Par complex to reinforce polarity o RhoA promotes actomyosin cables o Cooperation between lamellipodia actin protrusions and actomyosin cables  Under a bit of tension, RhoA activity (actomyosin contraction) extends the conforma
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