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Midterm

CSB331H1 Study Guide - Midterm Guide: Pdz Domain, Adherens Junction, Cell Junction


Department
Cell and Systems Biology
Course Code
CSB331H1
Professor
Katherine Sodek
Study Guide
Midterm

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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 extracellular 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

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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
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