Introduction to MCB 252 Topic 29 Cell Junctions Prof David Rivier MCB 252 Spring 2015 MCB 252 Topic 29 Cell Junctions Lodish 20 0 20 2 and 5 5 Types of Cell Junctions First defined by EM I Anchoring junctions attach cells to each other or to extracellular matrix Adherens junctions cell cell via actin Desmosomes cell cell via IFs Hemidesmosomes cell ECM via IFs Focal adhesion cell ECM via actin Types of cell junctions II Occluding junctions function as barriers Tight junctions zonula occludens in vertebrates Septate junctions in invertebrates III Communicating junctions Gap junctions Synapses neurons Plasmodesmata plants Types of Anchoring Junctions Alberts 19 3 Focal Adhesion Original description of junctional complexes in epithelia Farquhar and Palade 1963 By EM characterized tripartite junction complex in numerous different types of epithelial cells mucosal epithelia of stomach gall bladder oviduct uterus glandular epithelia of liver pancreas stomach thyroid epithelia of pancreatic hepatic and salivary ducts kidney Lodish 20 10 Adherens Junctions Extracellular adhesion molecules Cadherins Linked to actin cytoskeleton via adaptor proteins see also Lodish Fig 20 10 Alberts et al Fig 19 15 Proteins that link Cadherin intracellular domain to the actin cytoskeleton Desmosomes Cell cell adhesions via intermediate filaments Alberts et al Fig 16 20 EM view Desmosomes What is the adhesion molecule Clue came from autoimmune disorders Pemphigus vulgaris blistering of skin and mucous membranes http tray dermatology uiowa edu PemVul01 htm Auto antibodies disrupt adhesion between epithelial cells treat cells w antibodies lose adhesion What is the antigen How would you figure it out Identified new Cadherin family member Desmoglein 3 Pemphigus foliaceous another auto immune disorder leading to blistering of superficial epidermis Auto antibodies directed against Cadherin family member Desmoglein1 Desmosome Structure Alberts et al Fig 19 11 Desmosome Components Alberts et al Fig 19 17 Extracellular adhesion molecules Desmogleins and Desmocollins the Desmosomal Cadherins Intracellular domain binding proteins Plakoglobin similar to Beta Catenin Plakophilin similar to Gamma Catenin Linkage between IFs and intracellular domain binding proteins Desmoplakin Anchoring Junctions Tight junctions Gap junctions For Each Description What do they do What are they made of What happens when they don t work Structures at Cell Cell or Cell ECM Junctions E cadherin catenins Desmosomal cadherins Desmoplakin and plakoglobin Adhesion belt Polarity of Epithelial Cells apical surface basolateral surface Tight junctions TJs prevent diffusion maintain polarity selective transport Alberts et al Fig 19 23 Transcellular transport see Lodish 11 6 Fig 11 30 for details of glucose transport from intestinal lumen to blood EM Views of Tight Junctions Lodish et al Figs 20 10 20 16 Focal connections Freeze Fracture EM View of Tight Junctions Honeycomb like networks of ridges and grooves sealing strands TJ strands between two epithelial cells Lodish et al Fig 20 16 Freeze fracture EM exposes proteins within the lipid bilayer Tight Junctions Alberts 19 26 Transcellular vs Paracellular Transport molecules move extracellularly through parts of TJs ions water small molecules Lodish et al Fig 20 19 uptake on one side and release on the other Control flow of solutes Role of Tight Junctions in Plasma Membrane Liposomes phospholipid bilayer vesicles made in vitro Fuse 2 different types of liposomes to apical surface of MDCK cells 1 fluorescent labeled outer leaflet 2 fluorescent labeled inner leaflet 1 or fuse 2 fuse MDCK cell apical signal Ca2 apical basal Ca2 apical basal apical basal Barrier Function of Tight Junctions inject electron dense tracer molecule pancreatic acinar cells secrete digestive enzymes e g amylase lipase DNAse RNAse proteolytic enzymes Apical Basolateral Alberts et al Fig 19 24 Identification of Tight Junction Components Biochemical approach 1 Start with tissue that has abundant tight junctions e g liver 2 Isolate membrane fractions enriched in tight junctions by density gradient centrifugation see Lodish Figs 3 35 3 Generate monoclonal antibodies that bind to tight junctions Tight Junction Components How do you convince yourself that your antibody recognizes a tight junction protein Immunogold EM Is this protein inside or outside of the cell First TJ protein ZO 1 zonula occludens 1 intracellular How would you find transmembrane components Immunoprecipitation identified ZO 2 ZO 3 neither is TM protein Start all over Identify new TM protein Occludin Occludin was detected over the points of membrane contact in TJs Immuno gold EM J Cell Biol 123 1777 1788 1993 How to Determine Whether Occludin is Required for Tight Junction Function in vivo Knockout Mouse The Goal YFG Replace one of the wild type copies of YFG with a null allele Cross heterozygous mice to create a homozygous mouse with two null alleles of YFG Knockout Mouse The Goal YFG Marker Gene Replace the coding region of YFG with a selectable marker gene Knockout Mouse Transfected DNA can integrate at random sites standard transgenic organism This is a relatively common event Or the Transfected DNA can Replace the Endogenous Copy of the Gene via Homologous Recombination This is a relatively rare event Gene Deletion Deletion by Homologous Recombination Marker Gene Knockout Mouse Knockout Mouse How to select for the cells in which the Occludin gene is replaced with a mutant allele a null allele in the face of the fact that most of the transformed DNA will integrate at random sites neor gene makes mammalian cells resistance to the drug G418 tkNSV makes mammalian cells sensitive to the drug ganciclovir Knockout Mouse YFG neor tkHSV Red Mouse DNA including YFG and regions upstream and downstream of YFG Blue neor gene Purple tkHSV gene Black plasmid DNA not homologous to any mouse DNA Knockout Mouse Lodish 5 40 Knockout Mouse Different Types of Transgenic Organisms Occludin was detected over the points of membrane contact in TJs Immuno gold EM J Cell Biol 123 1777 1788 1993 But Occludin knockout mice still make TJs What does that tell you Claudins 20 family members identified in humans Cell type specific expression Many are co expressed in the same tissue Ectopic expression can result in TJ formation in cells that lack TJs Different combinations of claudin expression lead to changes in paracellular permeabilities Two ways to measure paracellular permeability 1 Add