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Cell Junctions _ Cell Adhesion

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					Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

CELL JUNCTIONS AND CELL ADHESION
Reading Assignment: Molecular Biology of the Cell, by Alberts, Chapter 19, pp 1065-1078, 1080-1090 KEY CONCEPTS AND LEARNING OBJECTIVES 1. The major characteristics of connective tissue are distinct from those of epithelial tissue. a. Compare the relative density of cells and the relative amount of extracellular matrix within connective and epithelial tissues. b. Identify the component (extracellular matrix or cell junction) that bears most of the mechanical stress exerted on connective tissue and on epithelial tissue. Long-term connections between cells require the complex structures of cell junctions. a. List the three major classes of cellular junctions. Tight or occluding junctions (zonula occludens) seal cells of an epithelial sheet to create a permeability barrier. They serve a similar function in brain capillary endothelium. a. Explain the function of tight junctions in blood vessels and in the epithelial cells which line the intestinal lumen. b. Explain the importance of transcellular transport of nutrients across sheets of epithelial cells that are linked by tight junctions. c. Explain the process of paracellular transport. Plaque or anchoring junctions mechanically attach cells and their cytoskeleton to their neighbors or to the extracellular matrix, and they stabilize cells against mechanical stress. a. List the different types of anchoring junctions. b. For each type of anchoring junction, identify the class of transmembrane adhesive proteins and the cytoskeletal proteins to which the adhesive proteins attach. c. Predict how the strength of anchoring junctions is impacted by removing Ca2+. d. Illustrate the importance of desmosomes using a clinical example.

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

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Cell-cell adhesion is mediated by plasma membrane glycoproteins. a. Identify the plasma membrane glycoproteins that mediate cell-cell adhesion. b. Provide an example of the role of cadherins in development. c. Explain how the binding of cadherins can be regulated. d. Explain the role of selectins with the interaction of white blood cells and endothelial cells. Communicating or gap junctions allow small molecules and electrical signals to pass between interacting cells. a. Identify the transmembrane proteins that form gap junctions. b. Identify specific types of molecules that are able to pass through gap junctions and those that are unable to pass through gap junctions. c. Explain how the permeability of gap junctions is regulated.

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

CHARACTERISTICS OF CONNECTIVE TISSUE AND EPITHELIAL TISSUE

Figure 19 –1 A cross-sectional view of part of the wall of the intestine. This long, tubelike organ is constructed from epithelial tissue (red), connective tissue (green), and muscle tissue (yellow). Each tissue is an organized assembly of cells held together by cell– cell adhesions, extracellular matrix, or both. CONNECTIVE TISSUE 1. 2. 3. 4. Plentiful ECM Sparse distribution of cells in ECM Matrix is rich in fibrous polymers (e.g., collagen). Matrix bears most mechanical stress.

EPITHELIAL TISSUE 1. Scanty ECM - mainly a thin mat of basal lamina 2. Many cells. The cells occupy most of the tissue volume. 3. Cells are tightly bound together in sheets. 4. Cells bear most of the mechanical stress through junctional complexes that attach to the cytoskeleton.

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

LONG-TERM CONNECTIONS BETWEEN CELLS REQUIRE THE COMPLEX STRUCTURES OF CELL JUNCTIONS.

Cell Junction
1. Tight (Occluding) Junctions

Function
Seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them

2. Anchoring Junctions Adherens Junction

Desmosome

Hemidesmosome

Focal Contact/Adhesion

Joins an actin bundle in one cell to an actin bundle in a neighboring cell Cell-Cell Adhesion “Spot weld” that anchors the intermediate filaments in one cell to those in a neighboring cell Cell-Cell Adhesion Anchors intermediate filaments in a cell to the basal lamina or underlying extracellular matrix Cell-Matrix Adhesion Anchors actin in a cell to the underlying extracellular matrix. Cell-Matrix Adhesion

3. Communicating Junctions Gap Junction Chemical Synapse

Cell-cell junction which allows the passage of small water-soluble ions and molecules Facilitates a type of neurotransmission

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

TIGHT OR OCCLUDING JUNCTIONS (ZONULA OCCLUDENS) SEAL CELLS OF AN EPITHELIAL SHEET TO CREATE A PERMEABILITY BARRIER.

Functions 1. Seals cells to create permeability barrier Transcellular Transport Blood-Brain Barrier 2. Apical vs. Basolateral Protein/Lipid sorting Molecular Composition 1. Claudin Major transmemebrane proteins 2. Occludin Transmembrane Proteins 3. ZO Proteins Peripheral plasma membrane proteins. Attach to actin cytoskeleton.

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

Identification of Claudin (band 9) JCB 141: 1539-1550,01998

Fig. 2. Behavior of the occludin and nine guanidine- insoluble bands on sonication followed by sucrose density gradient centrifugation. (c) Fractionation of sonicated junction fraction. After sonication, the isolated junctions were fractionated by stepwise sucrose density gradient centrifugation. 0:25%, 25:30%, 30:34%, 34:38%, 38:42%, and 42:50% interfaces were collected, and subjected to SDS-PAGE followed by silver staining. The distribution of the occludin band (occludin) was compared with those of nine guanidine-insoluble bands (band 1-9), and only band 9 was copartitioned with occludin. Silver staining and accompanying immunoblots with anti-occludin mAb (Oc-1) revealed that occludin was mainly recovered at 25:30%, 30:34%, and 34:38% interfaces, where band 9 was also characteristically accumulated. Bars in c indicate molecular masses of 200, 116, 97, 66, 45, 31, and 21 kD, respectively, from the top. Bar, 0.2 µm

Fig. 5. Colocalization of FLAG-tagged claudin-1 and -2 with occludin in MDCK transfectants. Confluent cultures of MDCK transfectants expressing FLAG-claudin-1 or FLAG-claudin-2 (FLAG-tagged claudin-1 or FLAG-tagged claudin-2) were doubly stained with mouse anti-FLAG mAb (antiFLAG) and rat anti-occludin mAb MOC37 (anti-occludin). Images were obtained at the focal plane of the most apical region of lateral membranes by confocal microscopy. Both FLAG-claudin-1 and FLAG- claudin2 were precisely colocalized with occludin at tight junction regions. Bar, 10 µm.

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

ANCHORING JUNCTIONS MECHANICALLY ATTACH CELLS AND THEIR CYTOSKELETON TO THEIR NEIGHBORS OR TO THE EXTRACELLULAR MATRIX. THESE JUNCTIONS STABILIZE CELLS AGAINST MECHANICAL STRESS.

ANCHORING JUNCTIONS Table 19-2 Junction Transmembrane Adhesion Protein Cell-Cell Adherens Junction

Extracellular Ligand

Intracellular Cytoskeletal Attachment Actin Filaments

Intracellular Anchor Proteins -Catenin -Catenin Plakoglobin Vinculin -Actinin Desmoplakin Plakoglobin

Cadherins (E-cadherin)

Cadherin in neighboring cell

Desmosomes Cadherins (desmoglein, desmocollin) Cell-Matrix Focal Integrins Adhesions (Contacts)

Cadherin in neighboring cell

Intermediate Filaments

Extracellular matrix Actin Filaments proteins

Talin, Vinculin -Actinin Filamin Plectin BP230

Hemidesmosome Integrins

Extracellular matrix Intermediate Proteins Filaments

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

ADHERENS JUNCTIONS 1. Form an adhesive belt just below tight junctions 2. Cadherins: Ca2+-Dependent transmembrane adhesion molecules. Homophilic. 3. Anchor (Plaque) proteins (-catenin, -catenin, vinculin, -actinin) anchor cadherins to contractile actin 4. Required for tight junctions to form.

DESMOSOMES 1. Spot welds that anchor cells together via intermediate filament attachment 2. Desmosomal Caherins: Desmogleins and Desmocollins 3. Plaque Proteins (plakoglobin, desmoplakin) anchor desmosomal cadherins to intermediate filaments 4. Pemphigus: Auto antibodies against desmosomal cadherins cause blistering.

Tonofilaments: Bundles of cytokeratin intermediate filaments

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

FOCAL CONTACTS 1. Link Extracellular Matrix (ECM) to actin filaments so cells can “hang on” to surroundings. 2. Integrins: Transmembrane ECM binding proteins 3. Bind to actin indirectly via anchor proteins (talin, -actinin, filamin, vinculin).

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

HEMIDESMOSOMES (“Half Desmosomes”) 1. Distribute shear forces on an epithelium to the basal lamina (ECM) 2. Integrins mediate basal lamina adhesion 3. Anchor proteins link integrins to intermediate filaments via plectin

Nu, Nucleus De, Desmosome HD, Hemidesmosome LD, Lamina Densa JBC 150: 1149-60, 2000.

CELL-CELL ADHESION 1. Tissue Assembly a. Selective adhesion retains cells b. Can adhere in place or after migration

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

CADHERINS MEDIATE CALCIUM-DEPENDENT CELL-CELL ADHESION 1. Cadherins: Ca2+-dependent cell-cell adhesion molecules 2. Single pass transmembrane proteins. 3. Ca2+ binds cadherin repeats in extracellular domain to stabilize structure 4. Members a. E Cadherin: Epithelial b. N Cadherin: Neurons, heart, fibroblasts, skeletal muscle… c. P Cadherin: Placenta, epidermis, breast epithilium d. VE Cadherin: Vascular Endothelium

Homophilic Binding

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

L CELL FIBROBLAST CELL-CELL ADHESION ASSAY

J. Cell Biol.136: 1109-1121, 1997

Figure 6. LI-cadherin mediates aggregation of transfected L cells. Aggregation of LI-cadherin expressing L cells was analyzed in the presence of 2 mM CaCl2 (a), 2 mM EDTA (b) or anti-LI-cadherin pAb120 (c). For the disruption of the cytoskeleton (d), cells were preincubated with 1 µM cytochalasin D for 30 min at 37°C. LI-cadherin acted as a Ca2+-dependent cell adhesion molecule when expressed in L cells. Its function was not affected by the disruption of the actin cytoskeleton.

SELECTINS MEDIATE TRANSIENTCELL-CELL ADHESION IN THE BLOODSTREAM 1. Ca2+-dependent, transient cell-cell adhesion 2. Lectins: Carbohydrate-binding 3. Heterophilic 4. Cooperate with integrins for strong adhesion

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

MEMBERS OF THE IMMUNOGLOBULIN SUPERFAMILY OF PROTEINS MEDIATE CALCIUM-INDEPENDENT CELL-CELL ADHESION 1. Weaker, fine-tuned adhesion a. Example: N-CAM 2. Ca2+-independent.

Usually homophilic, but ICAM mediates heterophilic integrinmediated adhesion on endothelial cells

COMMUNICATING GAP JUNCTIONS ALLOW SMALL MOLECULES AND ELECTRICAL SIGNALS TO PASS BETWEEN INTERACTING CELLS 1. Connexins a. 4 pass transmembrane proteins b. 6 connexins form a functional pore: Connexon c. Permeability varies with connexin composition (14 genes in humans) 2. Function a. Electrically connect cells: Nerve cells b. Average small molecules throughout a tissue: Liver c. More specialized cells uncouple from cells with different cell fates: Embryogenesis 3. Regulation a. pH: Low pH closes pores b. Ca2+: High Ca2+ closes pores c. Extracellular signals: Dopamine closes pores
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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

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Cell Biology, BMSC 412 September 28, 2007 Cell Junctions and Cell Adhesion Mitch Denning, Ph.D.

Untreated

Dopamine-Treated

Lucifer yellow dye transfer in microinjected retina neurons

GENERAL ORGANIZATION OF JUNCTIONAL AND NONJUNCTIONAL ADHESION 1. Junctional Adhesion a. Thousands of low affinity interactions give strength (i.e. Velcro). b. Cytoskeletal linkage stabilizes and assists lateral clustering 2. Non-Junctional Adhesion: 10-20 nm plasma membrane spacing a. May initiate or be precursor to junctional adhesion

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