Cell Junctions _ Cell Adhesion - DOC by malj

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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.

2.     Long-term connections between cells require the complex structures of cell junctions.
       a.    List the three major classes of cellular junctions.

3.     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.

4.     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.

5.   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.

6.   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.   Plentiful ECM
                2.   Sparse distribution of cells in ECM
                3.   Matrix is rich in fibrous polymers (e.g., collagen).
                4.   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.




                                                  3
                                                                          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        Function
1. Tight (Occluding)     Seals neighboring cells together in an epithelial
Junctions                sheet to prevent leakage of molecules between
                         them

2. Anchoring Junctions
Adherens Junction        Joins an actin bundle in one cell to an actin bundle
                         in a neighboring cell
                         Cell-Cell Adhesion
Desmosome                “Spot weld” that anchors the intermediate
                         filaments in one cell to those in a neighboring cell
                         Cell-Cell Adhesion
Hemidesmosome            Anchors intermediate filaments in a cell to the
                         basal lamina or underlying extracellular matrix
                         Cell-Matrix Adhesion
Focal Contact/Adhesion   Anchors actin in a cell to the underlying
                         extracellular matrix.
                         Cell-Matrix Adhesion
3. Communicating
Junctions
Gap Junction             Cell-cell junction which allows the passage of
                         small water-soluble ions and molecules
Chemical Synapse         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 (anti-
                                                                    FLAG) 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- claudin-
                                                                    2 were precisely colocalized with occludin
                                                                    at tight junction regions. Bar, 10 µm.



                                                           6
                                                                    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      Extracellular        Intracellular          Intracellular
           Adhesion Protein   Ligand               Cytoskeletal           Anchor
                                                   Attachment             Proteins
Cell-Cell
Adherens     Cadherins        Cadherin in          Actin Filaments        -Catenin
Junction      (E-cadherin)     neighboring cell                           -Catenin
                                                                          Plakoglobin
                                                                          Vinculin
                                                                          -Actinin

Desmosomes Cadherins          Cadherin in          Intermediate           Desmoplakin
            (desmoglein,       neighboring cell     Filaments             Plakoglobin
            desmocollin)

Cell-Matrix
Focal       Integrins         Extracellular matrix Actin Filaments        Talin,
Adhesions                      proteins                                   Vinculin
 (Contacts)                                                               -Actinin
                                                                          Filamin

Hemidesmosome Integrins       Extracellular matrix Intermediate           Plectin
                               Proteins             Filaments             BP230




                                        7
                                                                             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




                                                        12
                                                                           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 integrin-
                                                             mediated 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.




14
                                                                       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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