1. Cell Junctions, Cell Adhesion & Extracellular Matrix September 4, 2018 Mitch Denning, Ph.D.

2. Epithelial Tissue Connective Tissue Scanty ECM Many cells
Cells are tightly bound together in sheets
Cells bear most of the mechanical stress through junctional complexes that attach to the cytoskeleton.
Plentiful ECM
Sparse distribution of cells in ECM
Matrix is rich in fibrous polymers (e.g., collagen)
Matrix bears most mechanical stress

3. Long-Term Connections Require Cell Junctions

4. Four Functional Classes of Cell Junctions

5. Tight/Occluding Junctions
(Zonula Occludens)
Functions
Seal adjacent cells to create a permeability barrier
Intestinal Epithelium
Endothelial cells: Blood-Brain Barrier (Inhibits drug delivery to brain)
Regulates Paracellular Transport
Leakage between cells
Apical vs. Basolateral sorting
“Fence” function

6. Paracellular Transport
Transcellular
Transport
of Glucose
Paracellular Transport
Transcellular Transport: Active transport of glucose from gut lumen into cells via Na-driven glucose symporter (active transport).
Sodium and glucose are transported in the same direction.
Passive diffusion of glucose to blood via GLUT4? Channel (facilitated diffusion). Sealing and fence functions required.
Paracellular Transport: Transient breach in barrier to allow solutes and water around cells.

7. Tight/Occluding Junctions
Claudins are major tight junction proteins.
“Zip lock bag seal”

8. Anchoring Junctions Stabilize cells against mechanical stress
Mechanically attach cells and their cytoskeleton to their neighbors or to the extracellular matrix

9. Types of Anchoring Junctions

10. Adherens Junctions
Join an actin bundle in one cell to actin bundle in adjacent cell
Form adhesive belt just below tight junction
Cadherins: Ca2+-dependent transmembrane adhesion proteins
Anchor proteins connect cadherins to actin

11. Cadherins Mediate Calcium-Dependent Binding Between Cells
Homophilic/Homotypic
Adhesion
adaptor proteins
(catenins)

12. Desmosomes Spot welds that anchor cells together.
Attached to Intermediate Filaments (IF)
Desmosomal Cadherins are Desmogleins and Desmocollins
Plaque proteins attach desmosomal cadherins to IF.
Pemphigus: Auto-antibodies to desmosomal cadherins cause blistering, loss of skin integrity/barrier function
Tonofilaments:
Bundles of cytokeratin intermediate filaments

13. Desmosomes: Cell-Cell “Rivets”

14. Focal Contacts (Focal Adhesions)
Link Extracellular Matrix (ECM) to actin filaments
Allows cells to hang on to surroundings
Integrins: Transmembrane ECM binding proteins
Bind to actin indirectly via anchor proteins

15. Hemidesmosomes “Half-Desmosomes”
Distribute forces on an epithelium to the basal lamina (ECM)
Integrins mediate basal lamina adhesion
Anchor proteins link integrins to intermediate filaments

16. Cadherins Mediate Calcium- Dependent Cell-Cell Adhesion
Single-pass transmembrane proteins
Calcium-dependent adhesion

17. Cadherin-Dependent Cell Sorting
Homophilic Binding
E Cadherin: Epithelial
N Cadherin: Neurons, heart, fibroblasts, skeletal muscle
P Cadherin: Placenta, epidermis, breast epithelium
VE Cadherin: Vascular endothelium
Early Amphibian Embryo
Epidermal: Red
Mesoderm: Green
Neural Crest: Blue

18. Movie 19.1
GFP Cadherin

19. Selectins and Integrins Mediate Transient Cell-Cell Adhesion in the Bloodstream
Selectins are Lectins: Carbohydrate binding proteins
Transient, calcium-dependent adhesion
Heterophilic. Cooperate with integrins
E Selectin: Activated endothelial cells
Inside-out signaling: Leukocyte integrins become activated
Integrins bind adhesion molecules on endothelial cells

20. 19.2 Movie
Leukocyte Rolling

21. Communicating Gap Junctions
Sugars
(mono/disaccharides)
Nucleotides
Function
Electrically connect cells
Averages small molecules throughout tissue
More specialized cells uncouple
Connexins
4 pass transmembrane proteins
6 connexins form a functional pore: Connexon
Permeability varies with connexin composition

22. Signal-Relaying Junctions:
Synapse

23. The Extracellular Matrix
Hydrated network of protein and carbohydrate containing molecules
Abundant in connective tissues
Secreted by fibroblasts, chondroblasts, osteoblasts, epithelium
ECM bears most of mechanical stress

24. Glycosaminoglycans High negative charge attracts Na+ ions and water
• Unbranched polysaccharide chains composed of repeating disaccharide units.
• One amino sugar is usually sulfated and the other is a uronic acid
• Absorb large amounts of water: Occupy space
Heparin
Sulfate
Uronic Acids: Sugar acids where terminal carbon (#6) hydroxyl (OH) group is oxidized to carboxylic acid. [Glucuronic acid]
High negative charge attracts Na+ ions and water

25. (Hyaluronic Acid, Hyaluronate)
Hyaluronan
(Hyaluronic Acid, Hyaluronate)
Simplest GAG
Very long chain length: up to 25,000 disulfide units
No sulfated sugars
Synthesized from the basal side of an epithelial sheet
Creates a cell-free space into which cells can migrate: Embryogenesis
Diffusion of nutrients, metabolites, growth factors…
Resists compressive forces - Cartilage
Wound Healing
Promotes cell migration and proliferation
Wound Healing: Inflammation, granulation tissue, cell migration, angiogenesis, reepithelialization

26. Proteoglycans Glycoproteins
Glycosaminoglycans covalently attached to a core protein in golgi
At least 1 GAG
Very high carbohydrate content
Long, unbranched side chains
Regulate movement of molecules and cells
Chemical signaling between cells
Glycoproteins
Few, short, branched carbohydrate chains
Low carbohydrate content

27. Structural Proteins Collagens
The most abundant proteins of the extracellular matrix
25% of protein mass in mammal
Responsible for the strength of the ECM
Bones strength
Connective tissue integrity

28. Collagen Structure Triple-stranded, a-chains ~1000 amino acids long
Gly-X-Y repeat
Glycine – smallest aa
X: Proline
Y: Hydroxyproline
Vitamin C (Scurvy)
OH-Pro, OH-Lys
H-bonding
Lysine aldehydes
covalent intra-molecule and inter-fibril cross-linking
>40 different alpha chains can assemble into different collagen molecules.
Interchain OH-bonding.
Gelatin is hydrolyzed collagen

29. Synthesis of Collagen Fibrils and Fibers

30. 42 α chain genes assemble into ~40 types of collagen

31. Ehlers-Danlos Syndrome
Collagen Genetic Disorders
Osteogenesis Imperfecta
Defect in Type I collagen
Movie :”Unbreakable”
Ehlers-Danlos Syndrome
Mutations in collagen Types I, III, V
Collagen maturation/secretion
Both are Autosomal Dominant
OI: Decreased collagen synthesis, mutations, rearrangements
Samuel L. Jackson had OI

32. Elastin Imparts elasticity to tissues Super rubber band
Highly cross-linked
Major ECM of arteries
Fibrillin covers elastin fibers.
Fibrillin is essential for elasticity of elastin
Fibrillin Mutation
Marfan’s Syndrome: Aorta rupture
Mitral valve prolapse
Skeletal abnormalities
Tall stature

33. Fibronectin Adhesive ECM Glycoprotein
Binds cells to the ECM and guides cellular migration
Arg-Gly-Asp (RGD) binds integrins
Directs cell migration during development
RGD: Arginine-Glycine-Aspartate

34. Synthesized by cells on both sides.
Basal Lamina
Flexible, thin extracellular matrix mats that underlie epithelial cell sheets and tubes, surround fat, muscle, and Schwann cells
Functions as a molecular filter: Kidney glomerulus
Functions to orient/polarize cells: Hemidesmosome/Integrins
Important for cell survival: Anchorage dependence
Barrier to cell migration: Cancer
Guide for cell migration: Neuromuscular Junction
Around
Under
Between
Synthesized by cells on both sides.
Schwann Cells: Myelin producing cells of peripheral nervous system. Basal lamina serves as a scaffold for axon regeneration.

35. Composition of the Basal Lamina
Laminin
Adhesive protein which binds cells, collagen, GAG, PG
Fibronectin
Type IV Collagen
Perlecan (proteoglycan)
Nidogen (glycoprotein)
Heparin Sulfate (glycosaminoglycan)

36. Regeneration of Basal Lamina at Neuromuscular Junction

37. Integrins Focal Contact
Extracellular Matrix Receptors (Laminin, Fibronectin, Collagen…)
Low Affinity, High copy number
Binding is Ca2+ or Mg2+-dependent
Heterodimers of a and b glycoproteins
Overlapping specificity
Many bind RGD tripeptide
Cytoplasmic tails of b subunits link to cytoskeleton (usually Actin)
Focal Contact

38. Integrin Binding Specificities
INTEGRIN LIGAND DISTRIBUTION
a5b1 Fibronectin Ubiquitous
a6b1 Laminin Ubiquitous
a7b1 Laminin Muscle
aLb2 (LFA-1) Ig superfamily White blood cells
ICAM-1
a2b3 Fibrinogen Platelets
a6b4 Laminin Epithelial
hemidesmosomes
b1 subunits Partner with at least 12 a subunits

39. Regulation of Integrin Activity
Inside-Out Signaling
Integrins don’t bind ECM until activated by cells
Platelets activate b3 integrin to bind fibrinogen and aggregate.
T lymphocytes activate b2 integrins to bind antigen presenting cells.

40. Integrins Outside-In Signaling
Integrin binding to ECM activates intracellular signaling
Clustering of integrins at ECM contact sites
Focal Adhesion Kinase recruitment, clustering, activation
Similar to growth factor receptors
Responsible for survival/growth/polarization signals in epithelia attached to a basement membrane.