W3L1 Flashcards
Glycosaminoglycans
Chondroitin Sulphate
- Cartilage
Keratan Sulphate
- Cornea, cartilage and bone
Heparan Sulphate
- Ubiquitously expressed
- Binds ligands, binds water
- Involved in angiogenesis and tumor metastasis
Hyaluronate
- Connective tissue, epithelial and neural tissues
- Involved in inflammation, wound healing and tumor metastasis
For example,
- one protein core can have Keratan Sulphate
- another protein core can have Chondroitin Sulphate
- then the above 2 protein cores with their GAGs can bind onto Hyaluronate, therefore making it intermixed
Multi-Adhesive Proteins
Long flexible macromolecules found in ECM
bind to a variety of components (collagen, polysaccharides, cell surface receptors, growth factors, hormones, etc.)
Their major role is to attach cells to the extracellular matrix by cross-linking the matrix to the cell membrane.
e.g., Fibronectin
- important in wound healing and cell migration
- functions to help cells attach to other ECM components
e.g. Laminin
Integrins
Transmembrane proteins in cells that attach the cells to the ECM
‘Bridge’ between ECM and the cytoskeleton
Adhesive receptors
Bind the matrix as heterodimers
Integrin subtype, ligand and cell-type distribution
- α1β1
- ligand: Collagens
- Cell-type distribution: multiple cells - α3β1
- ligand: Laminins
- Cell-type distribution: multiple cells - α5β1
- ligand: Fibronectin
- Cell-type distribution: fibroblasts - α6β4
- ligand: Laminins
- Cell-type distribution: epithelial cells
Setting up 3D cultures of skin
- Collect normal tissues
- epidermis, oropharynx, esophagus, cervix - Isolate primary epithelial cells in tissue culture dish
- Single-step multiplex gene transduction
- can genetically modify the cell to test something - Transfer cells onto basement membrane with living stroma (ECM + fibroblasts) to establish 3D tissue
- If you just grow fibroblast on a dish, it will produce its own collagen and ECM
Keratin: protein found in epidermis
Collagen VII: fibril-associated, found in basement membrane (like Collagen IV)
Use 3D cultures to test anti-tumor compounds in vitro
Control
- Induce epithelial cells to be tumorigenic (genetically or with a carcinogen)
- induce cells to proliferate in the first image
- Epidermis gets thicker, it is neoplastic tissue. It continues to divide.
- Inducing cells to be tumorigenic will cause the epidermal cells to go through fenestrations in the basal lamina and enter the dermis
Chemotherapeutic drug
- add drug to block the epidermal cells from growing and invading the dermis. It works!
Epidermolysis Bullosa
Group of inheritable disorders
Incidence: 50 cases/ 1,000,000 people
Characterized by epidermal fragility with blistering and erosions
Disease with integrins
Shows up early in life, kids are called butterfly children
Weakening of the binding bw epidermis and dermis
Genetic modification and collagen injections are the treatments
There are different types of Epidermolysis Bullosa:
- Simplex basal Epidermolysis Bullosa: genes that encode intermediate filaments or plectin (binds intermediate filament to integrins) have mutations
- Junctional Epidermolysis Bullosa: mutations within the integrin gene, such as integrin alpha 6, integrin alpha 3, laminin 332
- Dystrophic Epidermolysis Bullosa: collagen VII mutation. Treatment is to inject collagen VII into skin of children
Cell Adhesion Molecules (CAMs)
CAMs are…
- Glycoproteins located at the cell surface
- Allows Tissue integrity: allow cells to associate with other cells and their surroundings (ECM)
- Cell-matrix interactions
a. Integrin, i.e. alpha5beta1
- heterophilic binding - Cell-cell interactions (Proteins will reach from one cell to a protein on another cell (could be the same protein or a different protein type))
a. selectin, i.e. P-selectin
- Found in platelets, allowing cells to aggregate
- Like to bind to glycoproteins
- heterophilic binding
b. Ig-superfamily, i.e. NCAM
- Some heterophilic and some homophilic binding
c. Cadherins, i.e. E-cadherin
- homophilic binding
Homophilic- association between two identical molecules
Heterophilic- association between different types of molecules
CAM: Selectins
Divalent cation dependent (magnesium, calcium)
Carbohydrate binding
- E-selectin (found on endothelial cells)
- L-selectin (found on leukocyte)
- P-selectin (found on platelet)
Play an important role in host defense mechanisms
CAM: Ig Superfamily
Calcium independent
Characteristic immunoglobulin domains
- Homophilic; 2 identical Ig CAMs binding to each other, for e.g., two N-CAMs (neural cell adhesion molecules)
- Heterophilic, for e.g. binding of Ig family member, I-CAM (intercellular adhesion molecule), to lymphocyte function-associated antigen-1 (LFA-1), a T-cell specific integrin
CAMs: Cadherins
Calcium dependent
Can have lateral or intercellular associations
- Cadherins can cluster together aka oligomerize on 2 separate cells, and then reach across the 2 cells to attach together. This increases strength compared to just 1 cadherin on each cell
Subclasses
- P-Cadherin; placental
- N-Cadherin; neural (also expressed in mesenchymal cells)
- E-Cadherin; epithelial
Homophilic interaction
CAMs: Properties affecting cell-cell adhesion strength
Examples apply to any CAM, not just cadherin
- Binding affinity
- i.e. how strong will 1 cadherin bind to another cadherin on a diff cell. Depends on the molecules involved - Spatial distribution
- i.e. how many cadherin can you bunch up and oligomerize on one cell, vs another cell - Activity state of adhesion molecules
- some CAMs can be phosphorylated on the intracellular portion, which changes its conformation on the outside of the cell to allow something to bind; this is activating the protein - External forces surrounding the cells
- the more stress you put on a cell, the greater the chance of breaking the bond from one cell to another cell
