ECM and Cell Interactions Flashcards
What fibers (fibrous structural proteins) do you find in the proteoglycan gel of ECM?
Collagen (strengthen, organize ECM)
Elastin (elasticity)
What is Ground Substance?
hydrated gel that resists compression, permits rapid diffusion of nutrients, metabolites, and hormones between blood and tissue
Proteoglycans (proteins and LOTS of GAGs) - resist compression/fill spaces
Glycoproteins (sugars and LOTS of proteins) - Adhesive glycoproteins (fibronectin, laminin, entactin)
What structures are the glycoproteins related to?
Fibronectin - CT
Laminin, Entactin - Basal Lamina
What are the characteristics of the ECM in different kinds of tissues?
Bone and Teeth: Calcification
Corneal Stroma: Transparency
Tendons: Rope-like
Cartilage: Shock-absorption
What are the functions of the ECM?
Scaffold to stabilize physical structure/resist forces
Influences cell behavior
What happens to epithelia if it is not anchored to the basal lamina?
It will undergo apoptosis.
What are the functions of Fibrous Proteins?
- Give strength or flexibility
- Water insoluble
- construct CT, tendons, and bone matrix
Describe Collagen.
Triple Helix of alpha chains
Main components: glycine, lycine, proline (glycine every 3rd residue is important)
Provides the tensile strength of tissue
What is important about the spacing of glycine residues in Collagen?
Giving a glycine every 3rd aa residue allows for H-bonding between glycine backbone and adjacent helix.
What functions do Proline and Lysine have? What are their alternate versions?
Proline (and Hydroxyproline): makes kinks, helps helix formation
Lysine (and Hydroxylysines)
How are Collagen chains synthesized?
(Collagen I)
- Preprocollagen sent to ER due to ER signal sequence
- signal sequence cleaved; Preprocollagen -> Procollagen (resists premature fibril formation)
- Hydroxylation of prolyl/lysyl residues, followed by glycosylation of hydroxylysines
- Disulfide bond forms at C terminus, aligning chains and initiating triple helix formation in a ‘zipper’ mechanism from C to N terminus
- H bonds between 3 procollagens: procollagen triple helix.
- Processing continues in Golgi, then procollagen secreted.
What compounds do the post-translational modifications of Proline and Lysine require?
Ascorbic acid (Vit C) and iron
Where do you find Hydroxyproline?
Collagen and Elastin.
What happens in the case of a Hydroxyproline deficiency? (Or Vit C)
Triple helix of collagen loses stability and will lose helix content at lower temperatures than normal.
Describe the pathology of Scurvy.
Defect: Vit C deficiency; weak CT
Symptoms: Bleeding gums, loosened teeth, Poor wound healing, poor bone development, anemia and fatigue.
Describe Collagen fibril formation.
- Peptidases remove N and C propeptides of procollagen, making collagen.
- self-assembles into collagen fibrils, strengthening collagen fiber
What are the Collagen families and what are the types associated with them?
Fibrillar: I II, III, V
Fibrillar-Associated Collagens (FACIT): VI, IX, XII
Sheet (Network)-forming Collagens: IV, X
Anchoring Fibrils: VII
Note: Type IV is only found in the basal lamina, and is also the only Collagen you can find in the basal lamina.
What is the pathology of Ehlers-Danlos syndrome?
Defect: collagen (fibrillar) synthesis; improper assembly of collagen
Symptoms: CT weakness -> hyperextensible fragile skin, joint hypermobility and dislocations.
Most common type: TYPE I (43%), very severe. Collagen I and V mutation.
What do FACIT Collagens do?
Mediate interactions of Collagen fibrils with other fibrils and ECM proteins, proteoglycans and GAGs
Binds surface of fibrillar collagens
What gives FACIT Collagens their flexibility?
Triple helix is INTERRUPTED by nonhelical domains, hence FACIT (Fibril-Associated Collagens with Interrupted Triple Helices).
What are Matrix Metalloproteinases (MMP)?
Enzymes that degrade collagen; they are important for remodelling the ECM
- MMP 1 (Collagenase I)
- MMP 2 (Collagenase IV)
What are the Collagen Defects/Disorders and what Collagen types are associated with each?
Osteogenesis Imperfecta - Collagen I
Ehlers-Danlos Syndrome - Collagen I, III, V
Alport Syndrome: Collagen IV
Goodpasture Syndrome: Collagen IV
Dystrophic Epidermolysis Bullosa: Collagen VII
What is the dominant ECM protein in the skin, lungs, uterus, and arteries?
Elastin: highly hydrophobic protein.
Differentiate between Elastin and Collagen.
Both Elastin and Collagen are rich in Proline and Glycine, but Elastin is NOT glycosylated and has NO hydroxylysine.
What is the precursor of Elastin? How does it transition into Elastin?
Tropoelastin (stretchy due to lack of secondary structures)
When tropoelastin is covalently cross-linked into fiber and sheet formation, tropoelastin is now elastin.
What covers the Elastin fibers?
A sheath of microfibrils, composed of glycoproteins (mostly Fibrillin)
Describe the pathology of Marfan Syndrome.
Defect: Fibrillin mutation that results in weak elastic tissue. (Autosomal Dominant)
Symptoms: Aortic root dilation, tall and thin skeleton, Funnel chest (pectus excavatum)
What is Elastase, and where would its function be INHIBITED?
Elastase = serine protease that works in Elastin degradation.
Tissues that need flexibility inhibit Elastase, like the lung. alpha1-antitrypsin (a1-AT) binds almost irreversibly to active site on elastase.
Describe the pathology of COPD (emphysema)
Defect: Mutated a1-AT; Elastase can freely destroy elastic fibers. (autosomal recessive).
Symptoms: Hyperventilation, Dyspnoea, Barrel-Chest (hyper-inflated chest)
What acts an inhibitor of a1-AT?
Smoke (smoking recruits neutrophils to lung, which accelerates degradation)
What are Glycosaminglycans (GAGs)?
Very long, unbranched chains of negatively charged repeating disaccharides. They covalently link to a core protein to form a Proteoglycan.
- most GAGs are made intracellularly and exocytosed.
Exception: Hyaluronan
What is special about Hyaluronan?
It is a unique Glycosaminoglycan whos structure consists of repeating sequences of identical NONSULFATED disaccharides.
- Huge chain length
- NO CORE PROTEIN INVOLVED
- Synthesized by enzyme on basal epithelial surface
What does Hyaluronan do?
- Produced during wound healing
- Lubricates joints (binds a lot of H2O)
- backbone for large proteoglycan complexes.
- FORMS THE GEL STRUCTURE OF VITREOUS HUMOR! (99% water with collagen fibrils suspended in the hyaluronan)
What is Aggrecan?
It is a Proteoglycan Aggregate in Cartilage. Its core is Hyaluronic acid, which lacks covalent bonds to proteins. Instead the core proteoglycans proteins associate with hyaluronic acid via Link Proteins.
Proteoglycan functions in the ECM consist of?
- form a porous, hydrated gel (fill space, resist compression)
- Structural support
- Cell signalling (bind growth factors)
- Cell migration
Describe the Basal Lamina and its composition.
It is a specialized part of the ECM. - Flexible and Thin - Its components are all produced by the epithelial cells resting on it. Composition: - Type IV Collagen (Major) - Laminin - Entactin/Nidogen - HSPGs (Heparan Sulfate Proteoglycan G)-Perlecan
What is Laminin?
It is an adhesive Glycoprotein that anchors Epithelial cells to the Basal Lamina. By binding to Collagen Type IV, it forms a scaffold.
It also binds HSPG, entactin, and integrins (this is where the cell membrane is bound).
What is the function of Perlecan (HSPG)?
It’s found in all basement membranes. It mediates cell attachment to basal lamina for endothelial cells and fibroblasts.
Along with Syndecan, Perlecan functions in angiogenesis and mitogenesis by binding/sequestering Growth Factors.
Describe the organization of the Basal Lamina.
- Collagen IV binds to laminin to form the scaffold.
- Entactin and Perlecan bind to both Laminin and Collagen IV, connecting the two networks.
What purpose do GAGs serve in the Basal Lamina?
In the glomerulus of the kidney, GAGs serve a s a size and charge-selective barrier. (GAGs possess high negative charge, which would mean proteins, which usually also have negative charge, cannot pass through GAG barrier)
Ex: Perlecan prevents macromolecules from passing from the blood into the urine.
Describe the pathology of Diabetic Nephropathy.
Defect: Dysfunctional basement membrane; Glomerular BM thickens (increased collagen, decreased HSPG)
Symptoms: Proteinuria, fluid retention, Hypertention, eventual Renal Failure.
What is the most common cause for Kidney failure in the world?
Diabetes Mellitus.
What are the functions of the Basal Lamina?
- Structural support for cells/Organizes cells/tissues
- Scaffold for tissue regen
- Filter (e.g. Kidney glomerulus)
- Determines Cell Polarity
- Influences cell survival, proliferation, differentiation
- Selective Barrier for Cell migration
Describe the general network of the ECM and its components. What are the two general organizations this network will fall under?
Fibrous Structural Proteins
- Collagens
- Elastin
- Fibrillin
Adhesive Glycoproteins
- Fibronectin
- Laminin
- Entactin
Gel of proteoglycans and hyaluronan
2 Organizations:
- Basal Lamina
- Interstitial Matrix
Give the function of Fibronectin.
Organizes the ECM and connects to all ECM.
- Exception: Type IV ECM, meaning Fibronectin does not bind to Basal Lamina
Note: Fibronectin forms dimers.
What are the binding sites on Fibronectin?
ECM component binding sites:
- Fibrillar Collagens
- Proteoglycans
- Fibrin
Integrin Binding site:
- RGD Sequence. (Arg-Gly-Asp)
What are the two forms of Fibronectin?
Soluble plasma Fibronectin: circulates in blood and enhances blood clotting/wound healing
Insoluble Fibronectin fibers: form on cell surface and deposit in ECM
How does Fibronectin influence cell movement/growth/organization?
Fibril formations between fibronectin fibers (difulside bonds) can cross-link the dimers and influence the organization of the actin network of the cell. The reverse is also possible.
What is an example of a Transmembrane Proteoglycan?
Syndecan
- binds Fibronectin and collagen (fibrillar: I, III, V)
Describe the structure and functions of Integrins.
Transmembrane proteins that form a/B heterodimers and bind to the ECM.
Their interactions with the ECM are like Velcro: many and weak so as to allow cell migration/movement.
What signalling function do Integrins serve?
Intracellular signalling in response to ECM. They contain adhesion-dependent activation of signalling proteins in the cytosol that regulate Anchorage-dependent Growth.
What are two types of anchoring junctions formed by Integrins?
Focal Adhesions
- integrins bind to actin stress fibers via adapter proteins, and to fibronectin
Hemidesmosomes
- integrins bind to intermediate filaments (keratins) via adapter proteins, and to laminin
What are the components of a Focal Adhesion?
Adapter proteins for integrins/actin:
- Vinculin
- Talin
Recall: A-actinin crosslinks actin fibers
Signalling protein:
- Focal Adhesion Kinase (FAK) -> regulates Rho GTPases
What are the functions of a focal adhesion? (Hint: you’ve done this before)
Cell motility
Signalling
What are the functions of a Hemidesmosome?
It is the strongest anchor between the cell and the ECM. NONMOTILE. Stability is its game.
Intermediate Fibers in cell embedded to plaques contain plectin (protein). Integrin proteins are linked to this plaque, and connect to ECM laminins. Collagen VII fibrils complete anchoring in the ECM.
What is the pathology of Bullous Pemphigoid?
Defect: Auto-Antibodies formed against hemidesmosomal protein.
Symptoms: Blistering, Subepidermal blisters, Presents over 60 years
Describe the function and reasons for Cell Migration.
- Often in response to external signals
- Important for embryogenesis, tissue repair, infection control
- Main motor is in the leading front (active polymerisation)
What are the pseudostructures (hint: podia) involved in Cell movement?
Lamellipodium: leading edge of moving fibroblast
Filopodia: Thin spike protrusions. Act as sensors.
What are the small G Proteins that regulate the actin cytoskeleton during cellular movement?
Rho: stress fibers and focal contacts
Rac: Lamellipodium
Cdc42: filopodia
What are the types of Cell-to-Cell Adhesions?
In order of appearance from apical side to basal layer.
Occluding Junction/Tight Junction
Anchoring Junctions
- Adhesion belt (cadherins)
- Desmosomes (cadherins)
Gap junctions (Connexin)
What are the general functions of Cell-Cell junctions?
- Provides strength and stability to cells and tissues
- Regulate movement of solutes/ions/water
- Convey info between cells
- Leukocyte Rolling
Give the specific functions for each Cell-Cell Junction type.
Occluding/Tight Junctions
- Selectively permeable barrier
Adherens Junctions
- mechanically attach cells and cytoskeletons
- adhesion belt
Desmosomes:
- mechanically attaches cells and their cytoskeletons (intermediate filaments)
- high tensile strength
Gap Junctions
- Cell communication
- permeability varies: blocks macromolecules (proteins, nucleic acids, polysaccharides)
Where are Tight junctions located? What additional proteins are involved?
Encircles apical ends of each cell and determines polarity of epithelial cells by separating the apical domain from the basolateral domain.
Transmembrane Adhesion Proteins: Claudin and Occludin (bind to intracellular proteins, which anchor to the actin cytoskeleton).
What are the two Inflammatory Bowel Diseases?
Chron's Disease (Skip Lesions) Ulcerative Colitis (continuous lesions)
What are Cadherins? What is required for binding Cadherins together?
Cadherins bind to cadherins of other cells to anchor two cells together. It requires calcium to bind subunits together.
In what kinds of tissue/organs would you find Anchoring Junctions?
Epidermis
Heart and Muscle
(they endure sever mechanical stress)
What binds the Cadherins of the Adherens Junctions to the actin cytoskeleton?
Linker Protein Catenins:
Cadherin - \Beta Catenin - Alpha Catenin - F Actin
What unique action can Adherens Junctions do?
Create invaginations of an epithelial sheet caused by organized tightening along adhesion belts in selected region of sheet, creating epithelial tube.
How are Desmosomes different from Adherens Junctions?
- Spot weld as opposed to Adherens “belt” junction
- anchored by Intermediate filaments, NOT F actin
- specific cadherins: desmoglein and desmocollin
- its anchor proteins are desmoplakin and plakoglobin.
Describe the pathology of Pemphigus.
Defect: autoimmune disorder OR genetic defect of desmosome cadherins
Symptoms: Blistering and raw sores on skin
What is the function of a Gap Junction?
Allows free passage of ions and small intracellular signalling between cells, mediating electrical and chemical ‘coupling’ between cells.
Describe the structure of a Gap Junction.
6 Connexin subunits comprise a single Connexon.
2 Connexons comprise a single channel
Few - Thousands of connexons per Gap Junction.
What does the “coupling” nature of the Gap junctions allow for in cells or specific tissues/organs?
Electrical coupling:
- Rapid spread of action potentials
- Synchronizes contractions in cardiac muscle/smooth muscle cells (transfer Calcium ions between cells)
Chemical Coupling:
- 2nd messengers can spread easily
- stimulates metabolic response (triggers secretion of contents across all secretory cells)
What regulates a Gap Junction’s channels?
Calcium ion levels.
High Calcium/low pH can close junction, limiting the spread of damage
What is the pathology of Charcot-Marie-Tooth neuropathy?
Defect: Mutation of Connexin 32.
Symptoms: progressive degeneration of peripheral nerves and Scwann cells, muscle weakness and atrophy, imparied deep tendon reflexes.
What are Lectins?
Carbohydrate-binding (on a glycoprotein) proteins that are Ca2+ dependent.
E.g: Selectins
What are the 3 Selectins discussed in class? Where are they found?
L-selectin (WBCs)
P-selectin (platelets)
E-selectin (activated endothelial cell)
How does Lymphocyte homing work?
L-selectin binds oligosaccharides on endothelial cells of lymphoid organs (glycoprotein = addressin)
What is Leukocyte rolling?
WBC will ‘roll’ across endothelium, E-selectins weakly binding to its oligosaccharides and activating its integrins. When Integrin strongly binds to ICAM (Calcium Independent CAMs) on endothelial cells, rolling stops and WBC enters the tissue (diapedesis).
What is the pathology of Leukocyte Adhesion Deficiency?
Defect: Beta 2 integring Deficiency; leukocytes don’t properly extravasate.
Symptoms: repeated life-threatening bacterial infections
What is the pathology of Glanzmann’s Thrombasthenia
Defect: platelet integrin complex (glycoprotein IIb-IIIa) missing; platelets fail to aggregate
Symptoms: increased bruising and bleeding.
Summarize collagen fibril formation.
- synthesize pro-alpha chain
- hydroxylate select prolines and lysines
- glycosylate select hydroxylysines
- self-assembly of three pro-alpha chains (hydrogen bonding)
- Procollagen Triple-helix forms
- Procollagen secreted
- Propeptides cleaved
- Self-assembly into fibril
- Collagen fibrils will aggregate to form collagen fiber.
Describe the pathology of Osteogenesis Imperfecta.
Defect: Type I collagen mutated
Symptoms:
- Type I: blue sclerae, bone fragility
- Type II: PERINATAL LETHAL; blue sclerae, severe fragility, skeletal deformity
- Type III: DEFORMING; blue-white sclerae, dentinigenesis imperfecta, bone fragility, short stature
- Type IV: normal sclerae, dentinogenesis imperfecta, mildly short, moderate skeletal fragility
What is the pathology of Alport syndrome?
Defect: mutated alpha5 chain of Type IV collagen (basal lamina).
Symptoms: nephritis and deafness, hematuria, proteinuria, hypertension.
What is the pathology of Goodpasture Syndrome?
Defect: auto-antibodies to Type IV collagen; inflammatory destruction of BM in kidney glomerulus and lung alveoli
Symptoms: Haemoptysis and glomerulonephritis (progressive renal failure follows)
Describe the pathology of Epidermolysis Bullosa (all 3 forms).
Defect:
- EBS (EB simplex): mutated keratin 5 or 14
- Junctional EB: mutated laminin, integrins, hemidesmosomal protein
- Dystrophic EB: mutated collagen VII
Symptoms: “Butterfly children” = very very fragile skin (minor friction/trauma results in recurrent blister formation)
What is the pathology of Achondroplasia?
Defect: constitutive expression of FGFR3 in chondrocytes, which restricts bone growth
Symptoms: Dwarfism
