Extracellular Matrix

ECM

Extracellular matrix - diverse group of moleculed secreted by cells and stay in close proximity of the cells that secreted them

ECM function

• enhance cell adhesion

• cell to cell specificity

• acting as barrier or filter

  • basal lamina - basement membrane

• promoting differentiation

• strength

• acting as charged surfaces

• bind to integrin receptors

• tumor microenvironment

• diseases

• tissue engineering - scaffolds

Type 1 ECM

Collagen film

• cells attack, divide, don’t multilayer

Type 2 ECM

collagen gel

• cells attack, divide, don’t multilayer

• cells don’t fully differentiate

Type 2 ECM + soak with fetal bovine serum (growth factors)

collagen gel

• cells attack, divide, don’t multilayer

• full differentiation, rolls up

Type 1 ECM cross linked

Collagen gel

• cells attack, divide, don’t multilayer

• cells don’t fully differentiate

• cross llinked the collagen gel

Stem cells require what?

special ECM-feeder layer

How do substrate dependent cells grow without cell culture disk?

Magnetic levitation

1. feed iron particles

2. lift cells up (EGTA/protease)

3. magnet - cells adhere to each other

ECM sponge experiment

sponge cell dissociation experiments

• demonstrate the regenerative capacity of sponges

• separate cells by forcing tissue through mesh (cheesecloth) or using calcium/magnesium-free seawater

• cells reaggregate into sponges

ECM embryo experiment

Embryo cell dissociation experiments

• breaking down embryos into single-cell suspensions using enzymatic or mechanical methods

  • Enzymatic: Trypsin, Accutase

  • Mechanical: trituration

Cell adhesion molecules (CAMs)

homophilic (cadherins, Ig super family)

heterophilic - integrin receptors

multi-adhesive (linker)

• fibronectin

• laminin

Cadherin - 3 major types

1. E = Epithelial (adherens)

2. N = Neural

3. P = Placenta

b. Dozen types - major adhesion molecule

All substrate dependent cells have them

cis/trans → strong

Ca2+ dependent

compaction

Are cadherins homophillic? - L Cell experiment

YES

cadherins are homophilic (bind the same type of cadherin)

• L-cells engineered with different cadherins (e.g., E vs N) only adhered to the same type and sorted into separate clusters, proving specificity.

• Requires Ca2+

What is the Ig superfamily (IgSF)?

Immunoglobulin → proteins with Ig-like extracellular domains.

A group of cell adhesion molecules with immunoglobulin-like domains that mediate cell adhesion and signaling.

Where are IgSF molecules like NCAM found?

Mainly on neural cells, where they mediate cell adhesion.

Why is NCAM important in embryogenesis?

Plays a critical role in morphogenesis, especially in neural development.

What happens if anti-NCAM is added in embryos?

Retinal ganglion cells fail to reach the optic tectum → shows NCAM is required for axon guidance.

What does the retinal ganglion cell experiment show?

NCAM is essential for cell migration and pathfinding during development.

What do Western blots of NCAM show in embryos vs adults?

Embryos: high PSA-NCAM (more polysialic acid)
Adults: low PSA-NCAM

What is the effect of polysialic acid on NCAM?

Adds bulky polysialic acid chains → decreases cell adhesion.

NCAM + PSA = what functional change? (embryo vs human)

Embryo = HIGH PSA → cells move

Adult = LOW PSA → cells stick

What are the main forms of the ECM?

1. Interstitial matrix (fluid-filled, disorganized)

2. Basal lamina (basement membrane)

3. Loose connective tissue

4. Dense connective tissue

What is the interstitium?

A fluid-filled network of interconnected spaces between cells, supported by collagen and elastin.

What is the structure of the interstitium?

Collagen-supported, fluid-filled compartments that are compressible and distensible.

Function of the interstitium

Acts as a shock absorber, supports fluid flow, and is a source of lymph.

Why is the interstitium considered organ-like?

It’s a body-wide interconnected network that supports structure, drainage, and transport.

Role of interstitium in cancer

Acts as a conduit for metastasis, allowing cancer cells to spread to the lymphatic system.

Role of interstitium in aging

Collagen stiffens over time → contributes to skin wrinkling and tissue stiffness.

Why is interstitium hard to see in histology?

Fixation drains fluid, making it look like empty spaces or tears.

What is loose connective tissue?

A spongy ECM (e.g., dermis) with elastin fibers and flexible structure.

What is dense connective tissue?

Highly packed ECM (e.g., tendons, bone) → strong and rigid.

What is special about elastin fibers?

They have an extremely long half-life (~74 years).

What is the basal lamina?

A specialized ECM layer secreted by epithelial and endothelial cells.

Where is the basal lamina located?

Between epithelial cells and connective tissue.

Function of the basal lamina

Provides structural support, cell anchoring, and acts as a barrier/filter.

Structure of the cornea (ECM example)

• Epithelium (dividing cells)

• Stroma (collagen + fibroblasts)

• Endothelium (non-dividing, regulates fluid balance)

Function of corneal endothelium

Maintains osmotic balance of the stroma → keeps cornea clear.

Interstitium vs basal lamina

Interstitium: fluid-filled, loose, transport

Basal lamina: structured, supportive, anchoring layer

Key properties of collagen

Most abundant protein, fibrous, insoluble, and forms a triple helix

What is special about collagen amino acid sequence?

Every 3rd amino acid is glycine

Major collagen types

Types I, II, III, IV ≈ 90% of collagen

Function of collagen

Provides strength and resists stretching

What modification is common in collagen?

High levels of hydroxyproline

What happens to collagen in disease?

Linked to cancer and structural defects

Where does collagen synthesis begin?

Rough ER (constitutive secretory pathway)

What are propeptides in collagen?

Located at N- and C- termini, help with assembly information, not signal sequence

Key modification in RER

Hydroxylation of proline → hydroxyproline

What is required for hydroxylation?

Vitamin C → deficiency causes scurvy

What happens outside the cell in collagen synthesis?

Lysyl oxidase forms covalent cross-links

What are glycosaminoglycans?

Not proteins, made of repeating disaccharides

Key properties, binding, function, and example of GAGs

Long, rigid, negatively charged

Bind cations (Na⁺) → create osmotic pressure for Diapedesis (extravasation)

• Binds to CD44 receptor

Hydrate tissue, resist compression, allow cell movement

Hyaluronan (hyaluronic acid)

Clinical use of hyaluronan

Injectable for osteoarthritis (SynVisc One)

What are proteoglycans?

Proteins + glycosaminoglycans

• Ex. syndecans

syndecans binding and function

Binds FGF (fibroblast growth factor)

• Controls hunger; overexpression → obesity

• Linked to cancer, obesity, arthritis, fibrosis

What is fibronectin?

A multi-adhesion protein, exists as a dimer

• Binds cells via integrins

• Important for morphogenesis

What is laminin?

A multi-adhesive protein (~500,000 Da), forms a trimer

• Self-assembles into sheets (basal lamina)

• Supports neuronal phenotype (“laminin loves neurons”)

What do integrins bind?

Many ECM molecules

• recognizes RGD (Arg-Gly-Asp) sequence

• Most require a cation

Low affinity (inactive) and high affinity (active)

How do melanoma cells metastasize?

Secrete collagenase → break through basal lamina

What happens if corneal endothelial cells are lost?

Leads to corneal opacity

Types of corneal transplants

Penetrating, lamellar, endothelial keratoplasty

ECM fillers

Hyaluronan (Juvederm, Restylane) and collagen (Zyderm)

Collagen vs GAGs

• Collagen = strength (resists stretching)

• GAGs = hydration + compression resistance

Collagen synthesis defect → result

Low vitamin C → scurvy → weak blood vessels