11. Extracellular Matrix

exam 3

Extracellular matrix (ECM)

• diverse collection or molecules secreted by cells 

    a. cell adhesion

    b. cell to cell specificity

    c. barrier = filter-bacterial laminal

    d. promote differentiation

    e. strength (ex: bones+tendens)

    f. turn on receptors - intergin receptors

    g. charged surfaces - hydration shell --> bind growth factors

    h. tumor microevnironemnt 

    i. diseases 

    j. used for medical application

    k. stem cell therapy 

    l. organoids 

How important is the ECM to tissue engineering - EpiDerm:

• what is the proper ECM?

1. Dry film Type 1 collagen (cell struck, partly differentiated)

2. Type 1 collegen --> gel (more differentiation)collagen

3. Type 1 collegen gel w/ FCS inhibitors (full differentiation)collagen

4. #3 but first cross x-linked w/ aldehyde (commercial product)

The Big Challenge: Develop methods to grow hESCs without mouse feeder layers

• Irradiated mouse fibroblast feeder layers are typically required for growing hESCs

• But this introduces animal proteins so frowned upon by the FDA due to the possibility of viruses in animal proteins

• New feeder cell-free plates and media have been developed

Nano 3D

•  a system where cells that rely on a surface to grow (substrate dependent cells) can be induced to form miniature, three-dimensional organs called organoids

• the cellular process of endocytosis (or phagocytosis), where a cell takes in a foreign object, such as an iron particle, and packages it into a vesicle that will be delivered to a lysosome for degradation or processing

classical experiment - sponges

•  intact sponge tissue is first separated into individual cells (some stained purple with "india ink"), then passed through a cheese cloth to completely dissociate them

• When these individual cells are allowed to settle and interact, they re-associate to form a functional sponge again, showing that cells inherently recognize and stick to each other

classical experiment - embryos

dissociated cells from different tissues, like the purple retina cells and the red liver cells, will self-sort when mixed, demonstrating that cells from the same tissue preferentially adhere to each other

cell adhesion molecules (CAM)

• Homophilic adhesion occurs when two identical CAMs on neighboring cells bind to each other, like two cells holding hands with the same type of molecule

• Heterophilic adhesion occurs when one type of CAM on one cell binds to a different type of molecule on a neighboring cell

• multivalent adhesion involves a third, separate bridging molecule that binds to and connects two CAMs on separate cells

cis vs trans

•  begins with trans binding, where molecules extending from Cell 1 interact with molecules extending from Cell 2 across the exterior space

• followed by the molecules on the same cell membrane associate laterally in a cis binding interaction

• The final structure shows a stable, interconnected array involving both cis + trans associations, which provides strong adhesion between the two cells

E-Cadherin is linked to the
cytoskeleton

• links the plasma membranes of two adjacent cells (Cell 1 and Cell 2) and, crucially, connects to the internal cytoskeleton

• span the plasma membrane and interact homophilically in the extracellular space

• On the cytosolic side, the E-Cadherin tails are tethered to the F-Actin cytoskeleton via a complex of adapter proteins

• β-Catenin, α-Catenin, ZO1, Vinculin, VASP, α-Actinin, and p120-Catenin, form an adherens junction that provides strong mechanical strength and stability to the tissue

cadlherins

•  a crucial family of Cell Adhesion Molecules essential for holding cells together, particularly in tissues that rely on a surface for structure

• strictly calcium dependent; if calcium is removed, they lose their function and cells fall apart

• bind to each other through cis/trans binding to link adjacent cells, and their binding specificity is so strong that their mechanisms are subject to competition between types

ex: E (epitellial),N(Neural),P(placenta)

are cadherins homophilic or heterophilic?

• Autophagy is the cell's "eating itself" process to recycle damaged components like mitochondria and peroxisomes, starting with the formation of a phagophore that matures into an autophagosome tagged by the protein LC3 before fusing with a lysosome

• this lysosomal function can be impaired, leading to non-inherited diseases like silicosis or by certain drugs like chloroquine and bafilomycin A1, which interfere with the degradation process

Ig (Immunoglobulin) supperfamily

• a representation of a molecule within this family that participates in hydrophilic binding

• N-CAM (Neural Cell Adhesion Molecule), where the "N" stands for neural

• These molecules are generally involved in cell-to-cell adhesion and recognition processes, particularly in the nervous system

N-CAM Functions

• Adhesion: They primarily function by mediating cell adhesion through homophilic interactions, which is essential for organizing cells and tissues

• Signaling: NCAM binding can activate intracellular signaling pathways, leading to changes in gene expression and influencing cell functions such as migration, proliferation, and differentiation

• Function in the nervous system: NCAMs are critical for neural development, including the formation of the olfactory bulb and hippocampus, neurite extension, axon guidance, and the formation and maintenance
  of synapses

Interstitium

• Layer of interconnected, fluid-filled compartments supported by collagen bundles and elastin

• Compressible and distensible – shock absorber around organs

• May explain how cancers metastisize

• Source of lymph

• Standard fixation procedures drain away the fluid and superficially looks like tears in tissue

• First seen with a probe-based confocal laser endomicroscope
  examining metastasis in a patient’s bile duct

• Lined by very unusual, flat cells – look like fibroblasts but function unknown and have little cytoplasm with an oblong nucleus

Axonal growth

• the axon of a retinal ganglion cell grows toward and targets the optic tectum

• The crucial role of N-CAM (Neural Cell Adhesion Molecule) is blocking N-CAM antibodies that blocks axonal targeting, indicating that N-CAM is essential for guiding the growing axon to its correct destination

western blots of N-CAM

•  The embryo's N-CAM is highly modified by PSA (polysialic acid), a large sugar chain that creates a substantial hydration shell around the molecule

•  makes the embryonic N-CAM larger and less adhesive

• the adult N-CAM, lacks or has significantly reduced PSA modification, resulting in a smaller protein that is more effective at promoting strong cell-to-cell adhesion

ECM - several forms

1. loosely ordered

2. basal lamin aka basement membrane/lamellar

3. loose connector tissue (ex: dermis of skin, conera/stroma)

4. dense connective tissue (ex: bones, tendons)

Corneal Transplant Types

• First cornea transplant was in 1905 – one of the first successful transplants of all tissue/organs

  • Penetrating keratoplasty – full thickness cornea in its entirety

  • Lamellar keratoplasty – limited to diseased areas only

  •  Endothelial keratoplasty – Descemet membrane and endothelial cell layer only

Elastic (Elastin) Fibers

• long half-life of 74 years

• primary role is to provide elasticity and resilience, allowing tissues like skin and blood vessels to stretch and recoil back to their original shape without damage

• stretching ability is essential for the function of the lungs, enabling them to store energy during inhalation and release it during exhalation

• a single elastin molecules can cross-link to form an elastic fiber that can readily stretch and then relax to return to its resting state

Collagen

1. most abundent animal protein 

2. fibrous

3. insoluble in the aqueous solution

4. triple helix

5. 12+ varieties 

6. every 3rd amino acid is glycine (gly-pro-met-gly)

7. proline and hydroxyproline 

8. resists stretching

9. disease states 

Basal Lamina

• specialized sheet of extracellular matrix that underlies epithelial cells

• The electron micrograph is situated between the basal surface of the epithelial cell's cytosol and the underlying connective tissue.

• includes an intertwined meshwork of Type IV Collagen, (a connecting layer of Laminin, and the linking glycoproteins Nidogen/Entactin and Perlecan)

• secreted by the epithelial cells to provide structural support, filtration, and cell adhesion

Collagen synthesis

1. RER - signal sequence

2. N - pro-petide, c-pro-peptide

3. RER - hydroxylation of proline(vitamin C dependent enzyme)

4. assembly + HSP 47

5. pro-collagen is secreted - outside of the cell

6. lose the pro-peptide 

7. final assembly (Lysyl Oxidase - covalent bond)

Scurvy

• lack of vitamin C 

  • vitamin C is a co-factor for proline hydroxylation

• bleeding 

• 1757 - scottish doctor says to suck on limes (limeys)

Glycosaminoglycans

• not proteins b/c:

1. repeating disachatides

2. length --> ums long (longer than a cell)

3. rigid molecules

4. bind to cations --> change osmotic pressure, increase hydration shell

5. resists compression

6. binds to receptor called CD44 + proteo-glycans 

7. extravasation

8. SynVisc - asteoarthritis

Proteoglycans

1. protein + glycosaminoglycan

2. Syndecans - can bind to FGF (growth factos)

3. overexpression of Syndecan 1 in mice --> obese (in hypothalamus)

Fibronectin

• is multi adhesive (bind to many different ECM molecules)

1. dimer

2. important to wound healing 

3. important to morphogenesis

Laminin

• multi-adhesive 

1. 800 kDa

2. self-assembly (molecules that requires little to no energy to make a sheet)

3. neuronal growth in vitro 

Integrin Receptors (Integrins):

1. ECM ink to integrins 

2. dimers - α, β subunits 

3. bind to Mg+, Ca+2

4. lower affinity than GICRs, RTK, etc

5. lower affinity --> easy detach from sunstrate 

6. higher in number 

7. "cross-talk" w/ RTKs and other like receptors 

8. undergo shift from low to high affinity state

9. binds to RGD sequence (tri-peptide)

10. cluster together in "adhesion plaques"

do integrins bind to the RGD sequence?

cells attachment requires a substrate w/ RGD

Leukoctye adhesion deficancy 

• lacking defetive β2 receptors

• par to no extravasation

• many infection

• hematopoietic stem cell transplant 

Duchene Musculr Dystrophy

• boys only

• "ECM disease"

• 1-7000 boys

• defects-dystrophin,laminin of other compnents 

Ehlers-Danlos Syndrome

• varity of ECM related disease

• hypermobility - unusal flexibility (1 in 10,000 to 15,000)

• classical - elastic skin

Osteogenesis Imperfecta (OI) - Brittle Bone Disase

Type 1:

•  most common (mildest)

• amount of collagen synthesized is low

Type 2: 

• most sever

• collagen helix not formed corrected

• death at birth