19: Cell Junctions and the Extracellular Matrix

Question 1

What are the two basic kinds of transmembrane adhesion proteins?

Answer 1

Cadherin: cell-cell

• Link to actin -> adherens junctions
• Link to IF -> desmosomes

Integrin: cell to matrix

• Link to actin -> actin-linked cell-matrix junctions
• Link to IFs -> hemidesmosomes

Question 2

With do cadherins bind with strong affinity?

Answer 2

Velcro-principle:
Bind to their (homophilic) partners with low affinity, but many weak bonds in parallel create a strong attachment.
Can be easily disassembled by separating the molecules sequentially.

Question 3

Which cadherin type is lost during epithelial to mesenchymal transition (EMT)?

Answer 3

E-type: epithelial cadherin
EMT occurs during
• normal embryonic development
• tissue repair and regeneration

Feature of invading and metastatic cancers

Question 4

Which adaptor proteins does the linkage of cadherins to the actin cytoskeleton depend on? Briefly describe the process

Answer 4

Inactive binding on cytosolic side via different catenins.
Process steps:
1. membrane protrusions initiate cell-cell contact. Activation of GTPase Rac (actin regulator)
2. Rac activity => actin and cadherin recruitment expands junction
3. Rac replaced by GTPase Rho => Actin remodeling (to linear) and myosin recruitment expands the adherens junction.

Pulling on a junction in one cell will increase the contractile force generated in the opposite cell.

Question 5

What are desmosomes?

Answer 5

Interactions between the intermediate filaments of adjacent cells.
Function: provide mechanical strength.
Abundant in tissues that are subject to high mechanical stress ex. heart muscle, epidermis
Type of IF depends on the cell type (ex. keratins in epithelial cells)

Question 6

What are tight junctions? What are the transmembrane homophilic adhesion proteins involved?

Answer 6

Seal gaps between adjacent cells, so molecules from basal lamina/medium above cannot leak across the sheet.
Have different characteristics regarding low molecular weight permissiveness.
Allow paracellular transport: transient modification of
tight junctions to modify the flow of AAs, etc.

Claudins - important for formation and function.
Occludins - limit junctional permeability

Question 7

How do tight junctions hinder transcellular transport?

Answer 7

Transcellular transport: TJs act as seals and fences =>
Must have specialized transporters on the apical and basolateral sides of the cells to enable movement of substances from the lumen into the ECM

Question 8

What are gap junctions?

Answer 8

Channels connecting neighboring cells.
Spanned by connexins (vertebrates) and innexins (invertebrates).
Connexin = 4 transmemb. domains.
Six connexins form a connexon (hemichannel). Does not need to be homotypic.
Connection between connexons on adjacent cells => continuous channel.
Combinations of connexons give different properties.

Not always open. Stimuli by voltage, memb. potential, pH, [Ca2+]…

Chemical coupling: Passage of inorganic ions and water-soluble molecules, not macromolecules.
Electrical coupling: flow of ions carrying electric charge

Question 9

Can you describe plasmodesmata?

Answer 9

Intracellular junctions between plant cells.
Desmotubules (cylindrical structure) run through the middle. Continuous with the smooth ER in the two cells, enabling lipid transfer.

Transports molecules of approx. the same size as gap junctions. Transport is regulated.

Can be removed when they are no longer required an inserted de novo.

Question 10

What is the role of selectins?

Answer 10

Calcium-dependent (C-type) lectin (cell-surface carbohydrate-binding protein) that mediates cell-cell interactions in the bloodstream.

L-selectin: on white blood cells
P-selectin: on platelet
E-selectin: on activated endothelial cells

Question 11

Can you describe the correlation between integrins and the immunoglobulin (Ig) superfamily?

Answer 11

Integrins bind to specific Ig-family proteins.

• ICAMs: intracellular cell adhesion molecules.
• VCAMs: vascular cell adhesion molecules.
• Heterophillic interactions: ICAM and VCAM
• Homophilic interactions: NCAM (N, neural)

Integrins = Ca2+ dependent
Ig = Ca2+ independent

Question 12

What three major macromolecules are the ECM constructed from?

Answer 12

Glycosaminoglycans (GAGs) - gel, migration, differentiation ++
- Large and highly charged polysaccharides that are usually covalently linked to protein in the form of proteoglycans.

Fibrous proteins - strength, organization
- Primary collagens

Glycoproteins - resilience
- Has asparagine-linked (N-linked) oligosaggharides.

Question 13

What is the gel-like “ground substance” of the ECM?

Answer 13

Highly hydrated, usually formed by proteoglycans, in which collagens and glycoproteins are embedded.
Resists compressive forces on the matrix, while permitting rapid diffusion of nutrients, metabolites, and hormones.

Question 14

Describe the structure of a glycosaminoglycan (GAG).

Can you name the four main groups?

Answer 14

Unbranched polysaccharide chains consisting of repeating disaccharide units containing one amino sugar (often sulfated) and usually a uronic acid.

Too stiff to fold, highly hydrophilic and negatively charged→ attract osmotically active Na+ ions → attract water into the matrix

Groups distinguished by sugars, linkage types, number and location of sulfate groups:

• Hyaluronan
• Chondroitin/dermatan sulfate
• Heparan sulfate
• Keratan sulfate

Question 15

Can you briefly describe proteoglycan synthesis?

Answer 15

Core protein component synthesized by membrane
bound ribosomes in ER -> Golgi
Addition of tetra-saccharide linker (primer)
Addition of polysaccharides by glycosyl transferases
in the Golgi, covalent modifications

Question 16

How does hyaluronan (hyaluronic acid) differ from the other types of glycosaminoglycans?

Answer 16

Has no sulfated sugars
All its disaccharide units are identical
Enormous chain length
Generally not linked covalently to any core protein.
Produced by enzyme complex at cell memb. surface

Insulates from compressive force and can force shape change during embryogenesis

Question 17

What are the main differences between glycoproteins and proteoglycans?

Answer 17

The proteins of proteoglycan are bound to polysaccharides with amino sugars.

Glycoproteins are often short and branched, proteoglycans are often long and unbranched with a larger carbohydrate percentage by weight.

Question 18

Can you characterize the collagens?

Answer 18

Major protein component of ECM.
Fibrous protein.
Long & stiff- triple-stranded α helical structure
Proline and glycine rich.
Often glycosylated
Can assemble into higher-order structures- fibrils (fibrillar collagens).
Fibrils are organised differently in different tissues.
Help ECM withstand tensile forces.

Question 19

How are collagen fibrils organized?

Keyword: fibril-associated collagens

Answer 19

Cells can regulate the disposition of the collagen molecules after secretion by guiding collagen fibril formation near the plasma memb.

Fibronectin (matrix glycoprotein) helps guide.

FACs mediate the interactions of collagen fibrils with one another and with other matrix macromolecules to help determine the organization. Bind to fibril surfaces. FACs are more flexible and do not aggregate with themselves.

Question 20

Can you describe elastin and its function?

Answer 20

Gives resilience to recoil after transient stretch.
Rich in proline and glycine, but not glycosylated.
Elastin core is surrounded by microfibrils => scaffold for elastin deposition.
Highly cross-linked to other elastin molecules, generating a network.
- Relaxed: random-coil conformation
- Stretched

Dominant ECM protein in arteries.

Question 21

What are the advantages with ECM proteins containing multiple binding sites? E.g., for matrix macromolecules and for cell surface receptors

Answer 21

Organizes the matrix and helps cells to attach to it.

Some also have binding sites for soluble growth factors

Question 22

What are the structure and functions of fibronectin?

Answer 22

Dimer consisting of two monomers cross-linked via disulfide bonds at their C-termini.
Exist in soluble form, and as insoluble fibronectin fibrils in the ECM (dimers are linked together via disulfide bonds). Can only assemble into fibrils on cell surfaces with fn.-binding proteins e.g., integrins.

Mediates cell-matrix interactions through different
domains. Binding to collagen, proteoglycans, integrins.

RGD peptide and synergy sequence are important for
integrin (has RGD receptors) binding

Question 23

What is the basal lamina and how is it structured?

Answer 23

Specialized type of ECM underpinning all epithelia and surrounding individual muscle cells, fat cells, and Schwann cells. Forms mechanical connection between these cells and the surrounding connective tissue.

Thin (40-120 nm), tough and flexible.

Synthesized by the cells on either side of it.
Precise composition varies between tissues/regions.
Typically contains the glycoproteins laminin, type IV collagen, and nidogen + proteoglycan perlecan. Some have fibronectin and type XVIII collagen.

Laminin - primary organizer of sheet structure. Self-assembles guided by interactions with integrins and proteoglycans. Different isoforms.

Type IV collagen - flexible, provides tensile strength.

Laminin and type IV collagen interact with other basal lamina components. Cell-surface receptors (e.g., integrins, dystroglycan) organize basal lamina assembly.

Question 24

What are the functions of the basal lamina/basement membrane?

Answer 24

Determine cell polarity
Influence cell metabolism
Organize the proteins in adjacent plasma membranes Promote cell survival, proliferation, or differentiation Serve as highways for cell migration - selective barrier.
Molecular filter in the kidney glomerulus

Question 25

Why is it important for cells to be able to degrade ECM in addition to making it? Which structures degrade matrix components?

Answer 25

Allow movement, cell division, and adaptation to
novel functional requirements.

Degraded by extracellular proteases that act close to the cells that produce them. Can be general or specific.

• Matrix metalloproteases
• Serine proteases

Question 26

What junctions do integrins participate in, and how are integrins structured?

Answer 26

Cell-matrix junctions. Can transmit signals in both directions across the membrane.

Structure:
2 noncovalently associated transmembrane glycoprotein subunits (α + β). N-terminal extracellular domains.
Ca2+ and Mg2+ dependent for affinity and specificity.
Inactive: folded state. Can be activated by intra/extra-cellular mechanisms through RGD seq. or adaptor proteins, respectively.

N-terminal domains bind specific seq. on matrix proteins or on other cells e.g., RGD seq. on fibronectin. Also binding sites in laminins and collagens.
C-terminal binds different proteins (adaptor proteins etc.) that form a linkage to the cytoskeleton.

Bind ligands with low affinity. Higher conc. of integrins increase binding strength: velcro principle.

Question 27

What are the most prominent cell-matrix attachment sites in epithelia?

Answer 27

Hemidesmosomes.
Specific type of integrin anchors the cells to laminin in the basal lamina (further attached to collagen).
Intracellular attachment is keratin intermediate filament via adaptor proteins plectin and BP230

Question 28

Which scaffold proteins are important for the assembly of mature cell-matrix juncitonal complexes?

Answer 28

Different scaffolding and signaling proteins e.g.:
Talin
Integrin-linked kinase (ILK)

Actin-binding proteins: assembly and organization of af.
Vinculin
α-actinin

Focal adhesion kinase (FAK) interacts with multiple components in the junction. Important in signaling.

Question 29

Can you describe anchorage dependence in relation to cell growth, proliferation, and survival?

Answer 29

Some cells might not grow and survive unless they are attached to the ECM.
Mediated mainly by integrins and the intracellular signals they generate.

Outside in signaling is often mediated by the recruitment of cell signaling molecules Focal Adhesion Kinase (FAK) and Src family kinases to sites of adhesion (focal adhesion).