ECM - Cell adhesions

Question 1

What are characteristics of cell adhesions?

Answer 1

-Transient anchorage
- Cell-cell recognition
- Typically before cell junctions are established (earlier in development)
- Partially overlapping molecules with cell junctions (cadherins, integrins)

*Observed by functional assays

Question 1

What are characteristics of cell junctions?

Answer 1

• “permanent” anchorage
• stability
• communication

*Cell junctions = all junctions from previous deck of cards, seen by electron microscopy

Question 2

What are cell adhesion molecules? (CAM)

Give an example of CAMs in the neural tube.

Answer 2

A subset of cell surface proteins that mediate the interaction between cells, or between cells and the extracellular matrix (ECM).
ex: cadherins, selectins, integrins

1. In the neural tube (composed of epithelial cells) there are cell that will become neural crest cells → crawl out to the surface of the neural tube as neural crest cells
2. Migration off of the surface
3. Aggregation in nuclei
4. Differentiation into peripheral ganglia (1st nerve cells)

Question 3

What different cadherins are expressed in different stucture/stages of development of the neural tube and neural crest?

Answer 3

*Different cadherins specifically expressed in different areas

1. Ectoderm (epithelial layer) → cells expressing E-cadherin
2. Neural tube → cells expressing E-cadherin in ectoderm + cadherin 6B in at junction of the neural tube budding out + N-cadherins in the neural tube
3. Ectoderm = E-cadherins, Neural tube = cells expressing cadherin 6B, N-cadherin and cadherin 7, Neural crest = cells expressing cadherin 7

*Immunofluorescence allow to see the change in pattern of gene expression → different groups of cells aggregate depending on if they express the same cadherins

Question 4

What agents are used for embryonic tissue dissociation?

Answer 4

Trypsin → protease

EDTA → chelates divalent metal ions → used to take Ca2+ away from cell adhesion molecules with its high affinity for Ca
Kd (Ca2+) ~ 1x10^-10M

Question 5

What experiment allowed to confirm cell adhesion molecules bring cells together?

Answer 5

1. Take embryonic tissue from liver and retina
2. Dissociate them with Trypsin and EDTA
3. Mix them
4. See how they re-associate together (cell adhesion molecules find each other)

Cell can be sorted out based on the type of cadherin they express (E- vs N-cadherin) or based on their level of E-cadherin → qualitative and quantitative differences
*Can be seen by labelling different populations of cells with different colours (fibroblasts)

Question 6

What are the different types/mechanisms of cell-cell adhesion binding?

Answer 6

Homophilic binding (preferred, most frequent)

Heterophilic binding (preferred)

Binding through an extracellular linker molecule (occasionnaly possible, really not favoured)

Question 7

What are the different families cell adhesion molecules? Which one is the exception?

Answer 7

1. Cadherins (E-cadherin) → homophillic interactions
2. Ig-superfamily CAM (N-CAM) → homophilic interactions
   - Extracellular domain = Type III fibronectin repeats + Ig domains (Ig domains interact with each other)
3. Mucin-like CAM → heterophilic interactions, glycoproteins that interact with the lectin domain of selectins
4. Integrin → heterophilic interactions (binds fibronectin), interact with fibronectin

*All Calcium binding sites except Ig-superfamily

Question 8

What are the common characteristics of the cadherin superfamily and the structural variety?

Answer 8

All have intracellular, TM, extracellular domains
- Extracellular domain has multiple cadherin domains (related to Immunoglobulin-fold)
- Intracellular portions varies → different intracellular ligands (signaling molecules, cytoskeletal proteins, etc.)
- Single pass membrane glycoprotein → 700-750 AA
- Exception = T-cadherin → no TM domain → GPI anchor

Question 9

Name different types of cadherins from the cadherin superfamily

Answer 9

1. Classical cadherin (E-cadherin)
2. Fat-like cadherins → larger extracellular portion (more cadherin domains)
3. Seven-pass TM cadherins (Flamingo) → G-coupled receptor + cadherin domains
4. Protein kinase cadherin (Ret) → signaling molecule as ligand??
5. Cadherin 23 → multiple cadherin domains
6. Protocadherins (important in CNS)
7. T-cadherin → GPI-anchored

Question 10

What are catenins?

Answer 10

They are intracellular anchor proteins essentila for binding of the cytosolic tail of cadherins to the actin filaments → essential for holding cells together
*For classical cadherins

Question 11

What components of the intracellular cytoskeleton do non-classical cadherins interact with?

Answer 11

They mostly interact with catenins to bind actin filaments (aderhens junctions)
Some form desmosomes by interacting with intermediate filaments → interact with different anchor proteins

Question 12

Explain the signal pathway of cell adhesion by classical cadherin interaction.

Answer 12

*Cadherin mediated
1. cell 1 and cell 2 have classical cadherins no their cell surface
2. Rac1-GDP sequestered in the cytosol by Rho GDI
3. Cadherin-mediated homophilic interaction occrus → Rac1-GDP dissociated from Rho GDI (unknown mechanism) → targeted to the membrane (in contact)
4. Cadherin activated PI3K → activates GEF → Rac1-GTP → positively regulates E-cadherin-mediated cell-cell adhesion + promote actin assembly → creates forces that push cell membranes together

Question 13

What is the role of p120?

Answer 13

It binds to classical cadherin cytoplasmic tail and regulates cadherin function
Not involved in linkage between cadherins and actin filaments

Question 14

What are selectins? (What is their structure)

Answer 14

White blood cells depend on selectins for moving from the blood stream to tissues (mediate blood stream cell-cell adhesions)

Calcium-dependent cell-surface oligosaccharide-binding proteins (lectins):
- TM protein
- At the tip of extracellular domains → EGF-like domain + lectin domain
- Binds oligosaccharids on target cells with lectin domains
- Unknown anchor proteins → interact with actin filaments
- Calcium dependent

Question 15

What are the 3 types of selectins?

Answer 15

L-selectin → Lymphocytes (WBC)
E-selectins → activated endothetial cells
P-selectin → platelets, endothelial cells that have been activated by inflammatory response

Question 16

What is the importance of endothelial cells in the blood vessels?

Answer 16

Similar ot epithelial cells as they form a tight layer
- Regulation of movement of blood cells/permeability
- Anchored on a basal laminal (same as epithelial cells)

Question 17

How do selectins and integrins mediate the cell-cell adhesion required for WBC to migrate out of the bloodstream into a tissue?

Answer 17

1. In lymphoid organs: endothelial cells express oligosaccharides recognized by L-selectin on lymphocytes
2. At inflammation sites: epithelial cells express selectins which recognize the oligosaccharides on lymphocytes (flag them to help)
3. Selectins bind lymphocytes by weak interaction → rolling of WBC on endothelial membrane → more interaction with selectins → slowing down of WBC (weak adhesions)
4. Strong adhesion when the integrins are activated (recognition of the protein) allow emigration of the WBC to the target tissue

Question 18

What type of interaction describes selectin and integrins with lymphocytes?

Answer 18

Heterophilic interaction:
- Selectin recognizes oligosaccharide on the glycoprotein and glycolipids
- Integrins recognize specific proteins

Question 19

What is one of the most important neural cell adhesion molecule and its characteristics? (N-CAM)

Answer 19

Ig superfamily:
- Ca independent cell-cell adhesion
- At least 20 forms of N-CAM can be generated by alternative splicing of RNA from a single gene
- For all N-CAM, extracellular part folded into 5 Ig-like domains
- Binds by homophilic interactions

*N-CAM → fine tuning of interactions during development and regeneration

Question 20

What are ressemblences/differences between Cadherins and Ig family members?

Answer 20

1. Frequently co-expressed
2. Cadherin adhesion is stronger vs N-CAM → fine tuning of interaction during development and regeneration
3. Like Cadherins, some Ig-like CAM can transmits signals to the cell interior (association with Src tyrosine kinase), other are TM tyrosine phosphatase
4. Both do homophilic interactions

Question 21

What is the structure of the extracellular part of Ig-like N-CAMs

Answer 21

1-2 fibronectin type III domains + multiple (~5) Ig-like domains

*Ig-like domains formed by disulfide bonds connecting the ends or each loop → disulfide bonds are catalyzed by protein disulfide isomerase in the ER

(Some have TM domains, some are GPI-anchored)

Question 22

What are 3 subclasses of CAM?

Answer 22

N-CAM → Neural cell adhesion molecules
I-CAM → Intercellular adhesion molecules
V-CAM → Vascular cell adhesion molecules

Question 23

Which integrins are present in C.elegans? In procaryotes? in plants? in fungi? in vetebrates (just subunits)?

Answer 23

2 alpha subunits + 1 beta subunit → 2 different possible integrins

No integrins in plants, fungi

Vetebrates → 18 a-subunits, 8 b-subunits

Question 24

What are the members of the mammalian integrin receptor family?

Answer 24

1.Recognize ECM molecules (cell-ECM) RGD receptors: b1* → aV, a5, a8 aIIbbeta3* aV → b8, b6, b5, b3* Collagen receptors: b1 → a1, a2, a4, a9, a10, a11 Laminin receptors: b1 → a3*, a6*, a7* a6b4 → connects to IF, not actin 2.Recognize IG superfamilly counter receptor (cell-cell interaction) Leukocyte-specific receptors: b2 → aM, aL, aX, aD b7aE *Some subunit can undergo alternative splicing → increase diversity **b1 synthesized in excess ***Most integrins recognized relatively short peptide motifs

Question 25

What are 3 types of cell interaction integrins act in?

Answer 25

1. Cell-matrix adhesion 2. Cell-cell adhesion 3. Cell aggregation

Question 26

What is specific of the beta4 integrin subunit?

Answer 26

Differs from all others → large intracellular domains → 1000 aa + connects to intermediate filaments

Question 27

What are the characteristics of intergins heterodimers?

Answer 27

- Type I TM proteins (C-term in the cytosol) - 90-160 kDa - Each subunit crosses membrane once - Most of each polypeptide is in the extracellular space (>1600 AA) - Short cytoplasmic domain (20-50 AA) - Ligand specificity depends on both subunits - Connected to actin via anchor proteins (except laminin → a6b4 → IF) - Alpha subunit often has Furin cleavage site connected by disulfide bond - N-terminus of both bind to divalent cations (Ca, Mg) fro stabilization, activation and interaction with ligand

Question 28

What is the integrin binding sequence on the the ECM proteins?

Answer 28

RGD = Argining-Glycine-Aspartic acid (D can sometimes be replaced by E) Binds Fibronectin, fibrinogen, laminin, some collagens → they have a this motif Apartic acid makes 1st contact with Calcium ion in the beta subunit → not sufficient for binding, need synergy sites

Question 29

What is required on integrin ligands (ECM proteins) for binding of integrins to ECM proteins?

Answer 29

BOTH FN10 and FN9 are required FN10 = RGD site → contains RDG integrin binding site → 1st contact is D with beta subunit FN9 = synergy site → makes contact with alpha subunit *Firbonectin is the best-characterized ligand in this regard

Question 30

What is the importance/role of aIIbβ3?

Answer 30

*Expressed on the surface of platelets aIIbβ3 integrin is activated by binding collagen IV or thrombin (outside blood vessels, platelet activation in forming clot) → activated aIIbβ3 integrin → binds to fibrinogen → mediates blood platelet aggregation/activation (cell aggregation, platelet are activated by the binding of integrin because it means contact with unusual cells → blood leaks) + blood clot formation *Fibrinogen is a hexamer →2 trimers of alpha, beta, gamma chains Its normally soluble in the blood → when it is bound to integrins → triggers assembly cascade → fibrin polymers (part of blood clot)

Question 31

What can cause Glanmann thrombasthenia? What is it?

Answer 31

It is a bleeding disorder cause by mutations in aIIb b3 integrins

Question 32

What are the steps of the fibrinogen assembly cascade?

Answer 32

Occurs when aIIbβ3 integrin binds fibrinogen: Firbinogen → exposed to thrombin → Fibrin monomer → fibrin dimer → fibrin polymer

Question 33

What are 2 ways integrin activation can occur? For what contexts are these activation process important?

Answer 33

1. via extracellular ligand binding → outside-in activation 2. from within the cell via the integrin cytoplasmic tail → inside-out activation Important for: - Cell aggregation → adhesion of circulating cells (platelets) to soluble matrix - Cell-cell adhesion (leukocytes and inflammation) *Unbound integrins (still dimers) diffuse freely in the plasma or on cell surface in inactive/non-ligand binding state *Activation involves separation of the 2 subunits cytoplasmic tails

Question 34

How does the integrin outside in activation occur?

Answer 34

1. Regulated via extracellular ligand 2. Binding to the bent inactive conformation (weak interaction) 3. Strainghtening of alpha and beta chains 4. Extracellular ligand will bind much stronger 5. Conformational change in beta subunit head domain 6. Separation of th entire alpha and beta subunit 7. Active integrin *Alpha and beta chains close together → inactive conformation (still binds to Ca) Outside in → binding of extracellular ligand → separation of cytosolic domain Ex of ligand = RGD motif

Question 35

How does the integrin inside out activation occur?

Answer 35

*Example = aIIbβ3 integrin 1. In inactive conformation → ionic salt bridge betwen alpha and beta subunits (cytoplasmic domain) → blocks the talin binding region of the beta subunit 2. Activated talin (head) binds to the beta subunit and disrupts the ionic salt bridge → separation of alpha and beta subunit → Activation

Question 36

How does activation of talin works? (For inside out activation of aIIbβ3 integrin)

Answer 36

Inactive Talin = folded on itself → Tail and head interact 1. Calpain (protease) → cleave the head domain of tailin → unfolds → active 2. Tailin interaction with PI4,5P2 (lipid in plasma membrane) → conformational change → head domain is still on, but unfolded active tailin

Question 37

What is the MW of Talin?

Answer 37

270 kDa

Question 38

What are disintegrins?

Answer 38

Block the function of some integrins: - Act as competitive inhibits for integrin-fibrin interaction (bind to aIIbβ3 to block firbinogen) + block blood coagulation - Inhibitors of angiogenesis Large group of snake venom proteins → cysteine-rich (disulfide bonds) + RGD sequence (in a loop that sticks out) - More than 40 different one → high homology - Small proteins → 40-100 AA long

Question 39

What can be a medical application for disintegrins?

Answer 39

1. Reduction of blood clots → reduce heart attacks, stroke 2. Reduction of angiogenesis to cancer cells → reduce growth by blocking transport of nutrients

Question 40

What is the affinity and avidity of integrins?

Answer 40

Low/moderate affinity High avidity → Found in integrin clusters *ligand binding promotes lateral diffusion and redistribution of integrins to focal complexes → clustering

Question 41

How does integrin-mediated finbronectin assembly occur?

Answer 41

*Fibronectin binds a5b1 integrins 1. Fibronectin dimer = soluble, compact conformation, bount to 2 integrins 2. Fibrin assembly = clustering of integrins-firbonectin → conformational changes + assembly into short firbrils Other explanation: a5b1 integrins bound to fibronectin on one side and actin filaments on other side → actin-myosin II contracts → opens up the compact fibronectin dimer → opens-up self-assembly sites → other fibronectin molecules can assemble → insoluble fibronectin network build up in the extracellular space