Cell Junctions and ECM (Unit 8) Flashcards
What is the basal lamina or “basement membrane”?
A thin, fibrous (mesh-like) and tough but flexible mat on the basal side of epithelial cells or wrapped around muscle and fat cells
What makes up the basal lamina?
Proteins and sugars secreted by local stroma and epithelial tissue
What are the 3 main components of the basal lamina?
Proteoglycans (perlecan)
Glycoproteins (Type 4 collagen, nidogen and laminin)
Collage 13
Fibronectin
What two things does the basal lamina separate?
Epithelial tissue and the surrounding connective tissue (stroma)
How do perlecan, type 4 collage and nidogen work together in the basal lamina?
They link laminin and collagen networks together
What is classical laminin?
A large and flexible heterotrimer held together with disulphide bonds that has multiple binding domains
How is classical laminin made into a sheet?
While bound to PM, Integrins (ex: dystroglycan) hold them in the right spots to create an organized sheet
What keeps blood macromolecules out of the urine in the kidney glomeruli?
Type 4 collagen and GAGs
What does the basal lamina do?
-Structure + Cell Polarity + organizing proteins near PM
-Cell Migration highway and filtering
-Influence: Survival, proliferation and differentiation
How does the basal lamina generally act as a filter in epithelial tissue
Keeps fibroblasts out
Meanwhile, macrophages and lymphocytes can use proteases to saw through
How does the basal lamina help with tissue regneration?
It usually survives breaks and thus acts as a scaffold
Why can fibronectin only assemble into fibrils in vivo?
It needs to link to actin via fibronectin binding proteins (usally integrin) so that it can stretch and reveal its binding site
They bind to eachother and recruiting molecules to make their fibrils
Why is fibronectin soluable in body fluids but not in the ECM?
In the ECM, dimers cross-link with more disulfide bonds
How do tissues prevent tears
They weave collagen in with elastic protein to gain a stretch ability (collagen also resists tensile forces)
What is a microfibril sheath?
A structure surrounding elastic fibers made of glycoproteins (mostly fibrilin)
They provide scafolding for elastic fibers but can also persist without them
What does fibrilin do?
Bind elastin and provides elastic fiber integrity in microfibrill sheaths
What is a microfibril?
Fibril of elastic proteins and other proteins
What is elastin?
A hydrophobic protein with lots of proline and glycine that cross-links using Lysine
The hydrophobicity allows for elastic properties and some parts of its chain have a loose coil that gives spring-like properties
They contain no glycosylation (no OH-Lys) but do have some OH-Pro
How is elastin made?
Tropoelastin is secreted and then cross-linked into a fiber close to the PM
Rose’s is constantly gaining and loosing collagen, what is wrong with her?
Nothing! Collagen turnover is healthy
Jen’s wounds are not healing, her blood vessels are fragile and her teeth are falling out, what is wrong with her?
The pro-alpha chains that are meant to become collagen cannot form a triple helix and are instead degraded.
This means no new collagen can be made
What kind of genetic diseases are collagen diseases typically?
Autosomal dominant
What is type 1 collagen?
The most common type, it is fibrillar and contains no interuptions
After aggregating into cable-link bundles, that look like a long rope, it can be seen with light microscopy
Found in skin and bone
What is type 4 collagen?
Collagen that forms networks
Its superhelix contains many interruptions and thus bends
Huge part of basal lamina
What are types 9 and 12 collagen?
Non-fibril forming
Helices are often interupted with short non-helice parts
They bind collagen fibrils to link them to eachother and the ECM (in charge of organization)
What drives collagen formation? Where does this happen?
-Self-assembly tendency
-Near the PM
What are the 8 steps of collagen assembly?
1) Make pro-alpha filaments
2) Proline and Lysines Hydroxylation (not all!)
3) Lysine glycosylation
4) Self-assembly
5) Triple helix forms
6) Leave secretory vesicle
7) Cleave propeptide chains
8) Full fibril forms (with self-assembly)
*Later aggregation to fiber
Why do collagen helices have propeptides?
To prevent fibrils from forming inside the cell
Why do we think we hydroxylate the Pro and Lys in collagen pro-alpha chains?
We think the OH stabilizes the triple helix via H-bonds
What two amino acids are plentiful in collagen?
What do they do?
Proline: Stabilizes helical structure of each class
Glycine: Lets chains pack together (exists every 3 a.a.)
How to we categorize GAGs?
Sugar linkages
Sulfation location and #
What is hyaluronan?
Most simple (no sulfation, tends to not be linked to protein core, identical disaccarides)
NOT exocytosed -> Secreted from enzyme complex on the PM
What are the 4 main GAGs
Keratan Sulfate
Heparan Sulfate
Chondrotin Sulphate (Dermatan Sulphate)
Hyaluronan
What are GAGs?
Unbranched disaccharide chains with high negative charge that attracts cation clouds that attract water (thus giving tissue compression resistance via turgor strength)
Their volume is a lot larger than their mass and they form gels at low concentrations
What cation is most prevalent in a GAG cloud?
Na+
What is a proteoglycan?
A heavily glycosylated (mostly O-linked) protein with at least 1 GAG sugar.
The core protein tends to have a LINK domain to help covalently link GAG
What are aggregan and decorin?
Proteoglycans
Aggregan has over 100 GAGs (in cartiledge)
Decorin has 1 GAG and binds collagen to regulate its fibrils
What are the main components of the ECM?
Proteoglycans
Fibrous Proteins (ex: fibrin, collagen, elastin)
Non-collagen N-linked glycoproteins
+ other matrix associated proteins that modify the behavior of the matrix
What are the two components of tissue
Cells
ECM
What type of microscopy do we need to see Gap Junctions?
TEM
How are GAP junctions made?
6 connexins form one connexon
2 connexons from 2 diff cells meet to form a channel
Multiple channels together forms a Gap junction
How do connexons come together to form their junction?
New ones exocytose and then diffuse till they stick to plaque (protein aggregation)
What can open/close a gap junction?
Change in pH
Voltage difference
Neurotransmitters
Are plaques and junctions static?
No! Plaques can be modfied, destroyed and reassembled
connexons are also always being removed from the core and added to the periphery
What is the main role of Gap junctions?
Mechanically and electrically connect cells by allowing the exchange of small molecules
Explain how the GAP junction turnover Experiment Works
What do proteoglycans do?
Resist compression and allow diffusion
(bc creates a hydrated gel-like substance)
What sugars typically make up GAGs?
1: GluNAc or GalNAc (usually sulfated)
2: Uronic Acid
What links GAGs to the core protein?
a linker tetrasaccaride that sticks to Serine (bc O-linked means link to Thr or Ser) and then they add the sugar one at a time
What is N-linked glycosylation?
Link to Asparagine
what are mesenchymal cells
unattatched epithelial cell precursors
they can migrate as individuals or stick in 3D clumps but they are only loosely organized
they may be surrounded by basal lamina
what are epithelial cells?
sheets of cells that move in harmony and have clear polar character
how do mesenchymal cells become epithelial cells
they start to express cadherin
what is cadherin?
Animal-defining
Calcium-dependent (bc needed to stabilize hing regions between domains - if destabilized, rapid degredation) Adhesion proteins (need it for binding affinity and rigidity) that form velcro-like anchoring junctions
with cadherins (but typically only those of the same type)
What are cadherin-cadherin junctions like?
A bunch of weak binding in parallel makes a strong attatchement that can easily be dissembled via sequential breaking on the sides
The junctions are symetrical and the cadherins bind at their N-termini tips
What links cadherin to the cytoskeleton?
Catenins and other acccessory anchor proteins
p120 and B catenin: link actin cytoskeleton in adherin junctions
y-catenin (plakoglobin): links intermediate filaments in desmosomes
What is the consequence of no cadherin?
Cells can’t act as one anymore,epithelial tissue is never formed from mesenchymal tissue, cells meant to be together don’t stick together
How do cadherins bind eachother?
Via homophilic interaction
How is the ECM regulated?
(AKA how are signals sent from cell to ECM and vise versa)
Via matrix receptors (transmembrane proteins linking the ECM to the cytoskeleton)
What is the main matrix receptor in animal cells?
Integrin
What kind of signal transduction does integrin do?
For what purpose?
Mechanical (associated with cell stress) to molecular
To modify the cytoskeleton
Describe the structure of integrin
Non-covalently linked heterodimers (where each combin has its own properties) with short C-terminal intracellular domains
Describe the following terms
Homo/Heterotypic
Homo/Heteromeric
Meric: Refers to the connexins in the connexons (either all the same or not)
Typic: Refers to the connexons in the channel
(either both the same homomeric connexon or 2 different homo or hetero connexons)
How does integrin interact in vs outside the cell?
Inside: With (mostly always) actin via talin (an accesory) but in epithelial, hemidesmosomes are more common
Outside: With specific A.A sequence on ECM proteins and sometimes other ligands on other cell surfaces (*dependent on the [ ] of Ca+ and Mg+)
They also bind other anchor proteins
How is integrin involved in Proliferation and Survival?
Anchorage Dependence is mostly mediated by integrins and their intracellular signals
Also multiple adhesions at widely separated sites force cells to spread over a large area which helps them to grow better (this is part of why tissues regen after injuries)
What is Anchorage Dependence?
-Many cells need to be attatched to ECM to grow/reproduce
-Muscle and epithelial cells apop without ECM
This ensures that cells only live and survive in the right situations (Cancer and metastasis are examples of mutations in this pathway)
What ECM proteins do integrins usually bind?
Laminins, fibronectin
What are hemidesmosomes?
Connects epithelial cells to basal lamina
Laminin (which binds type 4 collagen) lives in the basal lamina.
Laminin Binds Integrins which are in both the basal lamina and the cell.
Inside the cell, the integrins bind the anchoring protein plectin which then bind the anchoring protein dystonin (BP230)
Dystonin then binds keratin (an IF)
What kind of junction are most epithelial cell-matrix atatchments?
hemidesmosomes
What is inside-out activation?
Activation triggered by regulatory molecules inside the cell that activate talin’s ability to bind B chain of integrin to activate it
ex:thrombin binds GPCR which activates intracell signalling pathways that activates talin to activate integrim
What is the difference between active and inactive integrin
Only active integrin can bind ligans in the ECM and adaptor proteins in the cell
Which integrin dimer binds intracellularly?
Beta
How are adherin junctions assembled?
Cadherins cluster and then
Cortical tension is locally lost via local Rac Activation and Rho inhibition which relaxes actin-myosin bundles
Tension loss allows for expansion of the junction
What are Rac and Rho?
Monomeric GTPases that regulate the actin cytoskeleton
What does cortical tension do?
Keeps cells bulged instead of flopped
What are zonula adherins
also called adhesion belt. the typical type of adheren junction for epithelial cells
forms a continous adhesion belt beneath the apical surface allowing epithelial cells to act like one co-ordinated unit by being tethered to contractile actin
Which type of junction works like velcro?
cadherin
What is the difference between connective and epithelial tissue?
Connective:
-Lots of ECM usually with lots of collagen
-Strong attachement to its ECM (via cell-matrix junctions with cytoskeleton
-not many attatchements between the cells
-Bears most mechanical stress and provides support
Epithelial:
-Thin ECM mat on the basal lamina
-Tightly bounded sheets with continous cytoskletal bands due to junctions
-Linked to ECM with cytoskleton cell-matrix junctions)
Where in epithelial cells are tight junctions found?
Closer to apical side than to basal
What do tight junctions do?
Form a selectively permeable barrier
-keeps molecules from moving between cells and thus leaking back to OG space (thus spaces outside apical and basolateral side are separate areas)
-Keeps apical and basolateral proteins on their respective sides
Results in transcellular transport which requires specifc and different transporters on apical and basolateral sides
What is transceular transport?
Transport into one side of a cells membrane and then then out another side
What is paracellular transport?
Transport between cells via cell junctions
Are tight junctions perfect seals?
Depends on the cell type. There may be some paracellular transport
How can we visualize tight junctions with EM?
Put heavy metals in one area (ex: apical space) and watch how they can’t move into the other areas
What are the main 3 proteins involved in tight junctions?
Claudins, Occludins and Tricellulin
What are claudins?
Proteins that form the sealing strands of a tight junction.
Mice babies without them die shortly after birth
What are ocludins?
Non-essential tight junction proteins that limit permeability
What is Tricellulin?
A required tight junction protein that prevents leakage
Why do we think we have different types of claudin in different cell types?
So that paracellular pores of varying peremability levels and selection can be made
What are E, N and P-cadherin?
P: First found in placenta and epidermis
E: First found in epithalial cells
N: Found in Nerves first, also in lens and muscle
All of them are “classical cadherins”
What are desmosomes?
Organized buttons where Cadherins link to IFs via a dense adaptor protein plaque
Often found in tissues with high stress (bc IFs have lots of tensile strength)
What are junctional complexes?
A combination of apical sealing strands made by ZOs, adherens and desomosome junctions
What are ZO proteins?
Zonula Occludens (Intracellular scafolding proteins)
They have multiple bind sites for other ZOs, claudin, occludin, actin and signaling proteins,
What do ZO proteins do?
They bind other proteins and other ZOs
ZO proteins on the inside of 2 cells are linked by proteins (claudins and occludins) that bind their various intracellular domains and then bind on the outside
what proteins form the hemidesmosome plaque
plectin and dystonin
Explain Focal Adhesion Kinase
A cytoplasmic adaptor protein that phosphorylates Tyr residues
Part of the outside-in singalling:
Integrin tells talin to recruit FAK
Cluster of FAK cross-Ps to create docking site for other signalling molecules in the cell
*Note: Focal adhesions are Cell-ECM junctions that involve actin and allow for the transduction of signals
What is membrane ruffling?
When individual lamellipodium fails to adhere and is lifted up and carried backwards
How do cell adhesion molecules like integrin differ from normal cell-surface receptors that do signal transduction
They bind with lower affinity are are much more abundant
What do integrins do when they are activated?
Cluster and form a plaque ex: focal adhesions
How do cells rapidly change their adhesion to the ECM?
Inside-out or outside-in activation of integrin
Do you have the same ECM as your 7 year old self?
NO! it is constantly being turnedover
Why would we need to rapidly degrade the ECM?
Tissue repair!
Why would you degrade ECM?
Needed for:
-Cell division
-Cell travel
-WBC get to site of infection
-Escape from BL during normal growth
-metastasis
How is the ECM degraded?
Locally produced extracellualr proteases
-matrix metalloproteases: Depend on Ca+ or Zn+
-Serine proteases: highly reactive serine at their active site
Are ECM proteases general or specific?
Depends
How are ECM proteases carefully regulated?
-Local activation (they are secreted as an inactive precursor)
-Confiement by cell-surface receptors
-Secretion of inhibitors
What is the primary organizer of the BL
laminin
why is the mechanical role of BL important?
Epidermolysis bullosa
Other than under epithelial cells, where else might you find BL?
surrounding muscle cells in connective tissue
In between epithelial cells in urine holder and blood vessel endothelial cells
How does collagen resist tensile forces in all directions?
What determines their size and arrangement?
it is organized in different patterns
Connective tissue cells by guiding fibril formation near the PM
Fibril-associated collagens also mediate interactions of collagen fibrils with eachother and other matrix macromolecules
What is a glycoprotein?
A bunch of short sugar chains on a protein (vs proteoglycan which has long chains)
What determines Gap junction permeability
Its connexins
Where do tight junctions need to be to form sealing strands
Usually Apical to adherens and desmosomes
what does CPE do?
Cytotoxic toxin that disrupts claudin and forms pores in the PM
If you just use C domain, not cytotoxic but still INC paracellular permeability
where would you find lots of desmosomes
tissues subject to high stress
Where does cadherin link to catanin?
At its (cadherin’s) intracellular domain
If I have cells with lots of E cadherin and cells with less, how will they assemble?
The ones with more cadherin bundle in the center
What is the difference between adaptor and adhesion moleucles
Adaptor: in the cell
Adhesion: transmembrane
