L22 - Extracellular Matrix, Integrins and Cell Migration Flashcards
What components make up the ECM?
Fibronectin
Laminin
Collagen
Elastin
What is fibronectin?
Large protein
Secreted into extracellular matrix
What are the domains of fibronectin?
Heparin binding
Cell binding
Collagen binding
Self-association
What is laminin?
Large protein
Trimer
What are domains of laminin?
Integrin binding
Self-assembly
Perlecan binding
Coiled coil domain
What do integrins form?
Form heterodimers
Each monomer has a single transmembrane domain
What is the extracellular structure of integrins?
Cysteine rich domains
- Form disulphide bridges
- Alpha subunit is cleaved and held together by disulphide bridges
Matrix binding domain at amino end
- Binds to ECM and divalent cations (Mg and Ca)
- Activating function
What is the intracellular structure of integrins?
Talin, filamen and a-actin binding domain at carboxyl end
What is the composition of integrins?
18 α and 8 β subtypes
24 variants
What do integrins interact with?
The cytoskeleton
How do integrins bind?
Binding is a two-way process
Activated both intracellularly and extracellularly
Outside in activation – strong ligand binding
- Inactive integrin
- Extracellular domain folded up in an inactive state – not binding to ECM
- If they do bind to ECM the conformation changes
Inside out activation – strong talin binding
What are FAKs?
Focal adhesion kinases
When integrin binds to the ECM how does it activate FAK?
- Fibronectin interacts with integrin extracellularly
- Transduced intracellularly resulting in activation of FAK
- Results in tyrosine phosphorylation – recruitment/activation of multiple tyrosine kinases
- Results in polymerization of actin
What is FAK activated by?
Activated on formation of adhesions
What are FAKs important for?
Recycling focal adhesions to enable migration
What is attachment dependent cell death?
If some cell types are not adhered to appropriate substrate they will die
Act in anoikis - attachment-dependent cell death
How is mammalian cell motility brought about?
Actin based
What is the role of lamellipodium in cell motility?
The cell moves its self along by extending its lamellipodium in the direction of migration
- As cell moves it loses and create new adhesions
- Actin reorganisation
- Front membrane is being pushed out by actin polymerisation
- Actin polymerisation at plus end protrudes lamellipodium
- Action fibred are being added to at the leading edge
Actin forms a cortex in the cell which aids in?
Membrane tension
Why is a motile cell highly polarised?
Plus and minus end
Actin is continually recycled
Membranes are also being moved
What molecules helps in assisted tail retraction?
Myosin II
What two processes occur at the leading edge?
Ruffling
Actin transport
What is the role of ruffling at the leading edge?
Active fibres pushing up in different directions
Involved in sensing guidance cues
What is the role of actin transport at the leading edge?
Pushes out the membrane at the front
Cytochalasin binds to the plus end of actin filament and blocks more actin being added
What is the APR complex involved in?
Actin polymerisation
What is the APR complex made up of?
Made up of multiple proteins – Arp2 and Arp3
- Resemble the structure of actin
What is the role of the APR complex?
Nucleation of actin monomers to form a filament
- Arp2 and Arp3 bind to form a complex
- This complex binds to actin monomers on the minus end
- New actin monomers are then added to the plus end
What does Rac stimulate?
The formation of Arp2/3 complex
This then enables the cell to move forward – drives leading edge
What are important factor of focal contacts and motility?
Focal contact turnover, actin interaction and microtubules are all important
Why does recycling occur throughout the entire length of the cell?
Brings adhesion molecules to the front of the cell
What is the role of focal adhesions maturing?
Roles for action binding proteins, small G-proteins, myosin generating force
What are the two categories of focal adhesion?
Low density
- Rac1 kinase and cdcc42 kinase dependent
High density
- RhoA and actin-myosin interaction dependent
What focal adhesions are found at the front of the cell?
Low density adhesions immobile
Right at the leading edge
What focal adhesions are found at the back of the cell?
High density adhesions slide in the membrane
Why do extracellular signals interact with the cytoskeleton?
To direct movement
What are diffusible signals for motility?
Netrins
What are insoluble signals for motility?
CAMs
ECM
What are fibronectin trails?
Fibronectin is laid down by migratory cells for other cells to follow
What inhibits migration by fibronectin trails?
Antibodies to fibronectin
Injection of arginine, glycine and asparagine
What happens all around the cell during migration?
Membrane endocytosis
Deposited at the leading edge where exocytosis occurs
- Net membrane addition
If the membrane is fixed by focal contacts the cell will?
Move forward
What other roles does endocytosis have in motility?
Shaping chemotactic gradients
- Cell tries to reach towards higher level of signal
- To form gradient often have endocytosis of ligand
Confining signalling in migratory cells
- Confine activation to leading edge by endocytosis
Modulation of adhesive contacts and ECM
- Recycles by endocytosis to release adhesion
Polarisation of endocytosis
