6-9: Caswell Flashcards
How is Rac expression different in randomly migrating cells vs directional migration?
High Rac -> Multiple Protrusions -> RANDOM
Low Rac -> Focalised Rac Activity -> DIRECTIONAL
What are the three main types of directional migration?
Chemotaxis -> SOLUBLE external clues
Haptotaxis -> Matrix Ligands (e.g., collagen)
Durotaxis -> Physical cues/ECM sitffness/topologies
What mediates most steps in cancer metastasis?
Pairs of Chemoattractants and GPCRs (e.g., CXCR4 with CXCL12)
Fun side note: CXCL12 is the human version of sdf1a in Zebrafish!
Describe the two models of chemotaxis
Compass model (in a strong gradient with HIGH receptor occupancy at leading edge) - cells move continuously towards a chemoattractant, reorientating themselves like a compass
Bifurfaction + Bias (in a weak gradient with LOW receptor occupancy at leading edge) - cells reorientate more gradually, pseudopods split, direction is determined by the more stable pseudopod
Describe the general pathway by which a chemoattractant determines cell dynamics
Chemoattractant recognised by GPCR
-> PI(3,4,5)P3 activation
-> Rac activation (promotes polymerisation + protrusion)
Where Rac is less active (at the back of the cell) Rho dominates (they both inhibit each other)
Therefore, the Rac/Rho dominated areas of the cell are determined by where on the membrane GPCR activation is occurring
Describe the specific mechanism by which GPCR activation leads to Rac activation and migration
GPCR activation -> Gßy dissociation
-> Activates P13K
-> Converts PIP2 to PIP3
-> PIP3 and ßy synergise to activate P-Rex1 enzyme
-> P-Rex1 is a GEF which converts Rac-GDP to Rac-GTP
-> Rac promotes LAMELLIPODIA FORMATION
What do PI3K knockout experiments in Dictyostelium and Mus show about whether PIP3 production is essential for migration?
In Dictyostelium, migration is more “wobbly” but still occurs
In Mus, migration sometimes shorter, but still occurs
So PIP3 is PART of the answer, but not ALL - there are more ways to activate Rac
Describe the more direct pathway for Rac activation in Dicytostelium
Gßy -> ElmoE (a GEF)
-> ElmoE directly stimulates Rac activation
What happens involving the direct and indirect mechanisms of Rac activation when a cell is very close to a Chemoattractant?
They SYNERGISE -> PIP3 is highly localised, so migration is more direct and efficient
Describe how Rac/Cdc42/RhoA activity can be visualised as soon as a chemoattractant is “released” [EXPERIMENT]
The chemoattractant is initially caged, until released suddenly by UV
This is combined with FRET imaging of Rac, Cdc42 or RhoA:
RFP - PKN-RBD - RhoA - GFP
When RhoA is active, a conformational change brings RFP and GFP together, so RFP emits red light due to resonance energy
Describe the roles of Cdc42, Rac and RhoA in chemotaxis
Cdc42 = steering (inhibits RhoA and determines polarity)
RhoA = rear retraction
Rac = engine (drives lamellipodia via Arp2/3 activation) *activated last
How do cells sense the extracellular matrix (i.e. what structure)?
Cell Matrix Adhesion Complexes (CMACs)
How does the ECM differ from an in vitro environment (and how do CMACs accordingly differ)?
ECM in tissues is significantly softer than glass or plastic; CMACs are smaller but do still exist
What is the difference in terms of adhesion size and turnover between randomly and directionally migrating cells?
Random: small, dynamic adhesions with HIGH turnover
Directional: larger, stable adhesions with LOWER turnover
What happens if there is NO adhesion turnover?
No migration can occur - the cell is STUCK
Is there a linear relationship between cell adhesion and rate of migration?
NO - it is more like a bell curve, as adhesion is required for generating traction force and signals that promote actin remodelling, BUT very high levels of adhesion decrease motility
What are CMACs and what do they consist of?
They are multi-protein complexes that form the point of contact between the cell and the ECM
- Integrins (bind to ECM)
- Adaptor proteins e.g. talin, vinculin (link integrins to F-actin)
- Other adaptors and signals play various roles (e.g. FAK alters surrounding environment via phosphorylation, Paxillin binds GEFs/GAPs to promote actin polymerisation or Rho/contractility
Name the layers of a CMAC as you go further into the cell
- ECM
- Integrin ECD
[PLASMA MEMBRANE] - Integrin signalling layer
- Force transduction layer
- Actin regulatory layer
- Actin stress fibre
Describe the general structure of an integrin and how many varieties there are
They consist of an a and ß subunit, which Heterodimerise to form 24 integrins
There are 18 alphas and 8 ßs
Describe the general FUNCTION of an integrin
Priming: Talin (or similar signalling molecule) binds the cytoplasmic tail to prime integrins for ECM ligand binding
Activation: Extracellular Domain binds ECM ligands (which ones depends on the heterodimer), causing conformational change that activates the integrin
What is meant by “integrin clustering”?
Integrins bind ECM ligands, and naturally cluster around where those ligands are most concentrated
They then recruit adaptors to link to actin, and promote assembly of nascent adhesions
What are the three stages of adhesion complex maturation?
Nascent Adhesion
-> Focal Contact
-> Focal Adhesion
What are the possible next steps from Nascent Adhesions?
They can disassemble, OR:
They can recruit FAK + Paxillin
-> Paxillin recruits beta-Pix (a Rac-GEF) to activate Rac
-> Rac inhibits Rho and Promotes Actin Polymerisation
-> FOCAL COMPLEX
What are the next steps from Focal Contacts?
Focal Contacts continue to recruit adaptor proteins, which initially continue to activate Rac. Eventually, they change the signalling to activate RhoA rather than Rac (e.g., via p190-RhoGEF)
-> Once Rho is active, it activates ROCK and inhibits Rac
-> Contractility increases, causing tension and forming MATURE FOCAL ADHESIONS
-> Eventually, the cell rear shifts (Rear Retraction) and the migration cycle is complete
What is meant by “Signal Compartmentalisation” in the context of cell migration?
There are different signals, pathways and processes occurring at the front of the cell (where the lamellipodia are) compared to the trailing edge
Overall result of this: Actin Polymerisation, Rac and Cdc42 at leading edge, Contractility and Retraction, RhoA at trailing edge
Why do cells lacking FAK show defects in migration?
They actually generate MORE CMACs, not fewer. However, TURNOVER IS IMPAIRED, so they are stuck and can’t migrate
How does FAK promote CMAC turnover?
Through CALPAIN:
FAK recruits calpain2 (along with other adaptors)
-> Calpain cleaves Talin 1, breaking the link between integrins and the actin cytoskeleton
-> FAK also recruits MTs and Dynamin2, promoting clathrin-dependent endocytosis of integrins
Describe the process of endocytosis of integrins
Several different mechanisms - partly depends on where in the cell
At the back of the cell, FAK-Dyn2 complex recruits clathrin to promote endocytosis of integrins
At the front of the cell, Numb recruits clathrin
Rab21 binds DIRECTLY to the alpha subunit of ß1 integrins and induces apparently Clathrin-independent endocytosis(!)
{BR DETAILS FOR ESSAY}
Describe the actual mechanism by which Talin functions
The Talin head-domain (FERM) binds the Cytoplasmic Tails of ß-integrins, while its Tail Domain binds F-actin and Vinculin
Talin forms anti-parallel homodimers
Name and describe some of the other adaptor proteins involved in Integrin-Actin links (besides Talin)
Vinculin - binds talin, actin and others (these binding sites are normally masked by intramolecular head-tail domains
a-actinin - forms anti-parallel homodimers to link two AFs (part of the spectrin family of actin cross-linkers)
Kindlins - act synergistically with talins to activate integrins (note: they ALSO contain a FERM domain)
What is the overall name for the structure and interactions linking Integrins to Actin (and how many proteins are involved)?
The ADHESOME (around 180 interactions)
Give three examples of specific integrins (in terms of the a and ß subunits that comprise them) and which ECM ligands they recognise
a5ß1 -> Fibronectin
a1ß1 -> Collagen
a2ß1 -> Laminin
How does ECM ligand density affect a cell’s ability to migrate through the ECM?
Higher ligand density -> cells can form more CMACs and thus generate more traction and contractility (“CMACs win the tug of war with the lamellipodium”)
Therefore, higher density -> faster migration (up to optimum)
What drives Haptotaxis (and in what sense is it specific)?
ECM ligands - specifically, integrin receptors - and CMACs
Cells expressing fibronectin-binding integrins (for example) migrate towards fibronectin, but NOT laminin or vitronectin
What are the “key cellular players” in allowing cells to sense and respond to substrate rigidity?
- Force generator (actomyosin)
- Force transmitters (cytoskeleton and focal adhesions)
- Mechanosensors (maybe BR if time?)
What part of the cell is the KEY to allowing cells to “feel” force?
CMACs (specifically integrins) - point of contact between cytoskeleton and substrate
What are Catch Bonds (in general and in context of CMACs)?
Catch bonds are biological ligand-receptor bonds that become STRONGER when pulled apart by mechanical forces
In CMACs, actomyosin contractility applies a strong pulling force on CMACs, making ligand-binding tighter
(The more force applied, the stronger the bond)
Which type of Myosin is involved in Rear Retraction?
Non-muscle myosin II (NMMII or Myosin II)
Describe the basic structure of NMMII
Head: Mg2+-ATPase motor domains
Connected via Essential and Regulatory light chains (ELC and RLC) to the coiled-coil rod domain
Non-helical tail
Describe how Rho-A stimulates Rear Retraction (specific proteins involved)
Rho-A-ROCK signalling controls PHOSPHORYLATION of the Myosin Regulatory Light Chain (or MLC)
-> The coiled-coil rod domain unwinds
-> This allows formation of myosin filaments and activation of ATPase activity
How does Actomyosin Contractility relate to Adhesion Maturation (general principle)?
As Actomyosin Contractility exerts a force on adhesions, this promotes recruitment of further adaptors and signalling molecules (e.g. vinculin et al) which leads to maturation
How are vinculin and other adaptors able to bind to Adhesion Complexes when they are pulled and mature due to Actomyosin Contractility?
Some CMAC proteins (e.g., TALIN) can be stretched
-> As Talin stretches and unfolds, its Helix12 (and Vinculin-binding site) are exposed
Which experiment demonstrates what happens to Talin when it is pulled by actomyosin?
Attach the N-terminus of Talin to a glass slide via a Tag
Attach the C-terminus to a magnetic bead
Use a magnetic electrode to pull the C-terminus away from the N-terminus
-> Vinculin Binding site on helix 12 is exposed
What is meant by “Retrograde Flow” of actin?
When actin filaments slide back at the leading edge as they don’t have enough force to protrude the cell membrane forwards
What is the “CMAC Clutch” and how does it enable protrusion?
As AFs slide back at the leading edge (retrograde flow), CMACs transiently capture retrograde-moving actin
-> Forces are transferred to the substrate by integrins
-> Actin polymerisation continues - now the Clutch is engaged, polymerisation drives protrusion
Do cells migrate better on a soft or stiff matrix (and how do they sense the gradient)?
Soft - they pull/tug repeatedly on the ECM via CMACs to sample the environment and determine the stiffness gradient
In what two general ways do migrating cells overcome the barrier of the ECM in 3D?
DEGRADATION (invadopodia) and MORPHOLOGICAL CHANGES (squeezing through gaps)
What are the 4 kinds of cell protrusions seen in 3D migration?
Lamellipodia, Filopodia, INVADOPODIA, MEMBRANE BLEBS
Describe the general structure + function of Invadopodia
They are filopodia-like protrusions that degrade the underlying matrix
Describe the general structure + function of membrane blebs
They are protrusions generated by hydrostatic pressure, used by cells in amoeboid migration
Describe the role of invadopodia in migration
Invadopodia promote matrix degradation by co-ordinating delivery of proteases (specifically Matrix Metalloproteases such as MT1-MMP)
-> these cut through collagen fibres to allow efficient migration in 3D
Note: Invadopodia often form around points of restriction, and the nucleus must be deformed
Describe how collective cell migration generally occurs
Usually a leader cell (following steps 1-5 of individual cell migration) forming a microtrack, while keeping contact with the cells behind
-> all cells move together until individual cells start budding off by downregulating contacts
What must an epithelial cell do in order to become migratory?
Must acquire a Mesenchymal Phenotype by downregulating adhesion molecules (e.g. cadherins) and upregulating other molecules such as vimentin
-> Cells can transition between these states due to Plasticity
What is meant by plasticity?
Cells can transition between states and modes of migration (e.g. EMT/MET, Mesenchymal <-> Amoeboid, etc.)
Describe the differences between mesenchymal vs amoeboid migration
Mesenchymal uses lamellipodia/filopodia as its protrusions, and is protease-dependent; it is slower and uses stronger adhesions
Amoeboid uses membrane blebbing as its protrusions and is less dependent on proteases (less tissue damage); it is faster and uses weaker adhesions
What are two of the “various roles” played by Filopodia in migration?
- Sensing chemoattractant (familiar role)
- “Actin spikes” interact with ECM fibrils to generate protrusive force
What happens to the cell membrane when cells migrate quickly in 3D?
It accumulates at the rear of the cell (where membrane tension is low)
What does Flipper TR do?
It is a probe that can measure cell membrane tension
How is rear retraction activated in 3D mesenchymal migration?
- Caveolae form at the cell rear due to low membrane tension
- Caveolin-1 recruits Ect2 (a RhoA-GEF)
- Ect2 activates RhoA and Actomyosin
What do osmotic shock and addition of isotonic solution show about cell migration?
They show the importance of membrane tension (and lack thereof) in triggering rear retraction:
- Osmotic shock increases membrane tension at the rear, relocalises Caveolin-1 and prevents RhoA activation
- Isotonic solution re-establishes Caveolin-1 localisation and RhoA can be activated
What change allows Blebs to form in Amoeboid migration?
A decrease in cortical actin (i.e. actin linked to cell membrane)
Describe the process of membrane blebbing
- Cortical F-actin rupture
- Hydrostatic pressure causes bleb expansion
- F-actin begins to assemble at the bleb cortex
- Eventually, the bleb retracts due to RhoA, ROCK and Myosin
Describe the importance of actomyosin in allowing blebs to form
Actomyosin generates contractility at the rear and cortex of the cell (driven by RhoA-ROCK)
-> Where the membrane ISN’T taut, blebs form
Describe what is meant by “the nuclear piston”
- The nucleus can move somewhat independently within the cell
- Actomyosin contractility pulls the nucleus forward
- There is an increase in hydrostatic pressure in front of the nucleus (compartmentalised pressure)
- This hydrostatic pressure provides protrusrion force
- LOBOPODIA form at front of cell
Name some of the molecules required for Nuclear Pistoning
Actomyosin (ofc), IFs, Nesprin 3, Integrins, Vimentin
