2-5: Millard Flashcards
Describe the technique used to visualise Actin Filaments in cells
AND the technique for visualising Actin Dynamics in Migration
PHALLOIDIN binds F-actin but not G-actin -> fluorescently labelled Phalloidin can be used to stain F-actin in cells
GFP-ACTIN fusion protein shows actin dynamics
Are actin filaments NECESSARY for cell migration? How can this be demonstrated?
YES they are - if actin polymerisation is blocked by Cytochalasin D, lamellipodium collapses and cell can’t migrate in that direction
Describe the significance of photobleaching in our understanding of actin dynamics in lamellipodia
When GFP actin in a region near the front of the lamellipodium is photobleached, we see fluorescence recovery at FRONT of lamellipodium as new, non-bleached actin is incorporated (only at front) - all replaced within a minute
If the region BEHIND the lamellipodium is bleached, a wave of dark, bleached monomers appears at the front of the lamellipodium (reinforces first result)
Describe the dynamics of actin polymerisation observed in vitro (e.g., speed, concentrations etc)
Nucleation: occurs spontaneously, but slowly as it is kinetically unfavourable (formation of dimers/trimers/oligomers is slowest part of filament formation)
Elongation: rate of growth proportional to monomer concentration (occurs at both ends)
As monomers are incorporated into filaments, the monomer concentration falls; at Critical Concentration (around 0.1µM in phys. conditions) monomers are added/dissociate at same rate - equilibrium
Describe the dynamics of actin polymerisation observed in CELLS (e.g., speed, concentrations etc) and state why this is different from in vitro
Nucleation: rapid once triggered, but NOT spontaneous
Elongation: rapid but ONLY ONE END; branched network in lamellipodia
Monomer concentration is ~100µM so system NOT IN EQUILIBRIUM (hence rapid growth)
-> ACTIN BINDING PROTEINS control AF assembly/disassembly
Describe the role of profilin and why this explains the different actin dynamic in vivo vs in vitro
Profilin is an Actin Binding Protein that binds monomers, but NOT filaments (most G-actin in cell is bound to it)
Once bound, profilin changes the behaviour of actin:
1. It cannot bind to minus ends of AFs, can only incorporate into PLUS end
2. Readily exchange ADP for ATP
3. Cannot spontaneously nucleate new filaments
If profilin prevents spontaneous nucleation of new filaments in the cell, how does nucleation occur?
The Arp2/3 complex (in its active state) forms a filament “seed” into which profilin-bound actin monomers can be incorporated
Describe how Arp2/3 nucleation occurs
- Arp2 and Arp3 are activated (by various activators - see later lectures) and come together to form a filament “seed”
- Profilin-bound monomers can be incorporated into this seed
- Arp2/3 remains bound to the minus end, while the filament grows at the plus end
Note: Arp2/3 also binds to the sides of existing filaments (70 degree angle) leading to the branched networks seen in lamellipodia
What stops actin filaments from growing indefinitely once nucleated?
Capping protein (CP, or CapZ)! It binds to filament plus ends (usually within 1 second of nucleation)
How can polymerisation at the leading edge and depolymerisation at the minus end be experimentally visualised?
Photobleaching experiments:
-Use a strong laser to destroy fluorescence of GFP-actin at front of lamellipodium
-> newly incorporated fluorescent actin repopulates the entire lamellipodium within a minute
If AFs are capped at the plus end by capping protein, and at the minus end by Arp2/3, how can they be depolymerised?
ADF/Cofilin family (ADF = Actin Depolymerising Factor) promote depolymerisation of older actin
Describe the function of ADF/Cofilin proteins
(Exact mechanism unclear but thought to include the following)
- ADF/Cofilins are localised to cellular regions with high actin turnover, e.g. the leading edge of migrating cells
- They sever AFs and also increase the rate at which actin monomers “fall off” the pointed end
- ADF remains bound to monomers to prevent immediate reincorporation
How can ADF/Cofilins determine which sections of the actin network are “old” and need to be depolymerised?
ATP Hydrolysis acts as a molecular timer:
A few seconds after incorporation, ATP is hydrolysed to ADP, so sections of the network containing lots of ADP are older sections (and these are the sections ADF binds to)
If all dissociated monomers from AFs are bound to ADP, how can they be reincorporated at the plus end?
They bind to profilin, which catalyses the exchange of ADP for ATP
What are filopodia (structure and cellular function)?
Long, thin actin protrusions which act as “sensors” for the cell to explore its environment; they contain unbranched actin filaments, tightly bundled together
How do filopodia form?
The TIP COMPLEX (a group of many actin regulating proteins) associates with the barbed ends of some actin filaments, and prevents them from being capped
-> These filaments converge with each other, and are cross-linked by actin-bundling proteins e.g., FASCIN (shown by knockdown to be essential for filopodia stability)
- Filopodia grow until the tip complex dissociates
What are Actin Bundling Proteins? (And name an example found in each type of actin network)
They are proteins which cross-link and stabilise actin filaments in a network - they have at least 2 F-actin binding sites to allow this
alpha-actinin (in contractile bundles/stress fibres)
filamin (in gel-like networks in the cell cortex)
fascin (in tight parallel bundles in the filopodia)
Which proteins are found in the filopodia tip complex, and what do they do?
- Ena/VASP proteins are thought to prevent Capping Protein from binding plus-ends, and promote filament elongation
- Formins (e.g., dDia2, mDia2) nucleate unbranched actin filaments by binding to the PLUS END; they also promote elongation
What role do Ena/VASP proteins have OUTSIDE of the filopodia?
They regulate filament length in lamellipodial networks:
High Ena = longer filaments with weaker pushing force but faster advance
Low Ena = shorter filaments with stronger force but slower advance
Need the right balance of Ena vs Capping Protein for the environment
If filopodia form via convergence of existing actin filaments - but they can form in the absence of Arp2/3. How?
They can also be nucleated by FORMINS! E.g., DAAM
Knocking down BOTH Arp2/3 and DAAM prevents Filopodia, showing that at least one of these two is necessary
Describe the structure and components of contractile stress fibres
Long, unbranched actin filaments (nucleated by formins)
and the following actin-binding proteins:
- Myosin II (motor protein)
- a-actinin (cross-links filaments to stabilise)
- Tropomyosin (wraps around and prevents AFs from depolymerising)
How does profilin ensure that actin monomers can only incorporate into PLUS ends?
Profilin binds to the opposite face of the monomer from where the ATP-binding cleft is - thus blocking the side that could associate with minus ends, while leaving the plus-end-binding side exposed
Cell migration requries co-ordination of actin dynamics throughout the cell. How does this co-ordination happen?
The Rho-GTPase family are master regulators (Rho, Rac and Cdc42) as they regulate MANY Actin Binding Proteins
