1 (Cell motility+ actin) Flashcards
Describe the the different types of cell protrusion
Lamellipodia
* found on fibroblast and epithelial cells
* Behind the highly
dynamic lamellipodium is a more stable
lamella, which contributes to cell migration by coupling the actin
network to myosin II-mediated contractility and substrate adhesion
* extension via actin polymerisation by arp2/3
Pseudopodia
* found in neutrophils + amoeba
Filopodia
* More important as sensors, looking for guiding information/sites to attach
* Neurons use filopodia
* Has a structure called a growth cone which guides the filopodia to make sure it goes in the right direction
* Filopodium forms from a denser rod of parallel actin filaments
* Filopodia is often used by viruses as a bridge between cells
Invadopodia
* Actin polymerization couples with the
extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the
extracellular matrix. Aiding invasion through tissues.
* frequent in cancer metastasis
Differentiate between G-actin and F-actin, with context.
G actin are monomeric actin proteins, whereas F-actin is actin that has been polymerisied into a chain.
Polymerisation of G actin into f actin is found at the leading edge.
What molecule binds to f-actin but not G-actin
Phalloidin, from the death cap toadstall
What would be a good molecule to test if actin filaments are needed for protrusion assembly?
Cytochalasin D is used, which blocks actin polymerisation
How is the treadmilling nature of the lamellipodium shown in the lab?
Photobleaching using UV light, with polymerisation occuring at the leading edge, and shifting back.
What attach cells to the extracellular matrix, and what are they made from?
How would you show this?
What happens if focal adhesions don’t stick properly?
- Cells use focal adhesions to attach to extracellular matrix
- Focal adhesions are made from proteins and integrins
- you could use GFP-paxillin, a molecule which binds to focal adhesions
- Ruffles are created, where adhesions haven’t stuck down properly
What are stress fibres made from?
How are they used in cell motility?
How would you show that this is how they work?
Actomyosin cables, actin + myosin 2 that walk in opposite directions. They are anchored to the ECM via focal adhesions, the focal adhesions at the rear release, the actomyosin cables pulls the rear in.
You could use a myosin inhibitor like blebbistatin to show that it is myosin that pulls the rear in.
Contain:
* Long unbranched actin filaments
* Myosin 2, a motor protein
* A-actinin, crosslinks actin to stabilise
* Tropomyosin, prevents depolymerisation of actin
How do amoeboids migrate/form protrusions?
- Has a layer of actomyosin around the membrane creating pressure
- Actomyosin breaks forming a protrusion similar to lamellipodium but lacking in actin
Actomyosin then reforms inside the bleb
Describe Nucleation, elongation and steady state of acti in relation to rate.
What is the critical concentration?
Nucleation
* Formers of dimers and trimers during nucleation is kinetically unfavourable so is slow
* doesn’t occur in the cell without a trigger
Elongation
* Elongation rate is proportional to the amount of actin monomers
Critical concentration:
Where monomer addition = rate of monomer dissociation.
Usually due to low concentration of actin monomers, doesn’t really happen outside of vitro
What does profilin do?
Profilin binds to actin monomers, but not filaments.
- this makes polymerisation polar as profilin bound monomers can only bind to the plus ends
- profilin monomers swap ADP for ATP
Describe the role of ARP2/3 in actin polymerisation.
ARP2/3 binds to the side of existing actin filaments and nucleates
- Inactive Arp2/3 involves separated arp2 and arp3
- This makes it easier, as forming a dimer is often the hard part
- In the Active state the two come together
- Profilin-bound actin is the incorporated
Filament grows with the arp at the minus end
What stops actin filaments growing indefinetly after nucleation?
How are filaments then depolymerised?
- Capping proteins
- They are depolymerised by ADF/cofilin (actin depolymerising factor)
- ADF/Cofilin binds and severs ADP bound Actin, but not ATP actin
- Meaning the old actin molecules are targeted, so depolymerization occurs at the minus end
What do filopodia do?
Describe how filopodia form.
- Used by cells as sensors to explore the environment
- contain long unbranched actin filaments
Convergent Elongation
- Tip complex associates with a subset of actin filaments preventing them from being capped
- filaments grow and coalesce
- cross linked by actin bundling proteins, like Fascin
Tip Complex
- Ena/VASP proteins
- anti-capping
Actin bundling proteins
- Contain at least 2 f-actin binding sites
-
What is the role of Ena/VASP proteins?
What difference does the concentration of Ena have?
They form the tip complex and prevent capping.
-They regulate lamellipodia actin network
- Low Ena means shorter but stronger so it can push through a more viscous environment
- High Ena means longer filaments but weaker pushing force so can’t push through a viscous environment
What are formins, and what does it mean for actin filaments?
A family of proteins including DAAM and mDia. These can nucleate plus ends of actin filaments, meaning filopodia can form without Arp2/3 complex or even a lamellipodia network.
- formins make long unbranched actin filaments
What are stress fibres made from?
- Long unbranched actin filaments
- Myosin 2, a motor protein
- A-actinin, crosslinks actin to stabilise
- Tropomyosin, prevents depolymerisation of actin
See if you can remember what these all do.
Profilin
Arp2/3
Capping protein
ADF/Cofilin
Ena
Fascin
Formins
Myosin Il
Tropomyosin
a-Actinin
Check you can do these
(correlate to colour)
What is the role of Rho GTPases in actin network regulation?
Rho GTPases are molecular on/off switches
The active state is GTP bound, the inactive GDP bound.
IN the active state it can bind and regulate other proteins.
GEF (guanine exchange factor) is responsible for swapping GDP to GTP.
GAP (GTPase activating proteins) convert GTP to GDP.
Desrcibe the role of FRET in sensing when GTPases are active
FRET (fluorescent resonant energy transfer) as a biosensor
- These constituents form a singular protein
- If Rac is activated, GDP is swapped to GTP and binds to a protein
- This brings GFP and RFP close together which allows FRET so red fluorescence given off
Describe the process by which listeria move within cells
They induce actin polymerisation and get pushed around.
- uses ActA, a transmembrane protein in listeria
- it induces confirmation change bringing ARp 1 and Arp2 together to create the active Arp2/3 that nucleates actin polymerisation.
- aided by anti-capping proteins recruited by ActA
ActA has a:
* Proline rich domain that binds Ena proteins
* WH2 domain that binds actin monomers
* Acidic domain that binds Arp2/3
Describe the role of Rho GTPases as co ordinators of the actin cytoskeleton
Rho, Rac and Cdc42 are Rho GTPases.
- They regulate the activity of many binding proteins
- Rac and Cdc42 dominate the leading edge whereas Rho dominates at the rear
- Rac activates actin assembly and inactivates filament disassembly
Cdc42
* Cdc42 activates filopodia formation via both formin and Arp2/3 dependent pathways
* binds to formins mDia and reveals nucleation site
* Inactivates ADF/cofilin: Activates PAK -> activates Lim kinase -> inactivates ADF/cofilin
Rho
* stress fiber formation
* Rho activates ROCK which activates the motor activity of myosin 2
How do invadopodia differ from filopodia or lamellipodia in cell migration.
Actin polymerization couples with the
extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the
extracellular matrix. Aiding invasion through tissues.
Describe Wiskott-aldrich syndrome
It is recessive x linked disorder that causes immune deficiency and eczema.
This is caused by a mutation in the WASP gene. WASP family of proteins cont WH2 domains and acidic domains similar to that of ActA and can activate the Arp2/3 complex. This causes actin polymerisation.
How does WAVE activate Arp2/3?
WAVE is a human gene that is normally bound to an inhibitory complex.
Binding of Rac GTP releases WAVE from the inhibitory complex, allowing it to activate Arp2/3.
It is able to activate Arp 2/3 because it contains a WH2 domain which binds actin monomers and a acidic domain which binds ARp2/3.
How does Rac promote lamellipodia formation?
It can inactivate ADF/Cofilin (which depolymerises).
1) Activates PAK
2) Which activates LIM kinase (phosphorylates)
3) Which inactivates ADF/Cofilin
It can activate WAVE (which activates Arp2/3)
1) Binding of Rac-GTP to the inhibitory complex releases WAVE allowing it to activate Arp2/3
Describe the ways in which Cdc42 leads to filopodia formation
Activates N-WASP (which activates Arp2/3)
- N-WASP is usually in an autoinhibited conformation
- Cdc42 binds, which changes it into its active conformation, revealing WH2 site and acidic domain
- allows activation of ARP2/3, which nucleates filopodia formation
Activates Formin mDia
- Cdc42 binds to autoinhibited conformation of formin mDia, turning to active confirmation
- reveals nucleation site
Inactivate ADF/Cofilin (same way as RAC does)
1) Activates PAK
2) Which activates LIM kinase (phosphorylates)
3) Which inactivates ADF/Cofilin
What is Rho?
A family of gtpases.
They are master regulators of the actin. Molecular on/off switches. Rho, Rac and Cdc42 are all members of of the Rho family.
How does Rho promote stress fibre formation?
Rho activates ROCK which activates the motor activity of myosin 2.
Activate forming mDia and inactivate adf/cofilin also helps.