Actin In The Cytoskeleton Flashcards
Which three protein networks are present in the cytoskeleton, and what are their monomers
1) Microfilaments made out of actin
2) Microtubules made from aB-tubulin dimers
3) Intermediate filaments made from various different types of monomer
What is the cytoskeleton, which fibres make up the skeleton, and what are its functions.
1) The network of filaments extending throughout the eukaryotic cell
2) actin/microfilaments, microtubules and intermediate filaments
3)
- actin is responsible for cell movement, cell shape, muscle contraction, and separation of daughter cells at mitosis.
-organelle movement and distribution, vesicle transport, secretion and uptake are carried out with the help of actin and microtubules
-chromosome separation at mitosis is carried out with microtubules
-intermediate filaments are best suited to help cells and tissues resist stress e.g. mechanical stress
What are the physical properties of myosin and actin
- Myosins are motor proteins
- Most actin filaments are dynamic, rapidly changing length or organisation.
Some actin filaments are stable e.g. in microvilli.
Binding sites on actin are used by actin binding proteins to regulate actin organisation
Name and describe the three main cellular actin organisations
- Lamellipodium - branched and crosslinked actin filaments on the edges of the cell
- Filopodium - bundles of parallel actin filaments in the peripheral cell protrusions
- Stress fibres - antiparallel, and sometimes cross linked filaments of actin arranged into a contractile structure
What is the name and structure of the actin monomer
- G-actin
- -G-actin is a polypeptide that folds into four subdomains that generate two lobes separated by an ATP-binding cleft
Describe the structure of an actin filament (F-actin)
- An actin filament is composed of two strands of polymerised G-actin monomers wrapped in a helix with a (+) end and (-) end
- The ATP-binding clefts are all oriented in the same direction
- The end of the filament with an exposed ATP-binding cleft is the minus end
Explain the stages of actin polymerisation from G-actin monomers to F-actin
- Nucleation is the first stage, involving the slow polymerisation of G-actin
- Elongation is the second stage, involving rapid polymerisation of G-actin on the (-) ends
- The rate of gaining or losing monomers depends on the concentration of available G-actin
- Steady state is the third stage, and occurs when G-actin concentration drops to the point where addition of monomers = loss of monomers
Explain what Cc stands for and what a lower Cc on the (+) end signifies
- Cc is the concentration of G-actin at which addition of monomers at one end is equal to loss of monomers at the same end. (No net gain or loss at the end)
- -Generally Cc is lower at the (+) end than at the (-) end
-Above Cc there is a net addition of monomers, and below there is a net loss
-Indicating more addition at the (+) end relative to the (-) end, signifying a faster binding constant
-Therefore at low concentrations of G-actin, the (+) end grows faster than (-) end
Explain the reason why the association and dissociation rates at opposite ends of the filament are different
- Actin is added to the (+) end with ATP attached
- However actin is an ATPase meaning it can hydrolyse ATP into ADP + Pi
- The hydrolysis occurs relatively slowly but results in actin subunits nearer the (-) end containing ADP
- ATP-ADP bound actin have different conformations, which affect binding kinetics
- ATP-bound actin binds stronger to other actin monomers than does ADP-bound actin
- The (+) end therefore has a higher addition/association rate than the (-) end
Explain the process of actin treadmilling
- Actin treadmilling occurs because the rate of ATP-actin addition to the (+) end is higher than at the (-) end and the rates of dissociation are similar for both ends
- This is due to the different Actin binding capabilities of ATP bound and unbound actin
- Treadmilling at steady state results in preferential actin elongation at the (+) end and ADP actin disassembly at the (-) end.
Explain the roles of the three actin binding proteins in treadmilling
- Cofilin binds to ADP-actin at the (-) end, destabilising it and facilitating (-) end actin disassembly
- Profilin removes ADP and facilitates ATP recharging of actin
- Thymosin B4 sequesters away ATP-actin, controlling the concentration of free ATP-actin
Explain the stages of cell movement driven by the actin cytoskeleton
Initially the cell is attached to the ECM by integrins, forming focal adhesions
1. The first stage, extension, occurs when a change in actin assembly leads to extension of the membrane
2. Adhesion is the second stage, where the extended membrane forms a new adhesion
3. Translocation is the third stage, where the cells internal contents are shifted forwards towards the new adhesion via the interaction of myosin and actin
4. De-adhesion and endocytic recycling of integrins and membrane are the fourth stage. The focal adhesion is broken and the aforementioned molecules are recycled via endocytosis.
Explain the roles of cofilin and profilin in cell movement
- Actin assembly occurring at the leading edge pushes the membrane forward, stimulating cell extension
- This assembly is triggered by actin elongation at the (+) end
- Whilst cofilin destabilises and helps remove ADP-actin at the (-) end
- Allowing profilin to recharge ADP-actin to ATP-actin ready to be added to the elongating (+) end
Explain how branched actin filaments are formed at the leading edge
- Branches are nucleated due to the nucleation promoting factor (NPF) which facilitates Arp2/3 complex integration into F-actin
- Normal F-actin is synthesised but the Arp2/3 complex provides another site for actin elongation at 70 degrees to the main filament
Explain how focal adhesions are involved in cell attachment to the ECM
- Actin contractile bundles are connected to integrins, which connect the contractile bundles to the ECM
- When the cell is extending, contractile bundles put tension on focal adhesions
- Thus keep the cell attached to the ECM
