Cytoskeleton (J.B) Flashcards
What are the requirements for the cytoskeleton?
- Strong
- Very flexible/dynamic
What are the three core cytoskeletal filament proteins?
Why is a subunit based system?
- Allow for quick decisions to take place –> disassembly here and assembly on the other side.
Outline the structure of intermediate filaments.
Key feature –> fibrous/rope like property –> due to rope like units that wrap around each other.
These subunits are not polar –> no plus or minus end.
- They have a degree of dynamism but not as much as the others.
Where would you normally find a lot of intermediate filaments?
- Tissue that needs a lot of mechanical strength –> keratin filaments in the epithelial cells (skin).
- Nucleus –> find them in lamins of the nuclear envelope.
- Axonal –> neuronal strength
Outline the structure of actin filaments.
- Small monomer
- Four lobed square
- Plus end and minus end
- ATP binding site in the middle –> Binds to ATP hydrolyzes to ADP.
Outline the structure of tubulin microtubules.
- Dimer made of alpha and beta subunits
- Binds to GTP –> GTP hydrolysed to GDP.
- Forms more complicated microtubules –> structure with lumen in the middle –> tubule is thicker than actin filaments.
Where do actin filaments and alpha/beta-tubulin obtain energy for their dynamic activity?
- GTP is in the beta subunit –> alpha subunit is locked
Do they filaments and microtubules have polarity?
Yes, they have polarity –> plus (fast-growing) and a minus (slow growing) ends.
Outline the principle of polymerisation.
T form –> ATP/GTP bound –> charged state –> tendency for growth/polymerisation.
Hydrolysis from T to D
D form –> ADP/GDP bound –> tendency to shrink as it is less stable.
Allows for growth and disassembly.
Technically –> both ends can shrink or grow BUT the plus end has a greater affinity for growth and the minus end has a greater affinity for loss.
I.e –> if the filament needs to shrink –> minus end shrinks faster than the plus end.
What are the different phases of a dynamic filament?
- Nucleation phase (lag phase)
- Elongation (growth phase)
- Steady state (equilibrium phase)
What happens in the nucleation phase?
- Subunits Pool –> waiting for enough subunits to form a nucleus (platform for growth)
What happens in the elongation phase?
Growth –> subunits added
What happens in the steady-state phase?
The rate of gain equal rate of loss –> balance out –> occurs at the critical concentration.
Note!
There are two critical concentrations –> one for the plus end and the other for the minus end.
Minus end has a critical concentration where growth equals loss.
Plus end has a critical concentration where growth equals loss.
Explain the concept of treadmilling.
Note –> Actin filaments follow this process perfectly but microtubules are slightly different.
Remember both the plus and the minus ends have different critical concentrations.
Plus end has a much lower critical concentration (C.C) (i.e. 0.04 micromolar) –> has an affinity for growth –> when you go above the C.C –> plus end grows.
Minus end has a much higher C.C (i.e 0.18 micromolar) –> has an affinity for loss –> when you go below the C.C –> minus end shrinks.
So between the two C.C (0.04 and 0.18) –> the plus end will grow whereas the minus end will shrink –> this creates a treadmilling effect.
Note the growth/loss also depends on the state of the nucleotide (ATP/ADP or GTP/GDP).
What is dynamic instability in microtubules?
Remember hydrolysis if GTP to GDP (or ATP) is random —> stochastic process –> filaments age –> they more likely to have the hydrolysed form.
GTP cap present on growing end –> growth
However, when GDP is present at the tip –> increased instability –> rapid loss –> known as dynamic instability.
Why is it important for cells to be dynamic?
What are other names for the plus and minus end of the actin filament?
Plus –> barbed end
Minus –> pointed
When having filament what are the relative differences between ATP acting gain and loss on the plus and minus end.
Plus end –> Rate of addition –> 12 per second –> rate of loss 1.4 per second –> this means that the rate of addition is a lot higher than loss –> implying the plus end grows when in a pool of ATP Actin.
Minus end –> Rate of addition –> 1.3 per second –> rate of loss 0.8 per second –> this means that the rate of addition is slightly higher than loss –> implying the minus end grows when in a pool of ATP Actin.
This is counterintuitive as this would mean that both ends always grow –> This is because we haven’t taken into the consideration ATP hydrolysis to ADP.
When having actin filament what are the relative differences between ADP acting gain and loss on the plus and minus end.
Note –> the exact number aren’t important (numbers change) –> relative difference is.
Plus end –> rate of addition of ADP is 4 per second –> rate of loss of ADP is 8 per second –> this means that an ADP monomer is more likely to be lost from the end (2 times more likely).
Minus end – > rate of addition of ADP is 0.1 per second – > rate of loss of ADP is 0.3 per second –> this means that an ADP monomer is much more likely to be lost from the minus end than gained (3 times more likely)
What is the effect of ATP being preferentially gained and ADP being preferentially lost have on the actin filament?
- ATP is added to both ends
- However, over time the filament will hydrolyse ATP to ADP (random process)
- Since ATP is gained at the plus end continuously at a fast rate –> there will be an accumulation of ADP monomers present on the minus end –> ADP molecules are preferentially lost.
Results in this treadmilling effect –> preferential gain at plus end and preferential loss at the minus end.
Summarise the treadmilling effect.
Why is the critical concentration for the addition of ATP actin different between the plus and minus end?
This is due to the different rates of addition.
When the rate of addition is naturally higher –> need a lower critical concentration.
When the rate of addition is naturally lower –> need a lower critical concentration.
What is a problem that is caused by the lag phase?
Outline the role of nucleators.
- Spontaneous nucleation is slow –> so cells have to tell filaments where to form –> the factors that facilitate nucleation are called nucleators.
- Nucleators facilitate the localization and timing of filament formation
- Actin filaments and microtubules have their own unique classes of nucleators.
What are the two main nucleators for actin?
- ARP2/3 –> branched filaments
- Formins –> elongated filaments.
Structure and function of ARP2/3?
ARP –> actin-related protein –> similar to actin –> forms platform for actin growth.
ARP2/3 binds on the minus end of the filament and allow the plus end to grow.
There are many regulators for ARP2/3 –> ‘the regulators of the regulators’ –> WASP and Wave/Scar –> switch the system on and off.
Outline the steps by which ARP2/3 acts as a nucleator.
- WSAP or Scar facilitate the coming together of an existing strand and the ARP2/3 complex
- ARP2/3 holds the minus end of the filament.
- Allows the plus end to grow –> at 70 from the precursor filament.
Outline the structure of the formin nucleator.
- Forms dimer –> FH1 and FH2
- Key domain —> FH2 –> binds to 2/3 actin monomers at the plus end and facilitates the addition of new monomers by rocking backwards and forwards.
- FH1 –> binds to profilin –> likes to bind to ATP bound actin –> collects actin molecules.
- These filaments are not used as much for moving the cell (pushing the membrane) but act more like ‘highways’ which allow things to move.
Diagram of Formins in action.
How does nucleation occur on microtubules?
MTOC –> microtubules nucleate of gamma-tubulin –> like microtubules –> gamma-tubulin form complex where microtubules come from.
Structure of gamma-tubulin?
What are centrosomes and centrioles?
Structures of mammalian centrosome?
What is the concentration of actin in a real cell? Implications of concentration?
The function of monomer binding proteins?
Nucleators –> function to increase the number of filaments.
Monomer binding proteins –> function to prevent the formation of filaments –> especially important as due to the high concentration –> 99% of actin should be in the filament form –> but in reality, it isn’t.
The function of capping proteins?
Cap filaments and stop them from growing.
The function of filament bundling proteins?
- Sticking filaments together to form thick cables.
Difference between Actinin and fimbrin cross-linking?
What does filamin do to actin?
Benefits of having different types of cross-linkers?
Localise to a different region of the cell –> producing different properties.
The function of disassembly factors?
- Multifunctional –> switch on and off by phosphorylation (kinase and phosphatase).
- Cofilin can bind to filament –> twist –> twisting breaks the actin filament.
Role of MAPs in microtubule regulation?
Microtubule stabilizing MAPs
- Tau and related MAP proteins have multiple tubulin binding sites –> they bind along the protofilaments and stabilize the polymer –> prevent it from breaking down.
Role of katanin in microtubule regulation?
Role of TIPs in microtubule regulation?
+TIPs –> plus-end tracking proteins
Like to move to particular ends of the microtubule.
What are the three types of motors that exist?
Change in shape –> allow for the creation of force
Different motors –> different shape changes.
What are the principles behind a molecular motor?
ATP hydrolysis—> conformational change –> force
- Motor is anchored –> conformational change moves the filament (Power stroke in muscle cells)
- Track is anchored –> motor moves along the filament like a zip line
Structure of myosin motors?
Head provides conformational change –> which provides force.
Tail stabilise and anchor structure
Structure of myosin heads in muscle?
Myofibril –> made of bundles of myosin
Sarcomere –> single contractile unit in muscle
Myosin filament with the heads
Actin filament with binding sites for the myosin heads
Myosin heads can bind to actin –> undergo conformational change –> results in power stroke.
All of which occurs in response to Ca2+ by sarcoplasmic reticulum.
How many different myosin motors have been discovered?
14 different myosin motors have been discovered.
Myosin 2 –> muscle
The function of myosin 5?
Myosin 5 –> The great walker
Double head motor –> two myosin joined in a V shape
What are the different positions that myosin motor can find itself in? (Terminology –> cocked, power stroke, Rigor state)
Explain the step by step process by which myosin motors produce force.
Example –> contracting muscle.
Where do all myosin motors move to on an actin filament? What is the exception?
Exception –> Myosin 6
What is the role of myosin in cell division?
The contractile ring used in cytokinesis to split one cell into two daughter cells is composed of actin filaments, myosin II and several associated proteins.
Myosin heads pull with actin –> contracts the contractile ring –> divides the two cells
Structure of kinesins?
Cargo binds to the C terminal domains whereas the heads interact with the microtubules.
Outline the steps in the Kinesin ‘Hand over hand’ cycle.
Can kinesin motors travel in both directions along the microtubule?
Some can travel to the plus end while others travel to the minus end.
Structure of dyneins?
- ATPase ring –> barrel-shaped AAA ATPase –> 6 domains –> One binds an hydrolyzes ATP –> 3 bind but do not hydrolyze ATP –> last 2 do not bind to ATP.
- Stalk –> coiled-coil stalk protrudes from the catalytic domain and binds a microtubule.
- Tail –> binds to the cargo –> dynactin complex interacts with cargo.
ALWAYS move to Minus end.
Briefly outline the Dynein rachet like stroke
ATP hydrolysis –> cocking movement –> swinging tail –> converted to motion force.
How do kinesins and dyneins work together?
Kinesins move vesicle outs –> plus end-directed motor
Dyneins move vesicles inside the cell –> Minus end-directed motor
Cargo –> needs a signal which interacts with a specific motor –> helps direct the traffic of vesicles.
How do troponin and tropomyosin help regulate muscle contraction?
When troponin binds to Ca2+ it undergoes a conformational change which moves the tropomyosin which in turn exposes the binding sites.
What is the role of titin in muscle cells?
What is cytoplasmic streaming?
What is lamellipodium based movement?
- Extending forward using actin filaments
- Sticking down to a surface –> adhesive proteins
- De-adheres at the back of the cell
- Dissaembly of actin at the back
- Cell moves forward.
What are comet tails|? What are they used for?
Pathogens mimick the WASP, Wave or SCAR molecule needed for actin regulation –> pathogens hijack the filaments –> use it to travel around the cell.
Comet tail –> pathogens using the filaments to ‘rocket’ themselves around the cell –> useful as they can use it to move from cell to cell as shown in the diagram.
What are kinetochores?
