The Cytoskeleton (Actin and Microtubules) Flashcards
(21 cards)
What is the monomer that makes up microtubules?
Tubulin
How was F actin first purified?
First purified from rabbit skeletal muscle. And purified using its ability to polymerise and then depolymerise depending on the ionic concentrations of the solution.
How does ATP bind to actin?
ATP binds to the ATP binding cleft at 4 sites.
What is the physical structure of G actin?
Two lobes which have subdomains Ia, Ib, IIa, IIb and an ATP binding cleft in the middle.
Explain how affinity chromatography works.
Produce a column of immobilised DNAseI. DNAseI has a strong affinity to G actin so therefore you can purify G actin from cells due to their affinity to the DNAse depending on how much of it there is in the cell.
What is the structure of an actin fibre?
It is two strands of F actin wrapped around each other in a right handed helix.
In terms of an actin filament, what is L? How does it vary and why?
L is the length of one helix repeat. It is variable from 36-40nm because the helix allows torsional flexibility. L becomes 0 when the strands are parallel and there is no helix. The tighter the coil, the shorter L becomes and the looser the coil, the longer L is.
How many contacts with adjacent monomers does one actin monomer form in the helical F actin structure?
4 adjacent contacts. There are 4 binding sites for actin on each monomer. There are also 4 ATP binding clefts where ATP makes contact with the monomer.
What is the range of diameter of an active actin filament and what is this dependent on?
It ranges between 8.5-10nm and is dependent on L. The shorter L is, the tighter the coil and therefore the smaller the diameter.
What is myosin in relation to actin?
It is the molecular motor for actin.
What can you do to a proteolytic cleavage map of an actin monomer show about the domains of actin?
It can show which domains of actin bind to which proteins.
What is cofilin?
It is an actin regulatory protein which binds to both the C and N terminals. Cofilin disassembles actin fibres.
How do salts such as K+, Cl- and Mg2+ alter the ability of actin to polymerise? (Hint: two ways)
- Salt changes the G actin conformational shape to make it ore likely to polymerise by binding to other G actin monomers.
- Also, Mg can bind and displace the Calcium ions which disrupts the polymerisation process.
What must be present in solution in order for actin to polymerise?
- Salt which changes the conformation of actin, allowing it to bind to other monomers.
- An adenine nucleotide (ADP or ATP) to bind in cleft
What are three ways in which the polymerisation process of actin can be followed.
- Viscometry- solutions with more F actin will be more viscous.
- Sedimentation- more F actin will cause more pellet to form as the strands get intertwined. Use centrifuge and pellet forms if there is lots of F actin but not if there is only G actin.
- Prenyl binds to actin and absorbs different wavelengths of light depending on whether the actin is G or F (measure changes in absorption spectra).
What are the three phases of actin polymerisation?
Nucleation, elongation and steady state.
Steady state is an equilibrium.
Is ATP hydrolysis essential for polymerisation to occur?
No, if a non hydrolysable analogue of ATP is bound to cleft, polymerisation can still occur.
What are nucleides?
Oligomers. aggregations of actin monomers which once formed, can allow elongation at both ends to occur.
Why does the positive end of actin polymerise faster than the negative end?
THis is because addition of monomers at the minus end requires a conformational change
Describe the actin decoration experiment and what it shows.
Adding the myosin head to the actin filaments, down a microscope the actin filament looks like a series of arrow heads which demonstrates the polarity of the filament- one end is called the barbed end and the other the pointed end.
At which end (pointed or barbed) is the ATP binding cleft on actin?
The pointed end
