You are studying actin filament dynamics in a test tube that contains actin mono

Question

You are studying actin filament dynamics in a test tube that contains actin monomers and ATP. You observe that actin filaments are treadmilling when the monomer concentration is between 7 and 12 CU (concentration units). At 12 CU, one end is growing at a rate of 6 RU (rate units). At 7 CU, one end is shrinking at a rate of 3 RU.

A) What is the Cc(D) value?

B) What is the Cc(T) value?

C) At which monomer concentration is steady-state treadmilling achieved?

D) Instead of ATP, you add ATPS, a non-hydrolyzable form of ATP, into your reaction. For some reason, you can no longer observe treadmilling at any monomer concentration tested. Why?

E) In your reaction containing actin monomers and ATP, you add a 100-fold molar excess of profiling (relative to actin monomers). The monomer concentration is set at 15 CU. Explain what is happening at the two ends of the filament

F) After you make your observations in the experiment described in (E), you dilute the solution such that the monomer concentration is now 10 CU. Explain what is happening at the two ends of the filament after the dilution.

G) Instead of profiling, you add a 100-fold molar excess of thymosin (relative to actin monomers) in your reaction containing actin monomers and ATP. The monomer concentration is set at 20 CU. What is happening in your solution?

Explanation / Answer

The addition of ATP-G-actin to the ends of the previously formed actin filament can occur at both the ends at different rates. The addition of this molecule to (-) end is lesser than that to the (+) end. The two ends of the actin filaments have different critical concentrations for the growth of actin filaments.

Cc is the concentration of free ATP-G-actin where the rate of addition of the elements is balanced by the growth of the filament at the other end and by the rate of loss of monomers. Actin filaments grow above the critical concentration, Cc. Below this concentration, the shrinking of filament occurs or loss of actin subunits takes place.

A)

Cc(D) value is the concentration of the monomers where the monomers are not being used by the F-actin to polymerize until the critical concentration is reached. Here, Cc(D) value is 7CU.

B)

Cc(T) value is the concentration where the monomers are used by the G-actin-ATP to polymerize the actin filament until the critical concentration is maintained. Here, Cc(T) value is 12CU.

C)

Steady state treadmillling of actin polymerization is achieved when the concentration of monomers have reached the critical concentration, Cc. At Cc, the rate of addition of the monomers at one end of the filament will balance the removal of them at the other end of the filament. So, the concentration has reached a steady state and the dynamics of the actin polymerization has reached equilibrium. Here, it could be the average of 7 and 12, which is calculated as 9.5CU.

D)

The monomer G-actin-ATP is hydrolyzed to release ADP and Pi to form F-actin-ADP. This process is polymerization. In the case of depolymerization, F-actin-ADP combines with Pi to form G-actin-ATP monomers. So, if the reaction is supplied with ATPS, the actin polymerization does not occur. It stops.

E)

Profilin is the protein that helps in the binding of actin monomers to the filament. If 100-fold higher concentration of profilin is added to the reaction compared to the actin monomer concentration, the enzyme concentration increases to enhance the rate of reaction. The reaction occurs faster than before and reaches the critical concentration of 9.5CU from 15CU very soon. The rate of polymerization was initially 6RU at the +ve end as per the question. But, now the rate of polymerization would increase very much to bring back the concentration of the monomers quickly to the critical concentration. As the profiling only is increased, the depolymerization at the other end will not occur faster. So, the length of the filament keeps increasing will less removal at the other end (-ve end).

F)

If the monomer concentration is made 10CU by dilution, the profilin concentration remains the same (100-fold) and the rate of polymerization is higher as in the previous case. But, the time taken to reach the critical concentration will be further reduced. The rate of depolymerization is lesser compared to that of polymerization as the concentration of profilin is more. However, the steady-state dynamics is achieved very quickly than in the previous case.

G)

If the concentration of thymosin is increased by 100-fold in the current solution, then the rate of depolymerization increases leading to further increase in actin monomer concentration. The existing 20CU of the monomer concentration will be quickly updated to more than 20CU due to depolymerization and hence the polymerization also relatively increases. So, the process of depolymerization stimulates the occurrence of polymerization of actin filament and it goes on until the concentration of the monomers reaches the critical value of 9.5CU. This state is called as steady-state of actin dynamics. In the previous case, polymerization of actin filament is enhanced by the profilin while here, the enhanced polymerization is due to the increase in the concentration of actin monomers far above the critical value due to enhanced depolymerization.

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