Actin is a cytoskeletal protein that is conserved in all eukaryotes. Its polymerization reaction enables cells to exert forces that are useful for many cellular processes. The dynamics of actin polymerization in cells is maintained by the supply of energy, which is generated by the hydrolysis of actin-bound ATP molecules to ADP. In this work, we focused on the mechanism of actin copolymerization in the presence of ATP- and ADP-bound monomers. While it is known that ADP-bound monomeric actin can polymerize in vitro, the copolymerization reaction between ATP-actin and ADP-actin has never been studied in detail.
To address this problem, we used a fluorescent nucleotide, N6-(6-amino)hexyl-ATP-ATTO488, which binds to actin without affecting its polymerization kinetics. We compared the binding properties of this nucleotide and its ADP equivalent and found that the presence of the fluorophore did not affect the relative ability of these two nucleotides to bind actin. We then used these two fluorescent nucleotides in copolymerization experiments to quantify their relative incorporation into single filaments or organized actin structures.
Our results show that ATP-actin and ADP-actin copolymerize, and that ADP-bound actin monomers integrate into actin filaments in non-negligible amounts. For a large number of conditions studied, our results show that the probability of integration of an ADP-bound monomer is only about 3 times lower than that of an ATP-bound monomer. This result was surprising given that ATP has a 4-fold higher affinity for monomers than ADP, and that ATP-actin is known to polymerize much faster than ADP-actin.
To understand this result, we developed a stochastic model that reproduces this copolymerization reaction in silico. Our results show that the copolymerization reaction is more complex than expected. In particular, our results show that the state of the nucleotide bound to the terminal subunits of the filament is critical in controlling this reaction, allowing significant incorporation of ADP-bound monomers into actin filaments.
These results open new perspectives for understanding the role of actin-bound nucleotide during the polymerization reaction in the cell. While the scientific community has long believed that actin polymerization in the cell occurs exclusively from ATP-bound monomers, our results suggest that partial polymerization of ADP-bound monomers would be credible.