Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin

A novel microfluidic approach allows the analysis of the dynamics of individual actin filaments, revealing both their local ADP/ADP-Pi-actin composition and that Pi release is a random mechanism. The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi) that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds) and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments. Author Summary: Actin proteins assemble into microfilaments that control cell shape and movement by polymerizing or depolymerizing. These actin monomers can bind ATP or ADP molecules. The incorporation of an ATP-actin monomer into a growing filament results in rapid cleavage of ATP into ADP and inorganic phosphate (Pi), followed by a slower release of Pi. As a consequence, actin filaments are composed mainly of ADP- and ADP-Pi-actin subunits, which have different depolymerization kinetics and mechanical properties, and can be targeted specifically by regulatory proteins that affect filament function. Hence, the understanding of many cellular processes requires a knowledge of the ADP/ADP-Pi composition of actin filaments at a molecular scale. This has so far remained elusive because traditional studies rely on measuring an average over many filaments in solution. To address this issue, we developed a microfluidics setup to monitor individual filaments with light microscopy while rapidly changing their chemical environment. We find that depolymerization accelerates progressively and corresponds to an exponential ADP-Pi-actin profile in the filament, meaning that each subunit releases its Pi with the same rate. Our method also provides novel insight into the function of profilin, a protein important for regulation of actin dynamics in cells, thus demonstrating the method's potential in the functional analysis of actin regulators.