Abstract
Author SummaryActin 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.
Highlights
Actin-based motile processes are driven by the polarized assembly of actin filaments [1,2,3]
The understanding of many cellular processes requires a knowledge of the ADP/ADP-inorganic Phosphate (Pi) composition of actin filaments at a molecular scale
We developed a microfluidics setup to monitor individual filaments with light microscopy while rapidly changing their chemical environment
Summary
Actin-based motile processes are driven by the polarized assembly of actin filaments [1,2,3]. For Pi release, as for ATP c-phosphate cleavage, two main mechanisms have been considered since long ago [5] and continue to receive attention, notably from theoretical studies [10,11,12,13]: in the random model, each ADPPi-actin subunit releases its Pi with equal probability, while the vectorial model assumes that Pi can only be released from an ADP-Pi-subunit adjacent to an ADP-subunit (Figure 1A) In the latter model, an ADP/ADP-Pi boundary propagates toward the growing barbed end that displays a strict ADP-Pi cap, while the cap has a mixed composition in the random model
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