Actin Polymerization Overshoots and Hydrolysis as Assayed by Pyrene Fluorescence

¹ Washington University in Saint Louis

^* To whom correspondence should be addressed. E-mail: aec{at}physics.wustl.edu.

Submitted on October 3, 2007
Revised on November 25, 2007
Accepted on 17 March 2008

	Abstract

We investigate via stochastic simulation the overshoots observed in the uorescence intensity of pyrene-labeled actin during rapid polymerization. We show that previous assumptions about pyrene intensity that ignore the intensity dierences between subunits in dierent ATP hydrolysis states are not consistent with experimental data. This strong sensitivity of intensity to hydrolysis state implies that a measured pyrene intensity curve does not immediately reveal the true polymerization kinetics. We show that there is an optimal range of hydrolysis and phosphate release rate combinations simultaneously consistent with measured polymerization data from previously published severing and Arp2/3 complex-induced branching experiments. Within this range, we nd that the pyrene intensity curves are described very accurately by the following average relative intensity coecients: 0.37 for F-ATP actin; 0.55 for F-ADP+P_i actin; and 0.75 for F-ADP actin. Finally, we present an analytic formula, which properly accounts for the sensitivity of the pyrene assay to hydrolysis state, for estimation of the concentration of free barbed ends from pyrene intensity curves.

Key Words: Arp2/3 complex, branching, light scattering, severing, stochastic simulation