3.5. Pyrenyl-Actin Polymerization Assays

Actin polymerization assays were carried out as previously described [5,6]. Briefly, pyrene-labeled, gel-filtered Ca²⁺-actin (5% labeled; 2.5 µM final concentration) was pre-incubated with mDia1 or EnaΔL in the presence or absence of PFN1 and varying concentrations of actin oligomers or actin rings (0–200 nM) in reaction buffer (10 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 0.2 mM ATP, 0.5 mM DTT). Ca²⁺-ATP actin was then converted to Mg²⁺-ATP actin by the addition of 0.066 volumes of switch buffer (150 mM MOPS, pH 7.0, 3 mM ATP, 7.5 mM DTT, 4.5 mM EGTA, 1.5 uM MgCl₂) and incubation for 2 min. Polymerization was initiated by the addition of 0.33 volumes of initiation buffer (30 mM MOPS, pH 7.0, 0.6 mM ATP, 1.5 mM DTT, 3 mM MgCl₂, 150 mM KCl) and monitored at λ_ex = 365 nm and λ_em = 407 nm on the Infinite M1000 Pro plate reader (Tecan US, Inc., Morrisville, NC, USA).

Inhibition of polymerization by linear or circular actin oligomers was assessed by calculating the tangent slope of each pyrene fluorescence trace at 50% (40–60% interval) of maximum polymerization and fitting the obtained data to a binding isotherm [54].