The Tpm3.1 dimer brings TR100 into the filament leading to enhanced actin depolymerisation.
(a,b,d,e) Representative depolymerisation time course of 3 μM actin filaments (35% pyrene labelled) diluted 12-fold into F-actin buffer (100 mM NaCl, 10 mM Tris-HCl pH 7.0, 2 mM MgCl2, 1 mM EGTA, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT, 0.01% (v/v) NaN3) in the presence or absence of saturating amounts (5 μM) of Tpm3.1. Final concentrations of F-actin and Tpm3.1 are 0.25 μM and 0.42 μM, respectively. Tpm3.1 was pre-incubated with 1% (v/v) DMSO (a) or 50 μM TR100 (b) prior to mixing with F-actin (pre-saturation) compared to vehicle/drug added to Tpm3.1-coated F-actin (post-saturation; d and e, respectively). Depolymerisation curves are normalised to the initial fluorescence value. (c and f) Initial rates of depolymerisation, calculated from the first 600 s of depolymerisation, for F-actin alone or Tpm3.1/F-actin in the presence of vehicle or TR100. Rate data represents mean ± SEM, averaged from n = 4–6 replicates.