Carboxylated polybeads

Time-lapse TIRF microscopy movies were obtained using a iXon EMCCD camera (Andor Technology, Belfast, UK) fitted to an Olympus IX-71 microscope with through-the-objective TIRF illumination as described (Zimmermann et al., 2016 ). Mg-ATP-actin (10–20% Alexa-488 labeled) was mixed with a polymerization mix (10 mM imidazole [pH 7.0], 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 50 mM DTT, 0.2 mM ATP, 50 μM CaCl₂, 15 mM glucose, 20 μg/ml catalase, 100 μg/ml glucose oxidase, and 0.5% [400 cP] methylcellulose) to induce F-actin assembly (Winkelman et al., 2014 (link)). Where stated, formin or profilin was added to the polymerization mix prior to mixing with actin and initiating F-actin polymerization. The mixture was then added to a flow chamber and imaged at 10-s intervals at room temperature. For bead assays, Wsp1 and formin beads were prepared as previously described (Loisel et al., 1999 ). Carboxylated Polybeads (Polysciences, Warrington, PA) were coated with Wsp1 or unlabeled SNAP-FOR1(3P,FH2) and flowed into the TIRF chamber prior to initiating the reaction.