Fig. 3: Spa2(1-535) specifically nucleates and elongates ADP-actin.
From: Spa2 remodels ADP-actin via molecular condensation under glucose starvation
a The Fluorescence anisotropy profile of the indicated recombinant Spa2-N-terminal truncation variants (60 nM, Alexa 488-labeled) were titrated with increasing concentrations of ADP-actin monomer. Measurements from three biological replicates were plotted and fit with the Hill equation. b Representative time-lapse TIRFM images of actin filament severing by incubating 500 nM Spa2-truncation variants and 50 nM Cof1 with 0.1 μM F-actin derived from 0.5 μM ATP-G-actin with 10% Oregon Green 488 (OG 488) and 0.5% biotin labeling. c Quantification of the average actin filament signal intensity from multiple time points during 40 ~ 200 s (from top to bottom n = 34, 36, 35, 35 ROIs of 22 × 22 μm2). d Representative time-lapse TIRFM images of actin polymerization at the indicated time point using 3 μM ADP-G-actin (10% OG 488- and 0.5% biotin-labeled), with or without the indicated 10 nM Spa2 variants. e Comparison of nucleation efficiency at 5 min by measuring the seed number (from left to right: n = 22, 28, 29, and 29 ROIs at 64×64 μm2). f Quantification of the actin elongation rates and kymograph in d (left to right, n = 34, 36, 39, and 52 actin filaments). Whiskers represent min to max in e and f. Bars for b and d, 5 μm; Bar for f, 2 μm. Error bars, mean ± S.D. P-values were calculated using GraphPad Prism 6. Significance between two sets of data were determined by the one-way analysis of variance (****p < 0.0001, ***p <0.001, **p < 0.01 and specified, ns = not significant). Source data is provided as Source Data file.
