Journal: Nature Communications
Article Title: Myosin-based nucleation of actin filaments contributes to stereocilia development critical for hearing
doi: 10.1038/s41467-025-55898-8
Figure Lengend Snippet: A TIRFM visualization of actin filaments polymerizing on PEG-biotin-NeutrAvidin functionalized cover glass. Polymerization of 1 µM G-actin (20% TMR + 10% biotin labelled) was induced by KMEI (50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 10 mM imidazole, pH 7.0) at t = 0 s, in the presence of 25 µM ATP. Representative time-lapses shown for: 1 µM G-actin (top), 1 µM G-actin + 1 µM M15(wt) (middle), and 1 µM G-actin + 1 µM mutant M15(jd) (bottom). B Quantification of actin filament density shows delayed nucleation activity of MYO15A in the presence of ATP. n = 3 independent determinations. C Kymographs of actin filament elongation. D Barbed-end elongation rates for G-actin + KMEI (red, n = 69 filaments), G-actin + M15(wt) (blue, n = 94), G-actin + M15(jd) (green, n = 80). E Elongation rate data (from D ) re-binned before nucleation, G-actin alone ( n = 69 filaments), G-actin + M15(wt) ( n = 54), G-actin + M15(jd) ( n = 40). F Elongation rate data (from D ) re-binned after nucleation, G-actin alone ( n = 69 filaments), G-actin + M15(wt) ( n = 40), G-actin + M15(jd) ( n = 40). The G-actin + KMEI control data set (from D ) is reproduced identically as a comparator in ( E , F ). G Time-lapse of actin filament polymerization induced by KMEI at t = 0 s, with no ATP in solution. G-actin (ATP) monomers were prepared by desalting into ATP-free G-buffer. H Actin filament density shows nucleation activity of MYO15A is accelerated in the absence of ATP. G-actin + KMEI ( n = 4 determinations), G-actin + M15(wt) ( n = 5), G-actin + M15(jd) ( n = 5). I Barbed-end filament rates in the absence of free ATP. Reaction deadtimes were typically 50 s and included in quantification. TIRFM images are shown as inverted grayscale. G-actin + KMEI ( n = 40 filaments), G-actin + M15(wt) ( n = 40), G-actin + M15(jd) ( n = 47). All data are plotted as mean ± SD. Statistics were computed using two-way ANOVA with Dunnett’s multiple comparisons test ( B , H ), and one-way ANOVA (Kruskal–Wallis) with Dunn’s multiple comparisons test ( D , E , F , I ). Statistical significance is denoted by ****, P < 0.0001, ***, P < 0.001, **, P < 0.01. Scale bars are 10 µm ( A , G ). Data are from 3 to 5 experimental determinations ( A – F ), and 4–5 experimental determinations ( G – I ), using 2 independent protein preparations.
Article Snippet: Filament elongation rates were calculated from time-lapse TIRFM imaging sequences using kymographs generated in NIS-Elements Software (version 5.2, Nikon).
Techniques: Mutagenesis, Activity Assay, Control
