Journal: Communications Biology
Article Title: Crosslinked F-actin networks regulate load-dependent energy conversion
doi: 10.1038/s42003-026-09843-0
Figure Lengend Snippet: a Schematic of the fascin crosslinked actin network. b Time-lapse images of actomyosin contraction at 80 nM myosin and 6 µM actin with 0.6 µM fimbrin (left) or 6 µM fimbrin (right), with NADH fluorescence (bottom). NADH fluorescence over time normalized to the initial time point ( c ), and the boxplots showing ATP consumption rate ( d ) ( n = 3 droplets, N = 7 independent experiments in NADH only; n = 6, N = 3 in [Fimbrin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1). ( e ) Schematic of the filamin crosslinked actin network. ( f ) Time-lapse images of actomyosin contraction at 80 nM myosin and 6 µM actin with 0.6 µM fascin (left) or 6 µM fascin (right), with NADH fluorescence (bottom). NADH fluorescence over time normalized to the initial time point ( g ), and the boxplots showing ATP consumption rate ( h ) ( n = 3 droplets, N = 7 independent experiments in NADH only; n = 6, N = 3 in [Filamin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). i Actomyosin network contraction (left) at 80 nM myosin, [Fimbrin]/[Actin] = 1; black arrows indicate total displacement over 10 min, with local apparent strain fields shown on the right. Instantaneous power over time ( j ), and mean inferred mechanical power averaged over 10 min ( k ) ( n = 14, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1). l Actomyosin network contraction (left) at 80 nM myosin, [Filamin]/[Actin] = 0.1; black arrows indicate total displacement over 10 min, with local apparent strain fields shown on the right. Instantaneous power over time ( m ), and mean inferred mechanical power averaged over 10 min ( n ) ( n = 14, N = 3 in 0; n = 5, N = 3 in 0.01; n = 9, N = 6 in 0.04; n = 10, N = 6 in 0.1; n = 13, N = 5 in 1). o ATP consumption rate dependence on the concentration of various crosslinkers ( n = 6, N = 3 in [Fimbrin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1; n = 6, N = 3 in [Filamin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). p Mean inferred mechanical power vs. energy consumption rate on various crosslinkers (fimbrin: n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1; filamin: n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 9, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant. Scale bars, 10 μm.
Article Snippet: In the experiments assembling crosslinked actin network, fascin (CS-FSC01; Cytoskeleton Inc.), Fimbrin (Methods), α-actinin (AT01; Cytoskeleton Inc.), and Filamin (8312-01; Hypermol) were used at varied concentrations.
Techniques: Fluorescence, Concentration Assay
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![a Schematic of the <t>fascin</t> <t>crosslinked</t> actin network under varying fascin concentration. b Time-lapse images of actomyosin contraction at 80 nM myosin and 6 µM actin (left) with 0.6 µM fascin (center) or 2.4 µM fascin (right), with NADH fluorescence (bottom). c NADH fluorescence over time, normalized to the initial time point ( n = 7 droplets, N = 3 independent experiments in NADH only; n = 7, N = 4 in [Fascin]/[Actin] = 0; n = 11, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 8, N = 4 in 0.4; n = 10, N = 5 in 1). d Boxplot showing ATP consumption rate from ( n = 7, N = 4 in 0; n = 11, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 8, N = 4 in 0.4; n = 10, N = 5 in 1). e Actomyosin network contraction (left) at 80 nM myosin, [Fascin]/[Actin] = 0.1; black arrows indicate total displacement over 10 min, with local apparent strain fields shown on the right. f Mean compressive apparent strain of the actin network over time ( n = 7, N = 4 in 0; n = 9, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 7, N = 4 in 0.4; n = 10, N = 5 in 1). g Instantaneous power performed by myosin on the actin network over time ( n = 7, N = 4 in 0; n = 9, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 7, N = 4 in 0.4; n = 10, N = 5 in 1). h Mean inferred mechanical power averaged over 10 min in ( g ) ( n = 7, N = 4 in 0; n = 9, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 7, N = 4 in 0.4; n = 10, N = 5 in 1). i Mean inferred mechanical power vs. energy consumption rate ( n = 7, N = 4 in 0; n = 9, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 7, N = 4 in 0.4; n = 10, N = 5 in 1). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant. Scale bars, 10 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6796/pmc13106796/pmc13106796__42003_2026_9843_Fig3_HTML.jpg)
![a Schematic of the fascin crosslinked actin network. b Time-lapse images of actomyosin contraction at 80 nM myosin and 6 µM actin with 0.6 µM fimbrin (left) or 6 µM fimbrin (right), with NADH fluorescence (bottom). NADH fluorescence over time normalized to the initial time point ( c ), and the boxplots showing ATP consumption rate ( d ) ( n = 3 droplets, N = 7 independent experiments in NADH only; n = 6, N = 3 in [Fimbrin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1). ( e ) Schematic of the filamin crosslinked actin network. ( f ) Time-lapse images of actomyosin contraction at 80 nM myosin and 6 µM actin with 0.6 µM fascin (left) or 6 µM fascin (right), with NADH fluorescence (bottom). NADH fluorescence over time normalized to the initial time point ( g ), and the boxplots showing ATP consumption rate ( h ) ( n = 3 droplets, N = 7 independent experiments in NADH only; n = 6, N = 3 in [Filamin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). i Actomyosin network contraction (left) at 80 nM myosin, [Fimbrin]/[Actin] = 1; black arrows indicate total displacement over 10 min, with local apparent strain fields shown on the right. Instantaneous power over time ( j ), and mean inferred mechanical power averaged over 10 min ( k ) ( n = 14, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1). l Actomyosin network contraction (left) at 80 nM myosin, [Filamin]/[Actin] = 0.1; black arrows indicate total displacement over 10 min, with local apparent strain fields shown on the right. Instantaneous power over time ( m ), and mean inferred mechanical power averaged over 10 min ( n ) ( n = 14, N = 3 in 0; n = 5, N = 3 in 0.01; n = 9, N = 6 in 0.04; n = 10, N = 6 in 0.1; n = 13, N = 5 in 1). o ATP consumption rate dependence on the concentration of various crosslinkers ( n = 6, N = 3 in [Fimbrin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1; n = 6, N = 3 in [Filamin]/[Actin] = 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). p Mean inferred mechanical power vs. energy consumption rate on various crosslinkers (fimbrin: n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1; filamin: n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 9, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant. Scale bars, 10 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6796/pmc13106796/pmc13106796__42003_2026_9843_Fig4_HTML.jpg)
![a Boxplot showing relative duty ratio for α-actinin crosslinked network normalized by the mean of no-crosslinked condition. Duty ratio is estimated from the inverse of myosin ATPase activity ( n = 12 droplets, N = 6 independent experiments in 0; n = 10, N = 5 in 0.01; n = 11, N = 8 in 0.1; n = 17, N = 10 in 0.4; n = 11, N = 5 in 1). b Boxplot showing relative duty ratio for fascin crosslinked network normalized by the mean of no-crosslinked condition ( n = 7, N = 4 in 0; n = 11, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 8, N = 4 in 0.4; n = 10, N = 5 in 1). c Boxplot showing relative duty ratio for fimbrin crosslinked network normalized by the mean of no-crosslinked condition ( n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1). d Boxplot showing relative duty ratio for filamin crosslinked network normalized by the mean of no-crosslinked condition ( n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). e Relative inferred mechanical power, normalized by the mean of no-crosslinked condition, dependence on various crosslinker concentrations. Sample size is the same as ( a – d ). Inset is the boxplots showing relative power at [Crosslinker]/[Actin] = 1 ( n = 11, N = 5 in α-actinin; n = 10, N = 5 in fascin; n = 10, N = 6 in fimbrin; n = 11, N = 5 in filamin). f Summary of the crosslinker-induced load-dependence of inferred mechanical power. Fascin and filamin-crosslinked networks allow strongly load-dependent inferred mechanical power, whereas fimbrin and α-actinin-crosslinked networks allow weakly load-dependent inferred mechanical power. g Relative inferred mechanical power vs relative energy consumption rate on various crosslinkers (α-actinin: n = 12, N = 6 in 0; n = 10, N = 5 in 0.01; n = 11, N = 8 in 0.1; n = 14, N = 10 in 0.4; n = 11, N = 5 in 1; fascin: n = 7, N = 4 in 0; n = 9, N = 6 in 0.01; n = 6, N = 3 in 0.1; n = 7, N = 4 in 0.4; n = 10, N = 5 in 1; fimbrin: n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 11, N = 6 in 0.1; n = 6, N = 3 in 0.4; n = 10, N = 6 in 1; filamin: n = 6, N = 3 in 0; n = 5, N = 3 in 0.01; n = 9, N = 6 in 0.04; n = 9, N = 6 in 0.1; n = 11, N = 5 in 1). h Schematic summarizing crosslinker-induced control of inferred mechanical power and ATP consumption rate by myosin. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant. Scale bars, 10 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6796/pmc13106796/pmc13106796__42003_2026_9843_Fig5_HTML.jpg)