Figure 4.
Actin regulators are heterogeneous, display redundancy, and exhibit preferred subcomplexes.(A) Complexes of diverse composition generate FLSs in vitro. Example of three different actin regulatory proteins (TOCA-1, Cdc42•GTP, and Ena) at the membrane together with a 3D reconstruction of the z-stack of the FLSs growing from a supported bilayer. Note the diversity of regulatory protein combinations observed under the FLSs (colored image in the top right, FLSs highlighted with circles throughout the panel). Grid spacing and scale bars = 10 µm. Volume-rendered FLSs are shown by setting each voxel’s transparency according to its measured intensity (lighter indicates higher intensity). Numbered squares indicate the positions of the example areas displayed in G. (B) Matrix showing Spearman protein–protein intensity correlation and morphology–protein intensity correlation values calculated separately per field of view and averaged. See Fig. S3 for FLS n numbers and Table 1 and Table 2 for numbers of experiments. Gray boxes mean exact correspondence (Diaph3 and Fascin are both enhanced GFP tagged). (C) Correlations of protein immunostaining intensity with actin intensity, FLS tip complex area, FLS straightness, and length are similar to the correlations from tagged protein experiments in Fig. 2 B. FLS numbers: Ena, n = 1,176; N-WASP, n = 1,971; VASP, n = 698; and Fascin, n = 877. (D) FLS lengths are approximately exponentially distributed. The dashed line is a guide for the eye and indicates an exponential with characteristic length L* = 7.6 µm. FLS count, n = 117,365. (E) FLS base areas are approximately exponentially distributed. The dashed line indicates an exponential with characteristic area A* = 1.07 µm2. FLS count, n = 117,365. (F) Relative change in FLS length for enriched FLS with zero, one, two, or three observed proteins compared with the total population length mean, calculated for each field of view and subsequently averaged. Error bars indicate the 95% confidence interval of the mean. Data were pooled from all the different combinations of proteins. Zero, n = 579; one, n = 577; two, n = 566; three, n = 376. (G) Closeups of individual tip complexes (from the numbered examples in A) showing cases where one, two, or all three proteins are enriched in an experiment containing labeled TOCA-1, Cdc42, and Ena. Scale bars = 1 µm.

Actin regulators are heterogeneous, display redundancy, and exhibit preferred subcomplexes. (A) Complexes of diverse composition generate FLSs in vitro. Example of three different actin regulatory proteins (TOCA-1, Cdc42•GTP, and Ena) at the membrane together with a 3D reconstruction of the z-stack of the FLSs growing from a supported bilayer. Note the diversity of regulatory protein combinations observed under the FLSs (colored image in the top right, FLSs highlighted with circles throughout the panel). Grid spacing and scale bars = 10 µm. Volume-rendered FLSs are shown by setting each voxel’s transparency according to its measured intensity (lighter indicates higher intensity). Numbered squares indicate the positions of the example areas displayed in G. (B) Matrix showing Spearman protein–protein intensity correlation and morphology–protein intensity correlation values calculated separately per field of view and averaged. See Fig. S3 for FLS n numbers and Table 1 and Table 2 for numbers of experiments. Gray boxes mean exact correspondence (Diaph3 and Fascin are both enhanced GFP tagged). (C) Correlations of protein immunostaining intensity with actin intensity, FLS tip complex area, FLS straightness, and length are similar to the correlations from tagged protein experiments in Fig. 2 B. FLS numbers: Ena, n = 1,176; N-WASP, n = 1,971; VASP, n = 698; and Fascin, n = 877. (D) FLS lengths are approximately exponentially distributed. The dashed line is a guide for the eye and indicates an exponential with characteristic length L* = 7.6 µm. FLS count, n = 117,365. (E) FLS base areas are approximately exponentially distributed. The dashed line indicates an exponential with characteristic area A* = 1.07 µm2. FLS count, n = 117,365. (F) Relative change in FLS length for enriched FLS with zero, one, two, or three observed proteins compared with the total population length mean, calculated for each field of view and subsequently averaged. Error bars indicate the 95% confidence interval of the mean. Data were pooled from all the different combinations of proteins. Zero, n = 579; one, n = 577; two, n = 566; three, n = 376. (G) Closeups of individual tip complexes (from the numbered examples in A) showing cases where one, two, or all three proteins are enriched in an experiment containing labeled TOCA-1, Cdc42, and Ena. Scale bars = 1 µm.

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