Figure 4. Localization in the sealing zone of actin core and ring proteins.

(A) Representative RIM images of sealing zones co-stained for F-actin (red) and cortactin (green). (A’) Enlarged view of (A) where white crosses indicate the localization of actin cores. (A’’) Intensity profiles along the dotted line marked in (A’). (B) Representative RIM images of sealing zones co-stained for F-actin (red) and α-actinin1 (green). (B’) Enlarged view of (B) where white crosses indicate the localization of actin cores. (B’’) Intensity profiles along the dotted line marked in (B’). (C) Representative RIM images of sealing zones co-stained for F-actin (red) and filamin A (green). (C’) Enlarged view of (C) where white crosses indicate the localization of actin cores. (C’’) Intensity profiles along the dotted line marked in (C’). (D) Representative RIM images of sealing zones co-stained for F-actin (red) and vinculin (green). (D’) Enlarged view of (D) where white crosses indicate the localization of actin cores. (D’’) Intensity profiles along the dotted line marked in (D’). (E) Normalized intensity profiles of F-actin, cortactin, α-actinin1, filamin A, vinculin, paxillin, and talin (medians of 1080, 239, 265, 277, 299, 197 and 988 cores for each staining, respectively). Scale bars: 5 µm (A, B, C, D), 1 µm (A’, B’, C’, D’).

Figure 4—figure supplement 1. Analysis workflow for the 2D localization of proteins relative to actin cores with RIM technique.

Analysis workflow for RIM images of the sealing zone co-stained for F-actin (red) and vinculin (green). White crosses indicate the localization of actin cores. Intensity profiles along the marked dotted lines were analyzed. Similarly to the evaluation of the size of actin cores, the first step was to extract the coordinates of the cores thanks to the actin staining image. And in the same way, data was also extracted in order to assess the diameter of each core. Then, the local orientation of the sealing zone was evaluated by the user, in order to extract both actin and protein associated signals in the transverse direction. But these curves are not comparable as such, so thanks to the size-related data a normalization protocol was performed on the signals so that they were ready for statistical analysis. Scale bar: 5 µm.

Figure 4—figure supplement 2. Localization in the sealing zone of cortactin, α-actinin 1, filamin A and vinculin.

Gallery of RIM images of sealing zones co-stained for F-actin (red) and cortactin, α-actinin 1, filamin A, or vinculin (green). Scale bar: 5 µm.

Figure 4—figure supplement 3. Localization in the sealing zone of paxillin and talin.

(A) Representative RIM image of sealing zones co-stained for F-actin (red) and paxillin (green). (A’) Enlarged view of (A). (A’’) Intensity profiles along the dotted line marked in (A’). (B) Gallery of RIM images of sealing zones co-stained for F-actin (red) and paxillin (green). (C) Representative RIM image of sealing zones co-stained for F-actin (red) and talin (green). (C’) Enlarged view of (C). (C’’) Intensity profiles along the dotted line marked in (C’). (D) Gallery of RIM images of sealing zones co-stained for F-actin (red) and talin (green). Scale bars: 5 µm (A, B, C, D), 1 µm (A’, C’).