JCS03027 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Sequence of AF6i3 protein. Peptide sequences obtained by MALDI-TOF mass spectrometry are boxed. Red boxes mark peptides specific for AF6i3. The aa position at which the AF6i3-specific sequence starts is underlined.

• Supplemental Figure 2 -

  Fig. S2. Concentration of F-actin and formation of vinculin- and FAK-positive focal adhesions at the leading edge of migrating cells. Immunofluorescence analysis of marginal AF6i3-knockdown and control cells during wound healing assay. Detection was performed with phalloidin-TRITC (A), anti-vinculin antibody (B) and anti-FAK antibody (C). Bar, 20 μm.

• Supplemental Figure 3 -

  Fig. S3. Regulation of small GTPases Rac1 and Cdc42 in AF6i3-knockdown and control cells during wound healing assay. (A) Localization of Rac1 (red) and Cdc42 (green) as determined by immunofluorescence. Bar, 20 μm. (B) Activation of Rac1 after initiation of migration as determined by pull-down assay using PAK-PBD-agarose. Active Rac1-GTP bound to the PAK-PBD-agarose (top) and total Rac1 (bottom) were detected by western blot using anti-Rac1 antibody. (C) Active Cdc42-GTP (top) and total Cdc42 (bottom) detected in GDP- and GTPγS-loaded MCF10A cell lysates. Active Cdc42 could not be detected in AF6i3-knockdown or control cells after initiation of migration. (D) Western blot analysis of Rac1 protein levels in AF6i3-knockdown and control cells during wound healing assay.

• Supplemental Figure 4 -

  Fig. S4. AF6i3 knockdown decelerates cell-cell contact formation in Ca²⁺ switch assay. (A) Immunofluorescence images of AF6i3-knockdown and control cells 2 hours after the switch from 2 μM Ca²⁺ to 2 mM Ca²⁺. Staining was performed with indicated antibodies. Bar, 50 μm. (B) Western blot analysis of AF6i3-knockdown and control cell lysates during Ca²⁺ switch assay.

• Supplemental Figure 5 -

  Fig. S5. Proposed model of AF6i3-mediated stabilization of intercellular adhesion. We propose that AF6i3 stabilizes E-cadherin-dependent intercellular adhesion by increasing the interaction between E-cadherin and p120-catenin, as already published by Hoshino et al. (2005), and additionally by increasing the association of E-cadherin with the actin cytoskeleton. This function of AF6i3 depends on its interaction with the actin cytoskeleton. Thus, AF6i3 might function as an additional linker between the E-cadherin and cytoskeleton. This might occur by direct interaction between AF6i3 and α-catenin (Tachibana et al., 2000; Pokutta et al., 2002), or through their indirect association with ADIP (Afadin-DIL-domain interacting protein) / α-actinin, LMO7 (LIM domain only 7) / α-actinin or ZO-1 (Asada et al., 2003; Knudsen et al., 1995; Ooshio et al., 2004; Itoh et al., 1997; Yamamoto et al., 1997).

• Movie 1 -

  Movie 1. Time-lapse phase-contrast movie of MCF10A control cells during wound closure, starting 30 minutes after wounding, recorded for 4 hours. Frames were collected at 3-minute intervals. Display rate is 10 frames/second. Two of the frames are included in Fig. 4A.

• Movie 2 -

  Movie 2. Time-lapse phase-contrast movie of AF6i3-knockdown cells during wound closure, starting 30 minutes after wounding, recorded for 4 hours. Frames were collected at 3-minute intervals. Display rate is 10 frames/second. Two of the frames are included in Fig. 4A. Note the gaps appearing between the AF6i3-knockdown cells.