Supplemental Material

This article contains the following supporting material:

• Movie 1 - Mena displays two patterns of localisation at cadherin adhesive contacts.

  hE-CHO cells transiently expressing EGFP-Mena were allowed to adhere to hE/Fc-coated substrata for 30 min before being imaged by time-lapse fluorescence microscopy.

• Movie 2 - Time course of Mena displacement by Cytochalasin D.

  Cells transiently expressing EGFP-Mena were allowed to adhere and spread onto hE/Fc-coated substrata before addition of CytoD (25 nM).

• Figure 1 - Mena accumulates with cadherin in newly forming contacts.

  MCF-7 cell were grown to confluence (untreated) or treated with EGTA (2 mM, 30 min) to disrupt cadherin-based cell-cell adhesions. After chelation of extracellular calcium (0 min), the medium was replaced with DMEM supplemented with 2mM CaCl₂ for 20, 30 or 40 minutes before being prepared for fluorescence microscopy. All samples were pre-extracted in buffer containing TX100 before fixation and co-staining for Mena and E-cadherin. Both Mena and E-cadherin were lost from cell contacts as cells retracted from each other (0 minutes) but rapidly co-accumulated at newly forming contacts upon addition of calcium (20, 30 and 40 minutes). Strongest co-staining was seen at 40 minutes.

• Figure 2 - E-cadherin expression suffices to recruit Mena to cell-cell contacts.

  Parental CHO (pCHO) cells and CHO cells expressing human E-cadherin (hE-CHO) were grown to confluence, fixed and immunostained for Mena and either E-cadherin (to identify cell-cell contacts between hE-CHO cells) or F-actin (to identify contacts between pCHO cells). Mena showed strong co-accumulation with E-cadherin at cell-cell contacts in hE-CHO cells. In pCHO cells Mena localised at the tips of actin bundles as well as the free margins of the cells but failed to accumulate at cell contacts to any extent comparable to that seen in hE-CHO cells.

• Figure 3 - Localization of Mena with potential Mena-binding proteins at cadherin homophilic adhesions.

  (A) hE-CHO cells were transiently transfected with EGFP-IRSp53 and plated onto hE/Fc for 90 min. IRSp53 showed precise co-accumulation with Mena at the outer margins of cells (arrowheads) but did not accumulate with Mena in macroclusters (arrows). Regions in the boxed areas are shown in the far right-hand panel. (B) hE-CHO cells were allowed to adhere to hE/Fc for 90 min, then were fixed and stained for vinculin and Mena. Both proteins localised strongly to macroclusters (arrows), but vinculin was observed only intermittently with Mena at the outer margins (arrowheads). Note the red Mena staining at the very outer margin in the detailed overlay image (Detail, right panel)