Supplemental Material

This article contains the following supporting material:

• Figure 1 - Fluorescently labeled α1 integrin mediates adhesion-related functions normally. (A) Phosphorylation of FAK tyrosine 397 in wild type 3T3 cells and cells stably expressing either α1-GFP (A1G) or wild type human α1 (HA1) integrin. Cells were kept in suspension for 1 hour and allowed to spread on collagen IV (CN) or FN (FN) for 1 hour and pY397 FAK compared to the suspension control (S). The membrane was reprobed with anti-FAK monoclonal antibody (MAb) (Transduction). (B & C) 3T3 cells were transiently transfected with α1-RFP (B) or α1-GFP (C), allowed to spread on collagen IV for 1 hr and stained with anti-vinculin MAb (B) or with rhodamine-phalloidin for F-actin (C) and examined by epifluorescence microscopy. Cells transfected with either α1-GFP or α1-RFP displayed normal focal adhesion and actin stress fiber formation on collagen IV, while untransfected cells (C, cell bottom right) failed to spread. Bar, 10μm.

• Figure 2 - Cytoplasmic FA proteins colocalize to integrin containing projections at early spreading times. TIRF microscopy of A1R cells fixed approximately 15 minutes after spreading began on collagen IV and counterstained for (A) FAK. (B) TIRF microscopy of A1R cells co-expressing GFP-talin fixed 15 minutes after spreading began on collagen IV. Merged images are overlays of integrin (red) and the respective cytoplasmic focal adhesion protein (green). Bar, 10μm. Both endogenous and GFP tagged cytoplasmic focal adhesion proteins colocalize to integrin-containing filopodial projections at early stages of cell spreading.

• Figure 3 - Diagram of projection visualization in the evanescent field of TIRF microscopy. Since TIRF microscopy uses evanescent wave illumination for excitation of the fluorophores, only portions of the projections (Figure 3B) that lie within approximately 100nm of the substratum are illuminated. As the cell periphery is brought closer to the substratum, more of the projection is illuminated.

• Figure 4 - Expression level of stably infected reporter constructs. Western blots of A) talin, B) FAK and C) paxillin in parental NIH3T3 cells compared to A1R cells expressing either A) GFP-talin, B) GFP-FAK or C) GFP-paxillin. Expression levels of all cytoplasmic FA reporter constructs are similar to their endogenously expressed wild type counterparts.

• Movie 1 - Movie of normal fibroblast spreading. A1R cells were allowed to spread on collagen IV. Live cell spreading was followed by TIRF microscopy. Frames were taken every 90 seconds and a total of 29 frames were collected. The time of the frame is in the bottom right corner recorded in minutes. Note the long filopodial projections, which eventually become focal adhesions.

• Movie 2 - Movie of GFP-FAK during fibroblast spreading. A1R cells also expressing GFP-FAK were allowed to spread on collagen IV. Live cell spreading was followed by TIRF microscopy. Frames were taken every 60-90 seconds and a total of 35 frames were collected. The time of the frame is in the bottom right corner recorded in minutes.