Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - Co-localization of endogenous zyxin with RFP-zyxin at tails regardless of the expression level. Three NRK cells expressing different levels of RFP-zyxin are shown after fixation and staining with antibodies against zyxin. Immunofluorescence staining colocalizes with RFP-zyxin regardless of the level of RFP-zyxin expression. Bar, 20 µm.
• Supplemental video 1 - Retrograde fluxes of zyxin in NRK cells. Time-lapse recording corresponding to figure 1. Note the swinging of focal adhesion tails. Interval, 5 sec. Total recording time, 122 min.
• Supplemental video 2 - Concomitant flux of actin and zyxin in NRK cells co-transfected with EGFP-actin and RFP-zyxin. Interval, 2 min. Total recording time, 60 min.
• Supplemental video 3 - FRAP analysis of RFP-zyxin, showing both a flux and rapid recovery of fluorescence. Time-lapse recording corresponding to figure 3G. Interval, 2 min. Total recording time, 5 min.
• Supplemental video 4 - Flux of actin filaments as detected by photobleaching. Time-lapse recording corresponding to figure 3F. Interval, 5 sec. Total recording time, 4.5 min.
• Supplemental video 5 - Retrograde fluxes of EGFP tagged N-fragment of tensin in an NIH3T3 cell co-transfected with EGFP-tensin N-fragment and RFP-zyxin. Interval, 2 min. Total time, 310 min.
• Supplemental video 6 - Effect of Y-27632 on the focal adhesion flux. Treatment of a NRK cell expressing RFP-zyxin with 50 μM Y-27632 causes strong inhibition of focal adhesion fluxes, while focal adhesions reduce to small dot-like structures. Interval, 2 min. Total time, 86 min.
• Supplemental video 7 - Increased focal adhesion fluxes in cells immobilized on patterned substrates. NIH3T3 cells were plated on square islands of stiff polyacrylamide. Note that prominent elongated fluxes are concentrated at four corners of the island. Interval, 2 min. Total recording time, 128 min.
• Supplemental Video 8 - Concentration of phosphotyrosine at focal adhesions. NRK co-transfected with RFP-zyxin (upper panel) and YFP-dSH2 (lower panel). Interval, 2 min. Total recording time, 62 min. Bar, 20 µm.
• Supplemental Video 9 - Involvement of tyrosine kinase activity in the formation of zyxin fluxes at focal adhesions. Treatment of an RFP-zyxin transfected NRK cell with genistein causes the inhibition of zyxin fluxes without causing significant disassembly of focal adhesions. Genistein starts to inhibit zyxin fluxes at about 30 min after addition, and abolishes zyxin fluxes at 60 min post application. Interval, 2 min. Total recording time, 132 min. Bar, 20 µm.