Figure 1
From: Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM
Schematic of atomic force microscopy (AFM) combined with total internal reflection fluorescence (TIRF). (a) α-Catenin molecules (M1-M3 domain, residues 276–634, and M2-M3 domain, residues 385–634), with a GST-tag at the N-terminus and a His6-tag at the C-terminus, were chemically modified on coverslips at the C-terminus and were mechanically loaded using an AFM tip; simultaneously, full-length vinculin molecules (residues 1–1066), dissolved in solution, were observed by TIRF. Vinculin molecules modified with Alexa Fluor 488 dye were illuminated when they came into in an evanescent field in TIRF (cyan region, approximately 150 nm from the surface of coverslips). (b) Movement of the base of the AFM cantilever was regulated by a programmed piezo-height as follows: (i) Decrease the piezo-height with the velocity of 500 nm/s to approach the AFM tip to the α-catenin-modified coverslip and stop when the reaction force reaches 500 pN (the piezo-height is set to zero at this point); (ii) keep the piezo-height to wait for an interaction between the AFM tip and α-catenin; (iii) increase the piezo-height by 40 nm with a velocity of 500 nm/s to extend α-catenin; (iv) keep the piezo-height constant at 40 nm to hold α-catenin under tension and wait for vinculin recruitment; and (v) increase the piezo-height by 260 nm with the velocity of 500 nm/s to completely retract from the coverslip and examine the resistance force of α-catenin with or without vinculin recruitment.
