Figure 1

Src activity was down-regulated at the protrusion of a cell released from micropattern constrained space.

(A) A flowchart of the experiment: (a) coat comb-polymer on the surface of a PDMS gel membrane, coat fibronectin (FN) on the surface of a cover glass slide; (b) attach the PDMS gel membrane to the glass slide; (c) seed cells inside the micro-patterned wells within the membrane and start imaging; (d) peel-off the membrane during imaging; (e) observe the molecular activity during cell protrusion and polarization. (B) The schematic drawing of the Src biosensor, with its activation mechanism and membrane targeting strategy. (a) The biosensor contains an ECFP, a flexible linker connecting SH2 domain and a substrate sequence and a YPet. The biosensor substrate can be specifically phosphorylated by active Src kinase, subsequently binding to the intramolecular SH2 domain and causing a reversible increase of ECFP/FRET intensity ratio. (b) A KRas-tag was engineered to the C-terminal to insert the biosensor in the membrane microdomains outside of lipid rafts in live cells. (C) Visualization of ECFP/FRET emission ratio of Src biosensor at the initiation of constraint-released cell protrusion. Top panels show the ECFP/FRET emission ratio images; lower panels show the DIC images.