Fibrinogen anchors enables the subcellular micropatterning of a synthetic receptor fused to a cortical protein of interest. (A) Experimental scheme: NIH/3T3 cells stably expressing GBP-TM-mScarlet were allowed to spread on dual micropatterns of fibronectin/fibrinogen-Alexa647 and fibrinogen-biotin-ATTO490LS::streptavidin-GFP-GFP, and were then imaged live by TIRF microscopy. (B) Efficient relocalization of the GBP-TM-mScarlet construct onto an area defined by the extracellular GFP micropattern in live cells. (C) Quantification of the effects seen in B (mean ± SEM). Statistics were performed using a Student’s t test. n, number of cells analyzed. (D) Cells as in B were imaged by TIRF microscopy during spreading to evaluate the kinetics of GBP-TM-mScarlet recruitment onto the GFP micropattern. Fibrinogen and GFP micropatterns are outlined in blue and green dashed lines, respectively. (E) quantification of the effects seen in D (mean ± SEM; number of cells analyzed: 11 for control and 32 for Streptavidin-GFP-GFP). Scale bar, 10 µm. Ctrl, control; Strept., streptavidin.