Dynamics and controls of subcellular micropatterning of receptors. (A–C) Regular micropatterning does not allow subcellular micropatterning of receptors. (A) Experimental scheme. Stable NIH/3T3 cells constitutively expressing GBP-TM-mScarlet were allowed to spread on dual patterns of fibronectin/fibrinogen-Alexa647 and either GFP, fibrinogen-GFP (low degree of labeling of 0.5 mol GFP per mol fibrinogen), or fibrinogen-biotin::streptavidin-GFP-GFP, then imaged live by TIRF microscopy. (B) Only high-density GFP micropatterning via fibrinogen-biotin::streptavidin-GFP-GFP allows efficient relocalization of the GBP-TM-mScarlet construct onto an area defined by the extracellular pattern. Note that bottom panel corresponds to Fig. 6 B, reproduced here for convenience. Note also that the dynamic range of the GFP channel panels is not identical here. There is much more GFP when using streptavidin-GFP-GFP compared with using fibrinogen-GFP. (C) In contrast to when biotin-EGF was attached to the fibrinogen-biotin-ATTO490LS::NeutrAvidin sandwich (Fig. 7, A–C), direct micropatterning of biotin-EGF::streptavidin-Alexa555 (1 µg/ml) showed very weak, inhomogeneous, and nonspecific micropatterning, preventing micropatterning of the second, surrounding fibronectin pattern. Dashed line, region exposed to UV. (D and E) Dynamics of GFP-Notch relocalization by Delta micropatterns. (D) U2OS cells stably expressing GFP-Notch1 were allowed to spread on dual patterns of fibronectin/fibrinogen-Alexa647 and fibrinogen-biotin-ATTO490LS::NeutrAvidin::biotin-DLL4 and GFP-Notch fluorescence was imaged live during spreading by TIRF microscopy. Elapsed time in minutes:seconds. (E) Quantification of the effects seen in D (mean ± SEM; number of cells analyzed: 27 for control and 28 for Biotin-DLL4); see also Materials and methods. Scale bars, 10 µm. Fib, fibrinogen; biot, biotin.