Protein micropatterning

For micropatterning on PLL-PEG–passivated coverslips in an open configuration (all micropatterns except Fig. S3), clean room–grade coverslips (custom 25 × 75–mm size; Nexterion) were surface activated under pure oxygen in a plasma cleaner (PlasmaPrep2; GaLa Instruments) and then laid on top of a 200-µl drop of filtered PLL(20)-g[3.5]-PEG(2) (PLL-PEG; 100 µg/ml in 10 mM Hepes, pH 7.6; SuSoS) for 1 h in a humid chamber. Coverslips were then washed extensively in filtered MilliQ water and dried under a flow of dry nitrogen gas. Coverslips were then mounted in a sticky slide Ibidi 8-well chamber (80828; Ibidi) and pressed under a ∼4-kg weight overnight to stick the chamber to the glass. Then, per well of the 8-well chamber, 200 µl of BBTB was added (50 mM in 0.1 M sodium bicarbonate, pH 8.3) and exposed to micropatterned UV light using the DMD-UV arm of the micropatterning microscope (30–90-s exposure) after focusing on the glass/buffer interface. BBTB was subsequently removed by repeated dilution (12 washes with 600 µl of 0.1 M sodium bicarbonate, pH 8.3) before addition of 200 µl of fibrinogen anchor (100 µg/ml in 0.1 M sodium bicarbonate, pH 8.3, for a final concentration of 50 µg/ml). The fibrinogen anchor was allowed to adsorb to the surface for 5 min before washing by repeated dilution (12 washes). The surface was then quenched for 5 min with 0.5 mg/ml PLL-PEG (in 10 mM Hepes, pH 7.6). PLL-PEG was then removed by similar repeated dilution, so that the surface never dried. The protein of interest was then added in its buffer of choice (here 0.1 M sodium bicarbonate, pH 8.3, for GFP, biotin-BSA, and NeutrAvidin) at 5 µg/ml and allowed to bind to the fibrinogen anchor for 5 min before extensive washes and imaging. Alternatively, when proteins were directly micropatterned, the fibrinogen anchor in the above protocol was replaced by the protein of interest at a concentration of 50 µg/ml in 0.1 M sodium bicarbonate, pH 8.3 (except for fluorescent EGF, where biotinylated EGF complexed to streptavidin-Alexa555 [E35350; Invitrogen] was patterned at 1 µg/ml in 0.1 M sodium bicarbonate, pH 8.3). After a 5-min incubation, the surface was quenched and washed as above.

For sequential multiplexed micropatterning experiments on PLL-PEG–passivated coverslips (Fig. 1, C–E; Fig. 3; and Fig. S1, D–F), the protocol above was performed except that immediately after the PEG quenching step, BBTB was added to the chamber (50 mM in 0.1 M sodium bicarbonate, pH 8.3), and the whole process was repeated for a second fibrinogen anchor (and a third time for the triple patterning in Fig. 1, C–E). For Fig. 3, where fibrinogen anchors were used to subsequently bind to two proteins of interest, said proteins of interest were added together after micropatterning of both fibrinogen anchors (5 µg/ml each in 0.1 M sodium bicarbonate, pH 8.3, for 5 min) before extensive washes and imaging. All fibrinogen anchors that are not fluorescent were doped with 5 µg/ml fibrinogen-Alexa546 to image their respective micropattern and thereby align the next micropattern. For controls where one fibrinogen anchor was omitted, it was replaced with fibrinogen-Alexa546.

For dual micropatterning experiments where receptors were clustered to micropatterns (Fig. 6, Fig. 7, and Fig. S5), the above protocol was performed, with the small central region micropatterned first (50 µg/ml GFP, fibrinogen-GFP, fibrinogen-biotin-ATTO490LS, or fibrinogen-biotin-Alexa647), before PLL-PEG quenching and micropatterning of the second, outer region (50 µg/ml fibronectin plus 10 µg/ml fibrinogen-Alexa546 or fibrinogen-Alexa647). For relocalization of the GBP-TM-mScarlet receptor, micropatterns (with a central fibrinogen-biotin-ATTO490LS center) were incubated (or not) with streptavidin-GFP-GFP (10 µg/ml) and fixed with 0.5 mM dithiobis(succinimidyl propionate) for 20 min. After extensive washing, NIH/3T3 cells were added (20,000 cells/well) in serum-free DMEM for 30 min before washing into medium containing serum and imaging 30 min later. Alternatively, for live imaging of GBP-TM-mScarlet recruitment (Fig. 6), 20,000 cells were added per well, in L15 plus 20 mM Hepes, and imaged as they landed and spread on micropatterns. For relocalization of EGFR and GFP-Notch1, double micropatterns were incubated with NeutrAvidin (25 µg/ml) for 5 min before extensive washing and incubation for 5 min with biotinylated-EGF (1 µg/ml) or biotinylated-DLL4 (0.5 µM), respectively. Chambers were then extensively washed. HeLa cells were serum starved for 24 h before addition, in serum-free medium, to EGF micropatterns (20,000 cells/well). Cells were left to spread for 40 min before fixation. U2OS GFP-Notch1 cells, induced for 24 h to express GFP-Notch1, were added to DLL4 micropatterns in L15 plus 20 mM Hepes (20,000 cells/well) and imaged as they landed and spread on micropatterns.

For micropatterning of Kin1-biotin using the fibrinogen-biotin::NeutrAvidin “sandwich” (Fig. 4), PLL-PEG–passivated coverslips assembled into 8-well chambers were exposed for 90 s before the addition of fibrinogen-biotin-ATTO490LS (50 µg/ml in 0.1 M sodium bicarbonate, pH 8.3) as above. Micropatterns were then quenched with 0.5 mg/ml PLL-PEG for 5 min before addition of NeutrAvidin (10 µg/ml in 0.1 M sodium bicarbonate, pH 8.3, for 5 min) before washing and incubation with 5 µg/ml purified Kin1-biotin in 80 mM Pipes, 1 mM MgCl₂, and 5 mM ATP, pH 8.9, for 5 min. Alternatively, for direct micropatterning of Kin1-biotin, micropatterns were exposed to micropatterned UV light in the presence of BBTB for 90 s as above before addition of 5 µg/ml Kin1-biotin in 80 mM Pipes, 1 mM MgCl₂, and 5 mM ATP, pH 8.9. Micropatterns were then quenched with 0.5 mg/ml PLL-PEG for 5 min. 4 µl of GMPCPP MT seeds (see above) were then added to each well, in 200 µl of ATP-regenerating buffer (80 mM Pipes, 0.1 mg/ml κ-casein, 40 µM DTT, 64 µM glucose, 160 µg/ml glucose oxidase, 20 µg/ml catalase, 0.1% methylcellulose, and 1 mM ATP, pH 6.9), and imaged by TIRF microscopy. For direct micropatterning of Kin1-biotin, visualization of micropatterns was performed after imaging by addition of 1 µM streptavidin-Alexa647.

For micropatterning of Con A to bind to S2 cells (Fig. 5), surface-activated coverslips were passivated with 0.1 mg/ml PLL-PEG before being micropatterned using a quartz-chromium photomask and a deep UV light source for 5 min as described previously (Azioune et al., 2009). Exposed coverslips were then incubated with 200 µl fibrinogen alone (50 µg/ml fibrinogen and 5 µg/ml fibrinogen-Alexa546), Con A alone (50 µg/ml Con A and 5 µg/ml rhodamine–Con A), or fibrinogen–Con A (50 µg/ml fibrinogen–Con A and 5 µg/ml fibrinogen-Alexa546), all in 0.1 M sodium bicarbonate, pH 8.3, for 1 h. Coverslips were washed in 0.1 M sodium bicarbonate, pH 8.3, and assembled into an 8-well chamber. 100,000 S2 cells in low (1%) serum media were then added to each well for 1 h before incubation with SiR-tubulin (1 µM; Spirochrome; Lukinavičius et al., 2014) for 30 min to label cells. Chambers were washed with Schneider medium plus 10% heat-inactivated FBS and imaged by confocal spinning-disc microscopy.

For micropatterning on PEG-silane–passivated coverslips in a flow cell configuration (Fig. S3), clean room–grade coverslips (75 × 25 mm; Nexterion) were surface-activated under pure oxygen in a plasma cleaner and then incubated with PEG-silane (30 kD; PSB-2014; Creative PEGWorks) at 1 mg/ml in ethanol 96%/0.1% HCl overnight at RT with gentle agitation. Standard 22 × 22–mm coverslips were similarly passivated with PEG-silane, omitting the plasma-cleaning step. Slides and coverslips were then successively washed in 96% ethanol and ultrapure water before drying under nitrogen gas. The coverslip and slide were subsequently assembled into an array of six flow cells (∼15 µl each) using double-sided tape (AR-90880; Adhesive Research; cut with a Graphtec CE6000 cutting plotter). The flow cell chamber was then filled with BBTB (50 mM in 0.1 M sodium bicarbonate, pH 8.3) and exposed to UV light on the DMD-UV arm of the micropatterning microscope (3-s exposure) after focusing on the glass/buffer interface. The flow cell was subsequently washed with three flow cell volumes of carbonate buffer, and fibrinogen-biotin was then injected at 20 µg/ml in 0.1 M sodium bicarbonate, pH 8.3. After a 2-min incubation, the chamber was washed with three flow cell volumes of carbonate buffer, and NeutrAvidin (or NeutrAvidin-Dylight-550) was injected at 50 µg/ml in 0.1 M sodium bicarbonate, pH 8.3. The chamber was then washed with Hepes buffer (10 mM Hepes, pH 7.6) and quenched with PLL-PEG (0.2 mg/ml in 10 mM Hepes, pH 7.6) for 2 min. The chamber was then washed with Hepes buffer, and BSA-biotin-Alexa647 was added (10 µg/ml in 10 mM Hepes, pH 7.6) for 1 min, before extensive washing with Hepes buffer before imaging. For controls where NeutrAvidin was directly micropatterned, fibrinogen-biotin was replaced by NeutrAvidin/NeutrAvidin-DyLyte550 (50 µg/ml in carbonate buffer), and the chamber was directly washed with Hepes buffer, quenched with PLL-PEG, and incubated with BSA-biotin-Alexa647 as above.

In general, we found that buffer composition (Fig. S1 B) and avoiding drying of the surface (Fig. S4, A and B) were fundamental for ensuring reproducibility, selectivity, and homogeneity of micropatterning. Note that while the micropatterning buffer is important, the use of fibrinogen anchors allows one to bind the proteins of interest to the micropattern in virtually any buffer, as this binding step occurs after the micropatterning process.