Oil objectives

For time-lapse movies, cells were transfected with the relevant constructs. Cells were cultured overnight, and then replated on 35 mm glass-bottomed dishes coated with one of the gelatin substrates (see above), and cultured for 1–2 h to enable cell spreading. Images were acquired by DeltaVision (RT or Elite) microscopes, using 100x/1.3 or 60x/1.42 oil objectives (Olympus), or by a DeltaVision Elite microscope equipped with total internal reflection (TIRF) optics (Applied Precision, Inc.) using 100x/1.49 TIRF or 60x/1.42 oil objectives (Olympus). The system is equipped with a temperature- and CO₂-controlled environmental box.

For immunostaining, cells were plated at ~70% confluence on gelatin-coated coverslips or glass-bottomed dishes (see above) for varying time periods. Cells were fixed for 3 min in warm 3% PFA (Merck, Darmstadt, Germany), 0.5% Triton X-100 (Fluka-Chemie AG, Switzerland), followed by 3% PFA alone for an additional 30 min. After fixation, cells were washed 3 times with PBS and incubated with primary antibody for 1 h, washed 3 times in PBS, and incubated for an additional 30 min with the secondary antibody, washed again, and either mounted in Elvanol (Moviol 4–88; Hoechst, Frankfurt, Germany) or left in PBS for TIRF imaging or Z stack acquisition. Images were acquired using the DeltaVision Elite system (Applied Precision Inc., Issaquah, WA, USA), using 100x/1.3 or 60x/1.42 oil objectives (Olympus, Tokyo, Japan). Total internal reflection (TIRF) microscopy was carried out with the DeltaVision system, using 100x/1.49 or 60x/1.42 TIRF oil objectives (Olympus). Image analysis was performed using the UCSF PRIISM environment (http://msg.ucsf.edu/IVE), ImageJ software (rsbweb.nih.gov/ij) and Amira software (FEI) (http://www.vsg3d.com/amira/overview). Z stacks were deconvoluted by DeltaVision software (Applied Precision, Inc.), and analyzed by Imaris software (http://www.bitplane.com/go/products/imaris).