Apo tirf objective

Total internal reflection fluorescence microscopy (TIR‐FM) was performed as previously described.⁶³ Briefly, cells were mounted in PBS and imaged using a 60×, 1.49 NA APO TIRF objective (Nikon) mounted on a fully motorized Nikon Ti‐Eclipse inverted microscope with Perfect Focus System and coupled to an Andor “Diskovery TIRF/Borealis widefield illuminator” equipped with an additional 1.8× tube lens (yielding a final magnification of ×108). TIR‐FM illumination was achieved using a Diskovery Platform (Andor Technology). For live cell experiments, cells were maintained at 37°C during imaging. Imaging sequences were acquired using a sCMOS camera with 6.5 μm pixel size (pco.edge).

Cells were seeded on a gelatin-coated 22 × 22–mm cover glass (Corning; 2850-22) overnight and moved to fresh cell culture medium 30 min before being mounted on a 25 × 75–mm slide (Thermo Fisher Scientific; 3050). Imaging was conducted with a 60×, 1.49-NA Apo TIRF objective (Nikon) mounted on a Ti-Eclipse inverted microscope. Perfect focus was used during time-lapse imaging. TIRF penetration depth was ∼80 nm. Videos were acquired for 7.5 min at the rate of 1 frame/s for all live-cell imaging. For dual-color imaging, two sequential images of primary and secondary channel were taken within 1 s, and the video length was the same as single-channel imaging. Published cmeAnalysis software was used for CCP detection, tracking, and quantification (Aguet et al., 2013 (link); Jaqaman et al., 2008 (link); Loerke et al., 2011 (link)). More than 10 cells were imaged per condition, and the number of total analyzed tracks is indicated in the figure legends.

Transferrin receptor (TfnR) surface levels were measured using the anti-TfnR mAb (HTR-D65). ARPE-19 and H1299 cells (1.5 × 105 cells per well in a 6-well plate) were grown overnight on glass cover slips and further pre-incubated with 4 ug/ml of D65 in TfnR assay buffer (PBS4+: PBS supplemented with 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose and 0.2% bovine serum albumin) at 4°C for 30 min. After being washed with PBS⁴⁺, cells were fixed in 4% PFA for 30 min at 37°C, permeabilized with 0.1% Triton X-100 for 5 min and further blocked with Q-PBS (2% BSA, 0.1% lysine, pH 7.4) for 30 min. After three washes with PBS, cells were incubated with a 1:500 dilution of goat anti-mouse Alexa-568 labelled secondary antibody (Life Technologies) for 30 min, washed an additional three times with PBS before TIRFM imaging using a 100 × 1.49 NA Apo TIRF objective (Nikon, Japan) mounted on a Ti-Eclipse inverted microscope equipped with the Perfect Focus System (Nikon). Images were acquired with an exposure time of 150 ms for both channels using a pco-edge 5.5 sCMOS camera with 6.5 um pixel size. For inhibition studies, cells were initially pre-incubated in the presence of Akt inhibitor X (10 uM) for 30 min at 37°C, followed by pre-incubation with 4 ug/ml of D65 at 4°C for 30 min, in continued presence of the inhibitor.

Total Internal Reflection Fluorescence (TIRF) Microscopy imaging was conducted as previously described (Loerke et al., 2009 (link)). Cells were grown on a gelatin-coated 22 × 22 mm glass (Corning, #2850–22) overnight and then mounted to a 25 × 75 mm cover slide (Thermo Scientific, #3050). Imaging was conducted with a 60X, 1.49-NA Apo TIRF objective (Nikon) mounted on a Ti-Eclipse inverted microscope equipped with an additional 1.8X tube lens, yielding a final magnification of 108X. Perfect focus was applied during time-lapsed imaging. For EPI-TIRF imaging, nearly simultaneous two channel (488 epifluorescence/TIRF) movies were acquired with multi-dimension acquisition (MDA). Movies were acquired at the rate of 1 frame/s. cmeAnalysis was applied for CCP detection and tracking (Aguet et al., 2013 (link); Jaqaman et al., 2008 (link); Loerke et al., 2011 (link)). Variation could arise from the heterogeneity of cover glass by itself and the gelatin-coating.