The details on TIRF microscopy were described previously [57 (link)]. Briefly, using an inverted fluorescent microscope (IX-81; Olympus) with a 100X/1.45NA objective (Olympus), cells were excited using a combination of green (488 nm) and red (561 nm) lasers, passed through a LF405/488/561/635 dichroic mirror, and filtered emitted light was projected side-by-side on an electron multiplying charge-coupled device (EM-CCD) camera (DU 897; Andor). Using Andor IQ2 software, images of transfected cells were obtained at 5 Hz and 100 ms exposure times. Each day fluorescent beads (Invitrogen) were imaged in the green and red channels and superimposed by mapping corresponding fluorescent bead positions in both channels. The green and red images were subsequently transformed and aligned as described before [56 (link), 66 (link)]. All experiments were conducted at room temperature (~ 25 °C).
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