Apo tirf n a 1

The rotation of a polystyrene bead attaching to the γ-subunit was visualized under an inverted microscope (Ti-E; Nikon) equipped with 100× objective lens (Apo TIRF N.A. 1.49; Nikon), a condenser unit (LWD0.52; Nikon), LED (pE-100 660 nm; CoolLed), IR laser (YLM-2-1064-LP; IPG), an optical bench (RS2000TM; Newport), and three CCDs (CCD1 in Fig. 1A, CS8430i; Toshiba Teli, CCD2, Luca; Andor, and high-speed CCD3, LRH20000B; DigiMo). CCD2 and CCD3 were synchronized with TTL signal to identify the moment when optical trapping was turned on with a custom modification in CCD3. Dichroic mirrors, DM1 and DM2 in Fig. 1A, were custom made (Chroma Technology) and purchased (Asahi Spectra), respectively. A highly-stable customized sample-stage (Chukousha) was adjusted three actuators (SGSP-13ACTR; Sigma Koki). The optical system for 3-D tracking system was described previously^{14 (link),36} ; the calibration factor to determine z-position from Δx was set as 1 in all measurements. Most apparatuses except PCs and displays were compartmented in a custom-made thermostatic chamber (Nihon Freezer), and all operations were done from the outside of the chamber. The Measurements were done at 23 ± 0.2 °C. speed of camera was set as 30 and 500 f.p.s. for low and high-load, respectively.