Stage incubator

A quantity of 0.4 μM dynein was added to a 5 mg/ml BSA precoated flow chamber (Matsunami Glass, Osaka, Japan) and adsorbed onto the bottom for 5 min. A total of 10.5 mg/ml TMR-labeled tubulin and 7.64 mg/ml white tubulin were polymerized at a ratio of 1:40 and stabilized in 50 μM Taxol (Sigma)/BRB80 buffer (40 mM PIPES, pH 7.2, 0.5 mM MgSO₄, and 0.5 mM EGTA). The MT solution was diluted to 0.5 μM in ATP buffer (1 mM ATP and 100 μM Taxol) with oxygen scavenger (225 μg/ml glucose, 216 μg/ml glucose oxidase, 36 μg/ml catalase, and 1% 2-mercaptoethanol) and introduced into the flow chamber. Following confirmation of MT gliding by cytoplasmic dynein (0.5 μM), recombinant proteins (p80; 0.4 μM, Δ1-56 aa; 0.4 μM, p.G33W; 0.4 μM, p.S535 L; 0.4 μM, p.L540R; and 0.4 μM, BRB80) were added. MT gliding was observed via conventional inverted fluorescence microscopy (Olympus IX71, Tokyo, Japan) with an oil-immersion objective lens (UPlanSAPO, 100X, NA = 1.4, Olympus, Tokyo, Japan) and an EMCCD camera (ImagEM, Hamamatsu Photonics, Hamamatsu, Japan). Captured images were analyzed with AquaCosmos software (Hamamatsu Photonics, Hamamatsu, Japan). All experiments were performed at 37 °C with a stage incubator (Tokai-Hit, Shizuoka, Japan).

To analyze cellular in vitro migration by using a scratch assay, cells were seeded in cell culture chamber slides and incubated at 37°C under 5% CO₂ and 95% air atmosphere. When they reached 100% confluence, a straight artificial gap was created in the middle of the chamber. In order to smooth the edges of the scratch and remove floating cells, the cells were washed with medium several times. Afterward the medium was removed and replaced by medium supplemented with 1,8-cineol. Untreated cells were used as negative control. Photographs of the migrating cells were taken every 15 min by a microscope (Keyence, Osaka, Japan) with stage incubator (Tokai Hit, Shizuoka, Japan) as well as multipoint and time lapse function.

Live-cell imaging experiments were performed on an inverted Nikon Eclipse Ti–E confocal microscope equipped with a perfect focus system (Nikon), a CSU–X1–A1 Spinning Disc unit (Yokogawa), a Photometrics Evolve 512 EMCCD camera (Roper Scientific) and a Plan Apo VC 100× N.A.1.40 oil objective. Coverslips were mounted in a Ludin chamber (life imaging services) and maintained in culture medium at 37°C and 5% CO₂ in a stage incubator (Tokai Hit) during image acquisition.