Temperature controller

GMPCPP-MTs were polymerized using a mixture of 20 μM tubulin extracted from mouse brain, 1 mM GMPCPP (Jena Bioscience; catalog no.: NU-405L) and 4 mM MgCl₂ in BRB80, which was incubated for 2 h at 37 ˚C. Polymerized MTs were collected using an air-driven ultracentrifuge (Beckman; catalog no.: 340401), which were then resuspended in BRB80 with 20 μM taxol (Cell Signaling Technology; catalog no.: 9807), and stored at 37 ˚C. Part of MTs was treated with TTLL4 or TTLL7 for 2 h at room temperature of 37 °C. Then, MTs were immobilized on the surface of a cover glass using the tubulin antibody links (Sigma; catalog no.: T7816). Next, Kif5c-GFP was added into the flow cell in the imaging buffer (BRB80 supplemented with 1 mM ATP, 20 μM taxol, 80 mM d-glucose, 0.4 mg/ml glucose oxidase, 0.2 mg/ml catalase, 0.8 mg/ml casein, 1% β-mercaptoethanol, and 0.001% Tween-20). Images were recorded every 100 ms with a 50 ms exposure. The sample was kept at 35 °C using a temperature controller (Tokai Hit). Images were recorded using a TIRFM (Olympus).

To capture the localization of SAS-6-GFP in fission yeast, cells were cultured in minimal medium at 30°C, mounted onto a lectin-coated glass-bottomed dish (MatTek). Live cell imaging was performed on Nikon Eclipse Ti-E microscopes with Evolve 512 EMCCD camera (Photometrics) in a chamber maintained at 30°C by a temperature controller (Tokai Hit). Images were acquired as a Z-series (0.3 μm interval) and are presented as maximal intensity projections.