Ti e tirf microscope

Cover glasses were cleaned using 1 N KOH sonication and a plasma cleaner, and then the micro-chamber was built using adhesive tape^{19 (link)}. The observed glass surface of the microchamber was coated with 0.01% poly-L-lysine for 5 min. The carboxylic acid group in Vasa was strongly adsorbed onto the glass surface in the assay buffer containing the oxygen scavengers: 30 mM HEPES, pH 7.3, 150 mM potassium acetate, 5 mM magnesium acetate 0.1% NP-40, 500 µM TCEP, 10 mM TSY, 10 mM PCA, 12-fold diluted PCD (Pacific Biosciences), and 200-fold diluted RNasin^® Plus RNase Inhibitor (Promega). 5 µg/ml yeast RNA was included for the contribution test of exogenous RNA to the oligomer formation of Vasa. The fluorescent images illuminated by a 532-nm laser (Coherent, Sapphire) were acquired at a frame rate of 100 ms using an EMCCD camera (Andor, iXon+) equipped with a Nikon Ti-E TIRF microscope^{19 (link)}.

The assays were done as described before47 (link). Coverslips (12-545-F, Thermo Fisher, USA) and slides (10127101P, Shitai, Jiangsu, China) were used to make the flow chamber (containing four individual channels) for the gliding assays. Two-millimetre-wide double-sided tapes (200 MP, 3M (Minnesota Mining and Manufacturing), USA) were used to stick and isolate the flow channels. The surface was cleaned in acetone (Guoyao, China) followed by KOH (484016, Sigma-Aldrich, USA) with a sonicator (KQ500DE, Kunshan, China) and stored in water to keep it hydrophilic. A volume of 15 μl of the motor proteins (KIF5B or Zen4) were added to the flow chamber channels for 5 min. The motor-coated coverslips were blocked with 3 mg ml⁻¹ casein, followed by addition of 40 nM microtubules for 5 min. An energy system (0.5 mM ATP, 20 μM taxol, 10 mM DTT, and 1 mg ml⁻¹ casein), an ATP regeneration system, and an oxygen scavenger system were flowed into the chamber. Images of gliding assay were recorded every 500 ms using a Nikon Ti-E TIRF microscope under 640-nm laser excitation.