Tirf microscope

Stable U2OS cells expressing the red tyrosination sensor (A1aY1 with an N-terminal TagRFP-T) were treated with 10 µM nocodazole (Sigma-Aldrich; catalog no. M1404), 0.5 mM colchicine (Sigma-Aldrich; catalog no. C9754), or 1 µM vincristine on a microscope stage set at 37°C with 5% CO₂ for live-cell imaging. A time-lapse movie was recorded for microtubule depolymerization events on a Nikon TIRF microscope with a 1–3-s frame interval. More than 30-min movies were acquired for nocodazole and colchicine-treated cells, and 5–15-min movies were acquired for vincristine-treated cells to observe near-complete depolymerization events of microtubules. Cells were analyzed manually for plotting the parameters of polymerization, depolymerization, dynamic instability, and severing events from the complete frames of the movies. Graphs were plotted on GraphPad Prism6 software.

Bilayers were prepared by bursting liposomes on the glass surface using a glass-bottomed μ-Slide V1^0.5 chip from Ibidi. Also, 2.5 µL MgCl₂ at 500 mM was added into 122.5 µL buffer containing 50 mM HEPES (pH 7.4), 140 mM KCl, and 10% glycerol. Then, 125 µL extruded bilayer liposomes were added. Next, 60 µL MgCl₂-liposome solution was loaded into the channel of the ibidi chip and incubated for 40 min at room temperature. The channel was washed with the same buffer supplemented with 6 mM EDTA and then with buffer supplemented with 1 mM DTT. Depending on the purpose of experiments, MgCl₂ or CaCl₂ may be added in the buffer to the desired concentration. Then, 60 µL of 10 nM Munc13-1-Halo-Alexa488 were loaded into the channel and incubate with the bilayer for 60 min at room temperature. The channel was washed with the buffer supplemented with 1 mM DTT. The vesicle liposomes were diluted 30 times. Subsequently, 60 µL diluted vesicle liposomes were loaded into the channel and incubated for 5 min at room temperature. The channel was washed with buffer supplemented with 1 mM DTT.
The Ibidi chip was then mounted to the stage of a Nikon TIRF microscope. Bilayers, Munc13 particles on bilayers, and vesicles attached to bilayers were respectively imaged at room temperature with the TIRF microscope using the corresponding laser.

All experiments were conducted with a prism-type total internal reflection fluorescence (TIRF) microscope (Nikon) equipped with a 488-nm laser (Coherent Sapphire, 200 mW), a 561-nm laser (Coherent Sapphire, 200 mW), and two Andor iXon EMCCD cameras (28 (link),29 (link)). Flowcells and ssDNA curtains were prepared as previously described (28 (link),29 (link)). In brief, lipid bilayers were prepared with 91.5% DOPC, 0.5% biotinylated-PE and 8% mPEG 2000-DOPE. The ssDNA substrate was generated using rolling circle replication with a biotinylated primer, a circular M13 ssDNA template, and Phi29 DNA polymerase, as described (28 (link),29 (link)). The biotinylated ssDNA was injected into the sample chamber and attached to the bilayer through a biotin–streptavidin linkage. The flow cell was then attached to a microfluidic system and sample delivery was controlled using a syringe pump (Kd Scientific) (28 (link),29 (link)). For all two-color images, we used a custom-built shuttering system to avoid signal bleed-through during image acquisition. With this system, images from the green (GFP) and the red (mCherry) channels are recorded independently, these recordings are offset by 100 ms such that when one camera records the red channel image, the green laser is shuttered off and vice versa (28 (link),29 (link)).