BAR scaffolds drive membrane fission by crowding disordered domains

Wilton T. Snead; Wade F. Zeno; Grace Kago; Ryan W. Perkins; J Blair Richter; Chi Zhao; Eileen M. Lafer; Jeanne C. Stachowiak

A custom-built TIRF microscope was used to collect time-lapse movies of live cells. A 532 nm laser was used to excite mCherry, and a 635 nm laser was used for autofocus. An Olympus IX73 microscope body was equipped with a Photometrics Evolve Delta EMCCD camera and a Zeiss plan-apochromat 100x 1.46 NA oil immersion TIRF objective. The objective was heated to 37 °C using a Pecon TempController 2000-2 objective heater. The emission filter for the 532 nm laser was a dual bandpass filter centered at 583 nm with 37 nm width and 707 nm with 51 nm width, which minimized signal from the autofocus laser. Movies were collected at the plasma membrane just above the coverslip surface in 2 s intervals for 120 frames. Tube lifetimes and intensities were quantified from TIRF movies. Only movies of cells with similar expression level, acquired under identical imaging settings, were used for analysis. For the tube lifetime analysis in Fig. 4F , only tubes which appeared within the time course of imaging and departed before the end of the time course were included. For the tube intensity analysis, a single frame in the movie with the maximum number of tubes was selected, and the average tube intensity was measured along a straight line drawn on the tube. The mean intensity along an identical line on either side of the tube was also measured, and these values were averaged to estimate the local background intensity of the tube. The protein enrichment on the tube was then quantified as the ratio of the tube intensity to the local background, after subtracting the camera noise background from both values.