Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 1 -
  Caveolin transport during oocyte ovulation and early embryonic development. C. elegans stably expressing GFP-caveolin (a cargo of the ESCRT-mediated degradation pathway) in the germline were imaged live by confocal microscopy using a swept-field confocal microscope (Ti-E; Nikon). The majority of GFP signal is lost before the two-cell stage as a result of ESCRT-mediated transport of caveolin into the lysosome lumen, as documented previously (Wang and Audhya, 2014). Frames were taken every minute for 153 min.
• Video 2 -
  Caveolin degradation is impaired in embryos depleted of Vps32. C. elegans stably expressing GFP-caveolin (a cargo of the ESCRT-mediated degradation pathway) in the germline were imaged live by confocal microscopy using a swept-field confocal microscope (Ti-E; Nikon) after RNA interference–mediated depletion of Vps32. The GFP signal is stabilized in developing embryos lacking Vps32 as a result of a defect in ESCRT-mediated trafficking of caveolin to the lysosome lumen. Frames were taken every minute for 123 min.
• Video 3 -
  Docking of a Vps32 subunit onto the EM isosurface profile that was determined after two-dimensional class average analysis of Vps32 spiral filaments. This video simulation shows several attempts to dock a pseudoatomic structure of a Vps32 monomer onto the EM isosurface determined by cryo-EM experiments of Vps32 filaments.
• Video 4 -
  Tomogram of an individual spiral composed of truncated Vps32^1–190. Samples of purified, negatively stained Vps32^1–190 were imaged by electron tomography using an FEI tomography holder with maximum tilt angles of ±60° (Tecnai G² F30; FEI). Each frame corresponds to a digital section of the tomogram moving along the tomographic z axis (1° increments).
• Video 5 -
  Tomogram of a spiral pair composed of truncated Vps32^1–190. Samples of purified, negatively stained Vps32^1–190 were imaged by electron tomography using an FEI tomography holder with maximum tilt angles of ±60° (Tecnai G² F30; FEI). Each frame corresponds to a digital section of the tomogram moving along the tomographic z axis (1° increments).
• MATLAB scripts -
  A ZIP file is also provided that includes MATLAB scripts for particle picking, spiral interpolation, and spiral straighten developed for analysis of Vps32 filaments.