Figure 7.

Actin is enriched at the vertices of docked vacuoles. Purified vacuoles were either docked by the physiological Ypt7p-dependent pathway or clustered by cosedimentation as described in Wang et al. (2002). (A) Rhodamine-actin (4 μM, total rabbit muscle actin) was added at the beginning of the reaction, or Alexa Fluor 488–DNaseI (0.2 μM) was added to docked or cosedimented vacuoles (B). After a 30-min incubation at 27°C, vacuoles were fixed with 1% formaldehyde for 20 min at room temperature, sedimented at 3000 g for 2 min, resuspended in PS buffer, and subjected to microscopic analysis (as described in Materials and methods). Ratiometric images of representative clusters are shown. (C) Cumulative distribution plots of the ratios of pixel values (rhodamine:MDY-64 lipid label) obtained for each morphometric domain (vertex, boundary, and outside edge midpoints) and each treatment. Each curve represents an average of 410 individual ratio measurements on 110–115 vacuole clusters from three independent experiments.