Figure 8.
Assembly of actin patches on the internal membranes containing unrecycled Snc1p. (A) Localization of Abp1p-EGFP and mRFP1-Snc1p in the cdc50Δ erg3Δ mutant. The strain KKT158 (PGAL1 -3HA-CDC50 erg3Δ ABP1-EGFP) harboring pRS416-mRFP1-SNC1 was cultured in SDAW-Ura at 30°C for 12 h, followed by confocal microscopic observation. To distinguish between Abp1p-EGFP patches that appear to function in the normal endocytic pathway and those assembled intracellularly, the cell periphery and a position 500 nm distant from the plasma membrane are indicated with solid and broken lines, respectively (see text). Images are central focal planes. Bar, 5 μm. (B) The proportion of intracellular Abp1p-EGFP patches that showed colocalization with Snc1p structures. Intracellular actin patches (218 patches from 25 cells) were categorized according to their colocalization patterns with mRFP1-Snc1p. (C) Serial sections of an Abp1p-EGFP patch that was not colocalized with the Snc1p structure in one focal plane. Lower images represent four consecutive z-focal planes of an Abp1p-EGFP patch that did not show colocalization with mRFP1-Snc1p in a central focal plane (a square of the top merged image and the left most image of bottom images). These images were acquired in 2 s during which significant movement of actin patches was not observed. Representative images of 30 individual actin patches examined are shown. Cortical actin patches are distinguished as in A. Bars in top and bottom images are 5 μm and 100 nm, respectively.