Figure 7.

Motility of Late Endosomal Compartments after Filipin Treatment.

(A₁) and (A₂) Endosomes visualized by RabF2a-GFP. Endosomes moved actively in the apex and subapical region of growing root hairs (A₁). Filipin treatment for 15 min reduced movement of RabF2a-positive endosomes to Brownian motion only (arrowheads in [A₂]). Only a few individual endosomes continued to move in a restricted area and over short distances (arrow in [A₂]). Summary maximal projections from 15 serial confocal sections in fixed Z-position without averaging.

(B) Effect of filipin on the distribution of endosomal compartments visualized by the double FYVE-GFP construct. Endosomes are distributed mostly in the subapical region and in the cytoplasm-containing part of the root hair in control conditions (B). Distribution is not dramatically changed by filipin treatment (10 μg·mL⁻¹ filipin for 25 min). These compartments are not involved in the formation of filipin-positive apical complexes as documented in single a median section (B₁) and in whole z-projections (B₂).

(C) Confocal microscopy. Motility of endosomes visualized by the double FYVE-GFP construct after filipin treatment. Time-lapse imaging shows movement of FYVE-positive endosomes after complexation of sterols with filipin. Endosomes in the vicinity of the larger round vacuole reduced their movements (arrowheads). Endosomes arrived at the apical domain close to the region of clustered sterols became completely immobilized (arrows). Time is indicated in seconds.

Representative still image from Supplemental Movie 3 (DIC image in [A₁]), Supplemental Movie 9 (GFP image in A₁), Supplemental Movie 4 (DIC image in [A₂]), Supplemental Movie 10 (GFP image in [A₂]), Supplemental Movie 11 (GFP image in [B]), and sequence of images from Supplemental Movie 12 online (C). Bars = 5 μm in (A) to (C).