Internalization of sterols upon inhibition of Arp2/3 activity. (A) Localization of mCherry-D4H and F-actin marker GFP–calponin homology domain (CHD) upon depolymerization of F-actin by latrunculin A (LatA, 200 µM) or Arp2/3 inhibition by CK-666 (500 µM). (B) Time-lapse sequence of mCherry-D4H after CK-666 addition (upper panel) and wash-out after 1 h treatment (lower panel). (C) Quantification of the number of internal D4H-positive structures in CK-666–treated cells imaged every 5 min. Values are normalized to the maximal number of internal D4H dots in the z-stack. Error bars represent SD of seven independently tracked cells. (D) WT cells become resistant to AmB upon CK-666 treatment. The graph shows the viability of cells pretreated with CK-666 (or DMSO as control) for 1 h and then treated with a high dose (4 µg/ml) of AmB for another hour. n = 3 experiments. (E) Loss of filipin staining, but not PM GFP-Tna1 after 1 h CK-666 treatment. (F) mCherry-D4H in arp2-1 mutant cells grown at 25°C or 36°C for 6 h. (G) mCherry-D4H in selected endocytic (end4Δ, end4Δtld pan1Δapw), actin cable (for3Δ), eisosome (pil1Δ), and ER-PM attachment (scs2Δ scs22Δ) mutants treated or not with CK-666 for 1 h. The bottom graph displays the number internal dots in untreated cells in z-stacks (n ≥ 30 cells per strain). (H) mCherry-D4H relocalization upon energy depletion. Left: Cells were treated for 1 h with deoxyglucose (DG) and antimycin to deplete energy, and then incubated for 1 h more with CK-666. Right: Cells were precooled on ice for 30 min and then treated with CK-666 for 1 h on ice. Error bars show SD. Scale bars, 2 µm. CFU, colony-forming units.
Figure 2.

Internalization of sterols upon inhibition of Arp2/3 activity. (A) Localization of mCherry-D4H and F-actin marker GFP–calponin homology domain (CHD) upon depolymerization of F-actin by latrunculin A (LatA, 200 µM) or Arp2/3 inhibition by CK-666 (500 µM). (B) Time-lapse sequence of mCherry-D4H after CK-666 addition (upper panel) and wash-out after 1 h treatment (lower panel). (C) Quantification of the number of internal D4H-positive structures in CK-666–treated cells imaged every 5 min. Values are normalized to the maximal number of internal D4H dots in the z-stack. Error bars represent SD of seven independently tracked cells. (D) WT cells become resistant to AmB upon CK-666 treatment. The graph shows the viability of cells pretreated with CK-666 (or DMSO as control) for 1 h and then treated with a high dose (4 µg/ml) of AmB for another hour. n = 3 experiments. (E) Loss of filipin staining, but not PM GFP-Tna1 after 1 h CK-666 treatment. (F) mCherry-D4H in arp2-1 mutant cells grown at 25°C or 36°C for 6 h. (G) mCherry-D4H in selected endocytic (end4Δ, end4Δtld pan1Δapw), actin cable (for3Δ), eisosome (pil1Δ), and ER-PM attachment (scs2Δ scs22Δ) mutants treated or not with CK-666 for 1 h. The bottom graph displays the number internal dots in untreated cells in z-stacks (n ≥ 30 cells per strain). (H) mCherry-D4H relocalization upon energy depletion. Left: Cells were treated for 1 h with deoxyglucose (DG) and antimycin to deplete energy, and then incubated for 1 h more with CK-666. Right: Cells were precooled on ice for 30 min and then treated with CK-666 for 1 h on ice. Error bars show SD. Scale bars, 2 µm. CFU, colony-forming units.

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