First, we followed membrane internalization and vesicle-based transport to the vacuole using FM4-64, a lipophilic styryl dye that incorporates into the cell membrane, is internalized and reaches the vacuole through an energy- Tariquidar ic50 and temperature-dependent

transport mechanism. After 90 min in non-treated wild-type yeast cells, FM4-64 was entirely internalized and labelled the limiting vacuolar membrane (Figure 9A). Yeast cells treated with 60 μM dhMotC for 90 min were deficient in vesicle transport to the vacuole, as shown by residual fluorescent staining at the cellular membrane and accumulation of FM4-64 in small cytoplasmic vesicles (Figure 9A). Figure 9 DhMotC selleck chemicals interferes with endocytosis in yeast. Cells exposed to (A) FM4-64, a fluorescent endocytic marker staining the vacuolar CA4P solubility dmso membrane; (B) Lucifer yellow (LY), a fluid-phase endocytic marker accumulating in the vacuole. Cells were incubated with FM4-64 or LY in the presence of DMSO or 60 μM dhMotC and visualized after 90 min chase by fluorescence and phase contrast (PC) microscopy. In a second assay, we monitored the delivery of Lucifer yellow (LY),

a marker for fluid-phase endocytosis that accumulates in the vacuolar lumen. LY cannot cross biological membranes and, as a consequence, accumulation in the vacuole depends on vesicular transport. Untreated yeast cells displayed bright fluorescent 17-DMAG (Alvespimycin) HCl staining of the vacuole by accumulated LY, whereas after 30 min of treatment with 60 μM dhMotC, LY failed to enter the cells and could only be detected as weak staining at the plasma membrane (Figure 9B). The results from the FM4-64 and LY assays confirm

that dhMotC interferes with endocytosis. As mentioned, killing of yeast by dhMotC depends on the presence of functional mitochondria. To test whether the disruption of endocytosis in drug-treated yeast cells was also mitochondria-dependent, we used the FM4-64 assay to monitor endocytosis in ρ 0 petite mutants. We observed a disruptive effect of dhMotC on endocytosis in both ρ + and ρ 0 cells (data not shown). Based on these results we concluded that, unlike death induced by dhMotC, inhibition of endocytosis did not require functional mitochondria. We next examined whether motuporamines also inhibit intracellular membrane trafficking in cancer cells by examining effects on the internalization and degradation of epidermal growth factor (EGF) and its receptor (EGFR). Binding of EGF to EGFR at the plasma membrane leads to dimerization of EGFR, stimulation of its tyrosine kinase activity and initiation of downstream signaling cascades. The ligand-receptor complex is then downregulated via endocytosis and intracellular delivery to lysosomes for degradation [34]. MDA-MB-231 cells were incubated with fluorescently labelled EGF (FITC-EGF) for 1 h at 4°C, to enable binding of the ligand to its cell surface receptor.