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Results: 11

1.
FIGURE 1.

FIGURE 1. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Chemical structures of ATLs and edelfosine fluorescent analogues. Chemical structures of edelfosine (EDLF or ET), the related natural compound lysophosphatidylcholine (LysoPC), fluorescent edelfosine analogues PTE-ET, PTRI-ET, Et-BDP-ET, and Yn-BDP-ET, and the ATLs miltefosine (MLTF), and perifosine (PRIF).

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
2.
FIGURE 9.

FIGURE 9. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Edelfosine resistance of vps29Δ and vps35Δ mutants. Growth curves of wild-type (BY4741), vps29Δ (vps29), vps35Δ (vps35) knock-out mutants, and mutant strains harboring the corresponding cognate genes (vps29-YCplac111-VPS29 and vps35-YCplac111-VPS35) in SC medium containing 60 μm edelfosine. Data shown are mean values ± S.D. of at least six independent experiments.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
3.
FIGURE 10.

FIGURE 10. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Pma1p and edelfosine localization in wild-type S. cerevisiae and retromer mutants following drug addition. a, edelfosine-resistant retromer mutants vps29Δ and vps35Δ show the same pattern of ER localization as wild-type cells (WT) after treatment with PTE-ET. b, retromer mutants vps17Δ, vps29Δ, and vps35Δ show decreased vacuolar accumulation of Pma1p-GFP relative to wild-type cells (WT) after edelfosine (EDLF) treatment, as shown by the vacuole-accumulating stain FM4-64. DIC, differential interference contrast. Data shown are representative of three independent experiments.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
4.
FIGURE 3.

FIGURE 3. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Uptake of fluorescent analogues of edelfosine depends on LEM3. a, wild-type S. cerevisiae cells were incubated with the indicated fluorescent edelfosine compounds and imaged. b, lem3Δ cells were incubated and imaged as above. Because little to no intracellular fluorescence was observed in lem3Δ cells, higher exposition times had to be used, which accounts for the increased noise. Images shown are from representative experiments repeated at least three times. DIC, differential interference contrast.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
5.
FIGURE 4.

FIGURE 4. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Localization pattern of different fluorescent analogues of edelfosine in the endoplasmic reticulum of S. cerevisiae. a, PTE-ET localizes in the endoplasmic reticulum as shown by the organelle marker Elo3p tagged with GFP. b, same distribution pattern can be observed with PTRI-ET. c and d, Et-BDP-ET and Yn-BDP-ET also localize in the endoplasmic reticulum as assessed by their visualization around the nucleolus marker Sik1p tagged with red fluorescent protein (RFP) and close to the vacuole, as seen by differential interference contrast (DIC).

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
6.
FIGURE 5.

FIGURE 5. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Quantification of S. cerevisiae cells showing co-localization of PTE-ET with the ER markers Elo3p and Sec63p. a, cells carrying a Sec63-GFP (green fluorescence)-bearing plasmid, as a marker for ER, were incubated with PTE-ET (pseudo-colored red) and imaged. Areas of co-localization between ER and PTE-ET in the merge panels are yellow. The corresponding light microscopy images are also shown. b, fluorescence microscopy images of PTE-ET and the ER markers Elo3p-GFP and Sec63p-GFP were examined, and stained cells were quantitated for the subcellular localization of the edelfosine fluorescent analogue. For each experiment, 150–300 stained cells were analyzed. Data shown are representative or mean values ± S.D. of at least three independent experiments.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
7.
FIGURE 8.

FIGURE 8. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Effect of ATLs on Pma1p localization following ATL treatment in S. cerevisiae. a, association of Pma1p with detergent-resistant membranes from wild-type yeast cells untreated (Control) or treated with 15 μm edelfosine (EDLF), 2.5 μm miltefosine (MLTF), or 3 μm perifosine (PRIF) for 2 h in defined medium. Lipid raft isolation was performed by using Optiprep gradients. b, percentages of Pma1p associated with fraction 2 (containing detergent-resistant membranes) were determined by densitometry using ImageJ. c, fluorescence microscopy of a yeast strain expressing Pma1-GFP untreated (Control) or treated with the indicated ATLs as above. Data shown are representative of three independent experiments performed. DIC, differential interference contrast.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
8.
FIGURE 6.

FIGURE 6. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Edelfosine and Pma1p subcellular localization following drug treatment. a, edelfosine fluorescent analogue PTE-ET co-localizes with the tagged ER marker protein Elo3p-GFP. b, influence of decreased endocytosis on drug uptake was assayed by comparing end4 pep4Δ cells (kept at the semi-permissive temperature of 25 °C) and wild-type cells switched to 4 °C with wild-type cells at 30 °C. Cells were incubated in the presence of the endocytic markers lucifer yellow (LY) and FM4-64, and afterward with PTE-ET. c, GFP-tagged Pma1p is internalized to the vacuole after edelfosine treatment (EDLF) at 30 °C, and at a temperature blocking endocytosis (4 °C), this process, unlike drug uptake (see b), is impaired. Images shown are from representative experiments repeated three times. DIC, differential interference contrast.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
9.
FIGURE 7.

FIGURE 7. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Edelfosine and Pma1p localization to lipid rafts following drug treatment of S. cerevisiae. Aliquots of sucrose gradient fractions for the isolation of lipid rafts from membrane-enriched samples were analyzed for [3H]edelfosine (a) and Pma1p (b) distribution. Adjoining figures represent fractions of the same samples. a, [3H]edelfosine distribution in fractions of wild-type cells after drug treatment for 15 min (WT 15 min) and 360 min (WT 360 min). Underlined fractions correspond to lipid rafts. Drug accumulation in lipid raft fractions is even more remarkable in drug-resistant end 4pep4Δ cells (end4 pep4Δ 360 min). Inset, Western blot of the raft protein Pma1p in untreated wild-type yeast (WT 0 min), identifying fractions 4–6 as raft-enriched fractions. The position of Pma1p is indicated by an arrow. b, Pma1p (arrow) and Gas1p (arrowhead) distribution in sucrose gradient fractions obtained from the isolation of lipid rafts. Edelfosine alters Pma1p distribution relative to 15-min controls, decreasing its presence in lipid raft fractions. The resistant end4 pep4Δ strain manages to keep Pma1p in the edelfosine-enriched lipid rafts. Data shown are representative of three experiments performed.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
10.
FIGURE 2.

FIGURE 2. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Edelfosine-resistant S. cerevisiae screen and mutants affecting drug uptake. a, toxicity threshold of two wild-type S. cerevisiae strains (haploid BY4741 and diploid BY4743) and three single deletion mutants known to exert resistance to edelfosine following 48 h of incubation. b, growth of the complete set of haploid S. cerevisiae yeast deletion mutants in the presence of 60 μm edelfosine (EDLF). Each dot (red, deletion mutant; blue, wild-type) represents the growth of each yeast strain in the presence of 60 μm edelfosine for 72 h. The dashed line delineates strains considered resistant to edelfosine. c, functional distribution of the 262 genes found to cause resistance to edelfosine when deleted. d, uptake of [3H]edelfosine for three resistant strains found to have decreased drug incorporation relative to the wild type (WT, black solid bar). Each pair of bars represents a single gene deletion mutant (black patterned bar) alongside that mutant complemented by a centromeric plasmid carrying said gene (white patterned bar). Data shown are representative or mean values ± S.D. of at least three independent experiments.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.
11.
FIGURE 11.

FIGURE 11. From: Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

Proposed model for the mechanism of edelfosine cytotoxicity. a, essential proton pump Pma1p is associated with plasma membrane lipid rafts. b, edelfosine treatment causes the lipid raft to become disorganized. Edelfosine interacts with its core component ergosterol, and Pma1p dissociates from the raft microdomains. c, edelfosine induces internalization and vacuole-dependent degradation of Pma1p. Deletion of ESCRT-complex genes causes resistance to edelfosine. Pma1p could thus be degraded by ubiquitination (1), internalization by endocytosis (2), recognition of ubiquitin moiety by the ESCRT complex (3), leading to recycling of the ubiquitin and enclosement of Pma1p in lumenal vesicles of the MVB (4). Fusion of the MVB with the vacuole would lead to degradation of these Pma1p-containing vesicles (5). Vacuolar hydrolases could also degrade Pma1p. The retromer complex is essential for the effect of the drug. We postulate this complex is either withdrawing Pma1p from the endosome to the Golgi apparatus (GA) via retrograde transport (6) or is allowing the delivery of lysosomal hydrolases essential for the degradation of Pma1p (not shown). If any of these degradation pathways are impaired, a higher quantity of Pma1p could be available for recycling to the plasma membrane (7). d, both ergosterol and edelfosine are internalized. The drug accumulates in the ER, and ergosterol is directed to some hitherto uncharacterized compartment in the cytoplasm. e, pH homeostasis in physiological conditions is tightly controlled by Pma1p-mediated proton extrusion, V-ATPase proton sequestering, and mitochondrion-mediated proton pumping. Edelfosine decreases the availability of the first buffering mechanism, causing acidification. When edelfosine is added to cells lacking a functional V-ATPase, the loss of functional Pma1p leads to a greater cytosolic acidification resulting in hypersensitivity to the drug.

Álvaro Cuesta-Marbán, et al. J Biol Chem. 2013 March 22;288(12):8405-8418.

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