Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis - PubMed (original) (raw)

. 2016 Oct;17(10):1139-49.

doi: 10.1111/tra.12425. Epub 2016 Aug 8.

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Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis

Sarah R Elkin et al. Traffic. 2016 Oct.

Abstract

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low-density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 μm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.

Keywords: IC50; caveolae-mediated endocytosis; clathrin-independent endocytosis; mammalian cells.

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no conflicts of interests.

Figures

Figure 1

Figure 1. Ikarugamycin inhibits clathrin-mediated endocytosis

A) A semi-log plot of the effect of increasing concentrations of IKA on TfnR uptake in H1299 cells. IKA has an IC50 value of 2.7 µ

m

± 0.3. B) Effect of IKA on Tfn uptake in several different cell lines. The indicated cells were preincubated with 4 µ

m

IKA before uptake of TfnR was measured. Results are reported as amount of TfnR internalized after 5 min at 37°C as a % of untreated control cells. C) Under the same conditions as (B) IKA inhibits uptake of several CME specific receptors: TfnR (H1299), EGFR (H1299) and LDLR (ARPE-19). All experiments represent (n = 3, avg. ± SD).

Figure 2

Figure 2. Ikarugamycin is selective for CME

The effect of increasing concentrations of IKA on caveolae-mediated endocytosis (●), using albumin as ligand, clathrin-independent endocytosis using either CD44 (▲) or CD59 (■) as markers, or clathrin-mediated endocytosis using TfnR (□) as marker in H1299 cells pretreated for 3 h. Data are normalized to the extent of internalization after 5 min in control cells, which was 69 ± 24% for albumin, 23 ± 13% for CD44, 27 ± 16% for CD59 and 109 ± 28% for TfnR control. Two-tailed Student’s _t_-test were used to assess statistical significance. **p < 0.005; (n = 3, avg. ± SD).

Figure 3

Figure 3. Ikarugamycin inhibition of CME is rapid and partially reversible

A) H1299 cells were pretreated with 4 µ

m

IKA for increasing time and inhibition of TfnR uptake in the continuous presence of IKA was compared with control. B) Increasing concentrations of IKA were added without preincubation and TfnR uptake was measured after 5 min H1299 cells after 5 min in the continuous presence of IKA. C) H1299 cells were pretreated with 4 µ

m

IKA for increasing time periods then IKA was washed out for either 30 or 180 min. TfnR uptake was then measured and compared with control (no IKA treatment). All experiments represent (n = 3, avg. ± SD).

Figure 4

Figure 4. Ikarugamycin disrupts clathrin coated pit distributions

A) Immunofluorescence of control/untreated ARPE-19 cells or cells pretreated with 4 µ

m

IKA for 3 h probed with antibodies to CLC and AP2. B) Fractionation of ARPE-19 cells pretreated with 4 µ

m

IKA for 3 h or no pretreatment (control) probed for clathrin heavy chain (CHC) and AP1 and AP2, respectively. The cytosolic (C) fraction was diluted 4× compared with the membrane (M) fraction for western blotting. Na+/K+ ATPase and GAPDH were used as positive and negative controls respectively. C and D) Quantification of CHC and AP2 in membrane versus cytosolic fractions, respectively, plotted on a log scale as percent of total. Two-tailed Student’s _t_-test were used to assess statistical significance. *p < 0.05; **p < 0.005; ***p < 0.0005. All experiments represent (n = 3, avg. ± SD). E) Kymographs from time-lapse images of CLC in either ARPE-19 cells pretreated with 4 µ

m

IKA for 3 h or no pretreatment (control). Kymographs are 450 seconds and span 130 µm.

Figure 5

Figure 5. Ikarugamycin alters Golgi morphology

A) Immunofluorescence of ARPE-19 stably expressing eGFP-CLC either pretreated or not (control) with 4 µ

m

IKA for 3 h and probed with antibodies to adaptor protein 1 (AP1) and GM130. Scale bar = 25 µm. B) Electron microscopy (EM) images of either control or IKA treated cells. White arrows point to the Golgi in each condition. Scale bar = 0.5 µm.

Figure 6

Figure 6. The effect of ikarugamycin on cell viability

ARPE-19, HeLa and H1299 cells were treated with A) 4 µ

m

or B) 32 µ

m

IKA for increasing periods of time followed by measuring cell viability. All experiments represent (n = 3, avg. ± SD).

Figure 7

Figure 7. Ikarugamycin inhibition of CME is rapid and partially reversible at high concentrations

A) ARPE-19 cells or B) H1299 cells were pretreated with 10 µ

m

IKA for 15 min then IKA was either washed out for 3 h or not. TfnR uptake was then measured and compared with control (no IKA treatment). C) ARPE-19 or D) H1299 cells were treated with 10 µ

m

IKA for 15 min then IKA was washed out for 3 h or not followed by measuring cell viability. Two-tailed Student’s _t_-tests were used to assess statistical significance. *p < 0.05; **p < 0.005; ***p < 0.0005. All experiments represent (n = 3, avg. ± SD).

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References

    1. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422:37–44. - PubMed
    1. Cossart P, Helenius A. Endocytosis of viruses and bacteria. Cold Spring Harb Perspect Biol. 2014;6:a016972. - PMC - PubMed
    1. Mayor S, Parton RG, Donaldson JG. Clathrin-independent pathways of endocytosis. Cold Spring Harb Perspect Biol. 2014;6:a016758. - PMC - PubMed
    1. Cosson P, de Curtis I, Pouyssegur J, Griffiths G, Davoust J. Low cytoplasmic pH inhibits endocytosis and transport from the trans-Golgi network to the cell surface. J Cell Biol. 1989;108:377–387. - PMC - PubMed
    1. Heuser JE, Anderson RG. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989;108:389–400. - PMC - PubMed

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