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Supplementary Information Lipid Phase Separation and Protein-Ganglioside Clustering in Supported Bilayers Are Induced by Photorelease of Ceramide ǂ Daniel M. Carter Ramirez, a,b Young Ah Kim, c Robert Bittman, c and Linda J. Johnston a* a Measurement Science and Standards, National Research Council of Canada, Ottawa, ON K1A 0R6, CANADA b Department of Chemistry, University of Ottawa, Ottawa, ON K1N 6N5, CANADA c Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing, NY 11367, USA Electronic Supplementary Material (ESI) for Soft Matter This journal is © The Royal Society of Chemistry 2013

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Page 1: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Supplementary Information

Lipid Phase Separation and Protein-Ganglioside Clustering in Supported

Bilayers Are Induced by Photorelease of Ceramideǂ

Daniel M. Carter Ramirez,a,b Young Ah Kim,c Robert Bittman,c and Linda J. Johnstona*

a Measurement Science and Standards, National Research Council of Canada, Ottawa, ON K1A

0R6, CANADA b Department of Chemistry, University of Ottawa, Ottawa, ON K1N 6N5, CANADA

c Department of Chemistry and Biochemistry, Queens College of The City University of New

York, Flushing, NY 11367, USA

Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 2: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S1. Supported bilayers prepared from POPC/C16-Cer (9:1 molar ratio, labeled with 0.5 mol % DiI-C20) are stable to UV irradiation. Fluorescence images were recorded: (A) before UV irradiation; (B) aperture adjusted to irradiate a localized area of the bilayer; (C) after 10 min UV irradiation. Scale bar = 5 μm.

A B C

Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 3: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S2. Photolysis of 1 in POPC SUVs was conducted with 350 nm irradiation. Consumption of 1 was monitored by HPLC analysis of the caged coumarin’s fluorescence. (A) POPC/1 in 9:1 a molar ratio; (B) POPC/1 in a 98:2 molar ratio.

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Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 4: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S3. A POPC/1 supported bilayer labeled with 0.5 mol % DiI-C20 shows no phase separation before photolysis. (A) T0 min fluorescence image and (B) AFM scan. (C) T20 min post-AFM image. The bright features due to adsorbed vesicles show a different pattern after the AFM scan. Scale bar = 5 μm.

A B C

4 nm0 nm

Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 5: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S4. Domain disappearance as a function of pattern size. Each region of the irradiated bilayer shown in Figure 4 was re-imaged 5 min after the end of the photolysis. The images shown below are the post-UV images for the following irradiation times: (A) 2.5 s; (B) 5 s; (C) 15 s; (D) 30 s; (E) 1 min. The rate of domain disappearance increases with increasing irradiation time. Scale bar = 10 µm.

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Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 6: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S5. Photorelease of C16-Cer in a POPC/1 supported bilayer (95/5 molar ratio + 0.5 mol% DiI-C20) also results in the formation of ceramide-rich domains. (A) T0 min before UV; (B) closed iris showing the area to be irradiated; (C) following 5 s UV exposure; (D) T5 min post-UV. Scale bar = 5 μm. A B

C D

Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 7: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S6. Photorelease of C16-Cer in a POPC/1 supported bilayer (98/2 molar ratio + 0.5 mol% DiI-C20) does not afford ceramide-rich domains. (A) T0 min before UV; (B) closed iris showing the area to be irradiated; (C) following 5 s UV exposure; (D) 3 min post-UV. Scale bar = 5 μm.

A B

C D

Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 8: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

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Fig. S7. Correlated fluorescence and AFM imaging of a POPC/1 supported bilayer labeled with 0.5 mol % DiI-C20 after photolysis of the entire membrane surface confirms that the dye-excluded domains are stable. (A) T0 min before photolysis; (B) T52 min a second area of the bilayer following 30 min UV exposure of the entire sample; (C) T62 min AFM confirms the presence of gel-phase domains; (D) T75 min a fluorescence image taken following two consecutive AFM scans indicates a stable domain pattern. Scale bar = 5 μm.

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Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013

Page 9: Supplementary Information Lipid Phase Separation and ... · 0.5 1.0 1.5 0.0 1.0 2.0 3.0 4.0 5.0 Height (nm) Horizontal Distance (µm) Fig. S7. Correlated fluorescence and AFM imaging

Fig. S8. Photolysis of a POPC/1/GM1 bilayer (90/10/1 molar ratio labeled with 0.5 mol % DiI-C20) induces comparable morphological changes to those observed in the absence of GM1. (A) T0 min before UV; (B) closed aperture identifying the area to be irradiated; (C) following 2.5 s of UV exposure small, dye-excluded Cer-rich domains are observed in the area exposed to UV light; (D) T5 min the dark domains have completely disappeared; (E) a second 2.5 s photolysis in the same area recreates dark domains, most of which correspond in size and position to those in C. As before the domains disappear within minutes (not shown); (F) T20 min a similar distribution of domains is reproduced after a third 2.5 s photolysis. Scale bar = 5 μm.

A B C

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Electronic Supplementary Material (ESI) for Soft MatterThis journal is © The Royal Society of Chemistry 2013


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