Lipid Lateral Organization on Giant Unilamellar Vesicles Containing Lipopolysaccharides  Jakubs Kubiak, Jonathan Brewer, Søren Hansen, Luis A. Bagatolli  Biophysical Journal  Volume 100, Issue 4, Pages 978-986 (February 2011) DOI: 10.1016/j.bpj.2011.01.012 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Molecular structure of LPS-Re of E. coli (left). Schematic representation of the different LPSs used in this work (right). Gal, galactopyranose; Glc, glucopyranose; GlcN, 2-amino-2-deoxyglucopyranose; Hep, L-glycero-α-D-manno-heptopyranose; Kdo, 3-deoxy-α-D-manno-oct-2-ulopyranosonic acid; P, phosphate. For more details, see Caroff and Karibian (2). Biophysical Journal 2011 100, 978-986DOI: (10.1016/j.bpj.2011.01.012) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 Summary of the LPS incorporation procedure for four studied LPS strains. The x axis represents the starting LPS concentration as a fraction of the total amount of lipids in the sample. The y axis shows the concentration of LPS in the oligolamellar vesicles as a fraction of the total amount of lipids in the samples after completion of the purification procedure. The solid black line indicates an ideal 100% incorporation efficiency. These vesicles were used to form GUVs. The efficiency of LPS incorporation increases as the length of the polysaccharide decreases. Error bars are standard deviations. Biophysical Journal 2011 100, 978-986DOI: (10.1016/j.bpj.2011.01.012) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 Representative images of GUVs (false color representation) composed of E. coli polar lipid extract and LPSs: (A) LPS-smooth, (B) LPS-Ra, (C) LPS-Rc (above 10 mol %), (D) LPS-Rd (above 10 mol %), and (E) lipid A (above 5 mol %). The upper row shows fluorescence images (false color representation) of GUVs labeled with rhodamine-DHPE. The bottom row displays Laurdan GP images of the GUVs. For LPS-smooth (A) and LPS-Ra (B), no visible phase separation can be seen in the GUVs at any of the explored concentrations (up to 15 mol %). For deep rough LPS strains (Rc (C) and Rd (D)), the coexistence of domains of high GP values (close to 0.6 indicating presence of a gel-like structure) is observed above 10 mol %. Scale bars are 10 μm. Biophysical Journal 2011 100, 978-986DOI: (10.1016/j.bpj.2011.01.012) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Laurdan GP values of LPSs containing membranes as a function of LPS concentration: (A) LPS-smooth, (B) LPS-Ra, (C) LPS-Rc, (D) LPS-Rd, and (E) lipid A. (○ and ●) Laurdan GP values calculated from GUVs. Double points at high LPS concentration for C–E represent visible phase separation GUVs. These Laurdan GP values were calculated separately for each domain, liquid-like (○) and gel-like (●). Each point represents an average of 15–25 separate vesicles; error bars are standard deviations. (Δ) Laurdan GP values from the oligolamellar vesicles used to form GUVs (measured in a fluorometer in the concentration range where domains are not observed in the GUVs). Biophysical Journal 2011 100, 978-986DOI: (10.1016/j.bpj.2011.01.012) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 5 FCS experiments performed in GUVs containing LPS-smooth and LPS-Ra, respectively, doped with fluorescently labeled Alexa 488 LPS-smooth (0.001 mol % with respect to total lipids). The figure shows the corresponding LPS diffusion coefficient measured in GUVs (each point is an average of 15–20 separate vesicles; error bars are standard deviations). No microscopic phase separation was seen in these samples with either Laurdan GP or rhodamine-DHPE. Biophysical Journal 2011 100, 978-986DOI: (10.1016/j.bpj.2011.01.012) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 6 Sketch representing the different LPS-rich domain sizes observed in our experiments. LPS chemotypes (A: LPS-smooth and LPS-Ra; B: Rc and Rd2 (deep rough chemotypes of LPS)) are inserted into the lipid membrane (PE and PG, cardiolipin not shown). (A) The lateral organization of LPS-smooth (valid also for LPS-Ra) is represented for a low concentration of this lipid (the LPS domains in the sketch correspond to a diffusion twofold lower than that of the single LPS and are nanoscopic; see section 3 of the Supporting Material). (B) Formation of large membrane platforms (micrometer-sized) for the rough LPS chemotypes above 10 mol % LPS concentration. Biophysical Journal 2011 100, 978-986DOI: (10.1016/j.bpj.2011.01.012) Copyright © 2011 Biophysical Society Terms and Conditions