Dioleoylphosphatidylcholine dopc

Dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), diphytanoylphosphatidylcholine (DPhPC), and dierucoylphosphatidylcholine (di(C22:1)PC) were purchased from Avanti Polar Lipids, Inc. Alabaster, AL. Gramicidin A (grA) was generously gifted from O. S. Andersen, Cornell University Medical College. Bovine brain tubulin was obtained from Cytoskeleton (Denver, CO, USA). The peptide corresponding to the α-tubulin H10 domain sequence (residues 328–346), VNAAIATIKTKRSIQFVDW, was synthesized in an FDA/CBER peptide synthesis core (White Oak, MD, USA) and dissolved in DMSO. All other chemicals were analytical grade.

Large unilamellar vesicles containing NBD-phospholipids were prepared as described^{29 (link)}. 1-palmitoyl-2-(6-NBD-aminocaproyl)-PE (NBD-PE), 1-palmitoyl-2-(6-NBD-aminocaproyl)-PC (NBD-PC), and dioleoylphosphatidylcholine (DOPC) were obtained from Avanti Polar Lipids (Alabaster, AL). Fluorescently labelled phospholipid internalization experiments were performed as described^{29 (link),30 (link)}. Briefly, cells were grown to early logarithmic phase in SDA-U medium at 30 °C in the dark. After dilution to 0.35 A600 ml^-1, cells were incubated for 60 min at 30 °C with liposomes containing 40% NBD-phospholipid and 60% DOPC at a final concentration of 20 μM in the dark or under BL. Cells were then suspended in cold SD containing 20 mM sodium azide and 2.5% bovine serum albumin (BSA), incubated for 20 min, and washed with PBS. Flow cytometry of fluorescently labelled cells was performed on a FACSCanto II cytometer [BD]. For investigation of time response, overnight cultures were diluted to 0.2 A600 ml^-1 and incubated for a total of 3 h in the indicated condition (dark, BL 0.5 h, BL 1.0 h, or BL 3.0 h), in which the last 1 h was incubated with the NBD-PE liposome. The experiments were performed in three biological replicates consisting of 10,000 cells per sample. Significance for Fig. 3 was determined using a one-way analysis of variance with a Tukey’s test.

Plasmids pCI-Luc and pEFGP-N1 were prepared as previously described12 (link). Plasmid pCI-Luc comprises the luciferase reporter gene sub-cloned into the eukaryotic expression vector pCI (Promega, Southampton, UK) with transcription driven by the cytomegalovirus immediate/early promoter-enhancer13 (link). The plasmid pEGFP-N1 was purchased from Clontech (Saint-Germain-en-Laye, France). Peptide K₁₆GACSERSMNFCG19 (link) was synthesised by Zinsser Analytic (Maidenhead, UK), and dissolved in endotoxin-free water (Sigma-Aldrich, Dorset, UK) to 10 mg/mL. Liposomes consisted of 1-Propanaminium, N,N,N-trimethyl-2,3-bis(11Z-hexadecenyloxy)-iodide (DHDTMA iodide; Avanti Polar Lipids; Alabama, USA) with either dioleoylphosphatidylethanolamine (DOPE; Avanti Polar Lipids; Alabama, USA) or dioleoylphosphatidylcholine (DOPC; Avanti Polar Lipids; Alabama, USA) formulated at a 1:1 weight ratio, dissolved in sterile water to give a 2 mg/mL liposome suspension.