Technical Reference #1601

1601.

Functional implications of the influence of ABCA1 on lipid microenvironment at the plasma membrane: a biophysical study Zarubica; A.; Plazzo; A. P.; Sto¨ckl; M.; Trombik; T.; Hamon; Y.; Mu¨ller; P.; Pomorski; T.; Herrmann; A.; Chimini; G., Universite de la Mediterranee, The FASEB Journal, 23(1601), (2009)
Link To Paper

Abstract:
The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release ofcholesterol to plasmatic acceptors. The molecularmechanism is however unknown. We report here onthe biophysical analysis in living HeLa cells of theABCA1 li

Keywords:
FLIM; TfR; cholesterol; phosphatidylserine; FRAP

Materials & Methods:
Chemicals All chemicals for sample preparation as well as all solvents (CHCl3 CH3OH and CH3CN) were obtained in highest commercially available purity from Fluka (Taufkirchen Germany) and used as supplied. Plasmids cell culture and transfection HeLa Tet off cells (Clontech Mountain View CA USA) were grown in Dulbecco modified Eagle medium (DMEM) 10% fetal calf serum (Gibco Life Technologies Carlsbad CA USA) penicillin streptomycin and 1% sodium pyruvate (all from Invitrogen Karlsruhe Germany). Jurkat cells were grown in suspension below a concentration of 2 105 cells/ml in RPMI 1640 medium supplemented with 2 mM l-glutamine (both Biochrom Berlin Germany) 2 g/L sodium pyruvate and 10% fetal calf serum. Plasmids containing ABCA1 or its variants fused with enhanced green fluorescent protein (EGFP) enhanced yellow fluorescent protein (EYFP) or enhanced cyan fluorescent protein (ECFP) were generated in pBI vector (Clontech; ref. 12). Ced-7 cDNA (kindly provided by R. Horvitz Massachusetts Institute of Technology Boston MA USA) was engineered to include an EGFP tailpiece in pBI (24). All constructs were verified by sequencing. Transient transfections were performed on a 60% confluent monolayer of HeLa cells with 5 g of DNA in EXGEN 500 (Euromedex Souffelweyersheim France) (12). Transfection efficiency monitored at 24 h post-transfection by flow cytometry was consistently at 40%. Multiple simultaneous transfections were performed with 5 g of ABCA1 EYFP or its mutant (ABCA1MM) and 1.5 g of cationic probes (a kind gift of S. Grinstein University for Sick Children Toronto ON Canada). For fluorescence recovery after photobleaching (FRAP) experiments 5 g of ABCA1- ECFP or its mutant and 1 g of human transferrin receptor (hTfR) grafted with EGFP [a kind gift of D. Marguet Centre d’Immunologie de Marseille Luminy (CIML) Marseille France] were cotransfected. Surface binding of annexin V and ApoA-I was carried out at 60 h post-transfection as described previously (11). For treatment of Jurkat cells 0.5 106 cells were pelleted (5 min; 200 g). For activation with OKT3 antibody the pellet was resuspended in 25 l medium and 25 l OKT3 for 20 min at 37°C washed and incubated with 200 l medium containing 4 l TRITC-labeled anti-mouse IgG (diluted 1:100 Sigma Taufkirchen Germany) for 10 min. For sphingomyelinase (SMase) treatment the pellet was resuspended in 200 l medium containing 0.1 U/ml sphingomyelinase (from Staphylococcus aureus; Sigma) and incubated for 30 min at 37°C. Jurkat cells and the OKT3 antibody were kindly provided by Christian Freund (FMP Berlin Germany). Antibodies The molecules were detected with antibodies purchased as follows: flotillin-1 caveolin and LAMP 2 (BD Biosciences; Le Pont de Claix France; Pharmingen San Diego CA USA); -COP (Sigma); and calnexin (Stressgen Ann Arbor MI USA). Antibodies against hTfR were a kind gift of P. Pierre (CIML Marseille France). Protein blots were probed with horseradish perioxidase-conjugated secondary antibodies (Jackson Research Laboratories West Grove PA USA) and detected with ECL reagents (Amersham Little Chalfont UK). Giant plasma membrane vesicles (GPMVs) GPMVs or blebs were prepared from almost confluent HeLa cells either untransfected or expressing ABCA1 or ABCA1MM fused with EGFP or ECFP by chemically induced vesiculation as described previously (25 26). Cells grown in T25 flasks were washed twice with GPMV buffer (2 mM CaCl2 10 mM HEPES and 0.15 M NaCl pH 7.4). To each flask 1.5 ml of freshly prepared GPMV reagent (25 mM formaldehyde and 2 mM dithiothreitol in GPMV buffer) was added. Flasks were incubated for 1 h at 37°C while being slowly shaken (60–80 cycles/min). After incubation GPMVs that had detached from the cells were gently decanted into a conical glass tube and allowed to settle on ice. Before imaging R18 (octadecylrhodamin- B-chlorid; Invitrogen Karlsruhe Germany) was added at a concentration of 0.5 M to the GMPV suspension. Images of the equatorial plane of the blebs were taken at 25°C with temperature controlled with a water circulating bath. The relative domain size was calculated by approximating the vesicle to a circumference and calculating the length of the cord covered by the liquid-disordered (Ld)-like phase where 1 is the length of the whole circumference.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HeLa