Supplemental Data


Abstract
Full Text
Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells
David A. Zacharias, Jonathan D. Violin, Alexandra C. Newton, and Roger Y. Tsien

Supplementary Material

Methods

Mutagenesis. Primer sequences used for mutagenesis with the QuickChange (Stratagene) mutagenesis strategy. All residue numbers are as in (1) Care was taken not to disturb residues unique to any one spectral mutant (i.e., residue 203) when designing primers. The mutagenic codon is bold.

A206K top: 5'-CAG TCC AAG CTG AGC AAA GAC CCC AAC GAG AAG CGC GAT CAC-3'
A206K bottom: 5'-GTG ATC GCG CTT CTC GTT GGG GTC TTT GCT CAG CTT GGA CTG-3'
L221K top: 5'- CAC ATG GTC CTG AAG GAG TTC GTG ACC GCC GCC GGG-3'
L221K bottom: 5'-CCC GGC GGC GGT CAC GAA CTC CTT CAG GAC CAT GTG-3'
F223R top: 5'-CAC ATG GTC CTG CTG GAG CGC GTG ACC GCC GCC GGG-3'
F223R bottom: 5'-CCC GGC GGC GGT CAC GCG CTC CAG CAG GAC CAT GTG-3'
L221K/F223R top: 5'-CAC ATG GTC CTG AAG GAG CGC GTG ACC GCC GCC GGG-3'
L221K/F223R bottom: 5'-CCC GGC GGC GGT CAC GCG CTC CTT CAG GAC CAT GTG-3'

Recombinant protein expression and purification. For protein expression, cDNAs for all YFPs were cloned into pRSETB (InVitrogen) and transformed into E. coli (strain JM109) and grown to an OD600 of 0.6 in LB containing 100 mg/liter ampicillin, at which time they were induced with 1 mM isopropyl Greek Letter Beta-D-thiogalactoside. The bacteria were allowed to express the protein at room temperature for 6 to 12 hours then overnight at 4°C. Bacteria were then pelleted by centrifugation, resuspended in phosphate-buffered saline pH 7.4 and lysed in a French press. Bacterial lysates were cleared by centrifugation at 30,000g for 30 min. The His6-tagged proteins in the cleared lysates were affinity-purified on Ni-NTA-agarose (Qiagen). The purity of all expressed and purified proteins was analyzed by sodium dodecylsulfate, polyacrlyamide gel electrophoresis (SDS-PAGE). Spectrophotometric analyses of the purified protein were done to determine the absorbance and emission spectral properties, the extinction coefficient (as measured by chromophore denaturation) and quantum yield. Fluorescence spectra were taken with a Fluorolog spectrofluorimeter. Absorbance spectra of proteins were taken with a Cary UV-Vis spectrophotometer.

Analytical ultracentrifugation: Sedimentation equilibrium. Sedimentation equilibrium experiments were performed on a Beckman Optima XL-I analytical ultracentrifuge at 20°C using interference optics. Purified, recombinant proteins were dialyzed extensively against phosphate-buffered saline pH 7.4. Samples of protein (125 Greek Letter Mul) at concentrations ranging from 50 Greek Letter MuM to 700 Greek Letter MuM were loaded into 6-channel centrifugation cells with EPON centerpieces. Samples were blanked against the corresponding dialysis buffer and centrifuged at 8, 10, 14, and 20 � 103 rpm. Periodic measurements at each speed ensured that the samples had reached equilibrium at each speed. The data were analyzed by nonlinear least-squares analysis using the software package (Microcal Origin) supplied by Beckman. The goodness of fit was evaluated on the basis of the magnitude and randomness of the residuals, expressed as the difference between the experimental data and the theoretical curve and also by checking each of the fit parameters for physical reasonability. The molecular mass and partial specific volume were determined using Sedenterp v 1.01. These data are tabulated below and were factored into the equation for the determination of homoaffinity.

Supplemental Table 1.
MutantMolecular massPartial specific volume
wtEYFP26796.230.7332
His6 wtEYFP 30534.260.7273
His6 EYFPA206K 30593.370.7277
EYFPL221K30551.290.7270
His6 EYFPL221K 30549.270.7271
His6 EYFPF223R 30543.270.7270
His6 EYFPL221KF223R 30560.300.7267

Imaging. Images in each of three channels were captured before and after a 3-min photobleach (excitation at 525 to 565 nm): the CFP channel (ex 430 to 450 nm; em 470 to 490 nm) YFP channel (ex 490 to 500; em 523 to 548) FRET channel (ex 430 to 450: em 523 to 548). Only a single set of images following photobleach was used, because excitation of the sample at 430 to 450 nm causes partial regeneration of the ability of YFP to function as an acceptor. The 3-min time for bleach was determined empirically on our system to bleach YFP completely while doing minimal or no bleaching to CFP.

Cells were imaged on a Zeiss Axiovert microscope equipped with a cooled CCD camera (Photometrics, Tucson, AZ USA) controlled by Metafluor 3.0 imaging software (Universal Imaging, West Chester, PA). Excitation and emission filters were alternated in filter wheels (Lambda 10-2, Sutter, San Rafael, CA). Optical filters were obtained from Chroma Technologies and Omega Optical (Brattleboro, VT).

It was important to ensure approximately equivalent expression of both donor CFP and acceptor YFP constructs in all experiments. This was determined from calibration of each channel by measuring the intensity of YFP and CFP before and after photobleach, respectively of YC3.1, a fusion of CFP, calmodulin, M13, and YFP containing equimolar CFP and YFP (2). The calcium sensitivity of FRET in this construct was irrelevant, because the YFP intensity was measured with direct excitation of the YFP, and the CFP intensity was measured after photobleaching the YFP.

Cell fractionation. MyrPalm-mEYFP L221K and mEYFP-GerGer F223R stably transfected MDCK cells were selected with 200 ng/ml G418 (Gibco). Fractionations were performed as described (3, 4) with the addition of a Percoll gradient to purify plasma membrane from intracellular membranes (5, 6) before carbonate or detergent fractionation. Cells were homogenized and centrifuged at low speed to collect a post-nuclear supernatant (PNS). This was layered on top of 30% Percoll (Fluka) and centrifuged at 64,000g for 30 min; an opaque band was collected as plasma membrane (PM). One half of the PM was adjusted to 1% Triton X-100 (4°C) and the other half to 500mM Na2CO3, and both were sonicated and adjusted to 45% sucrose. These were overlaid with equal volumes of 35% and 5% sucrose and centrifuged for 16 hr at 285,000g. An opaque band at the 5%/35% sucrose interface was collected as either detergent-resistant membrane (DRM) (3) or caveolae-rich membrane (CRM) (4, 7) respectively in 0.5 ml. A 0.5ml volume was collected from below the 45%/35% sucrose interface as detergent-soluble membrane (DSM) (3) or noncaveolar membrane (NCM) (4). These fractions from each stable cell line were separated by SDS-PAGE (10%), transferred to polyvinyl difluoride, immunoblotted and visualized on film by chemiluminescence.


Additional References

Additional references discussing FRET and its applications in two-dimensional spaces may be useful (8-12). Further references regarding the structure and function of lipid rafts may also be of interest (13-15). Other relevant literature includes additional uses of FRET to examine lipid rafts in the exoplasmic leaflet (16, 17), and evidence for lipid raft function in T-cell activation (18, 19).

Supplementary References and Notes

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