Figure S1. A: Dimerisation of non-monomeric FPs is detected using FRET.
Comparison of FRET ratio as a function of surface density (ICFP) between cells
expressing CFP-GPI and CitFP-GPI , curve fit green lines, and cells expressing
monomeric mutants, mCFP-GPI and mCitFP-GPI , curve fit red lines. Fits are by
simple linear regression, dashed lines indicate 95% confidence intervals for the fit. B.
Direct determination of efficiency of FRET E, between mCFP-GPI and mCit-GPI.
FRET efficiency was calculated as the fractional increase in mCFP-GPI fluorescence
following photobleaching of mCitFP-GPI, corrected for photoconversion as described in
Supplementary Methods. Red lines show best fit by linear regression and 95%
confidence intervals for the fit. Negative values arise because of small changes in focal
plane during time taken for photobleaching, necessity for correction for CFP
photoconversion, and other sources of experimental noise. C-E. Quantification of
total GPI levels by binding of aerolysin toxin. C. COS-7 cells expressing high levels
of mCFP-GPI. D. Labelling of the same cells and neighbouring untransfected cells with
Alexa488-conjugated aerolysin (from Protox Biotech, Victoria, Canada). E.
Quantification of mean fluorescence intensities for mCFP-GPI and Alexa488conjugated aerolysin in COS-7 (green ) and Jurkat (red ). Imaging parameters were
different for the different cell types, so comparison between absolute values is not
meaningful.
Figure S2. Filter-sets and excitation for FRET imaging. Fluorescence intensities
from the three combinations shown are referred to in the text as ICFP, ICitFP and IFRET*.
The two COS-7 cells shown are expressing soluble, dimeric mCFP-mCitFP. Cell
perimeter is outlined.
Figure S3. Organisation of GPI-linked proteins is not different in ruffles or
protrusions from unstimulated Jurkat cells as compared with the rest of the PM.
Both FRET efficiency as determined by calculation of the ratio between mCFP-GPI and
mCitFP-GPI fluorescence (A) and the ratio between mCFP-GPI and LmCitFPGT46 (B)
were constant in all regions of the plasma membrane. Ratios were calculated on a
pixel-by-pixel basis and normalised so that maximum value is 254. Live cells at 37˚C.
Figure S4. A and B. Calculation of sensitised CitFP emission. A. Examples of
calibration data to allow correction of IFRET* for non-FRET signal, resulting in calculation
of IFRET (see Methods).  Cells expressing CFP alone.  Cells expressing CitFP alone
B. Comparison of FRET between CFP and CitFP expressed as a single protein  and
expressed separately in the same cell . FRET ratio is IFRET / ICFP. C. Residuals for
linear regression analysis of data in Figure 1C.  Control cells.  After cyclodextrin
treatment.
[1] Erickson, M.G., Alseikhan, B.A., Peterson, B.Z. & Yue, D.T. Preassociation of
calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living
cells. Neuron. 31, 973-85 (2001).
[2] Förster, T. Zwischenmoleculare energiewanderung und fluoreszenz Ann. Phys. 2,
55-75 (1948).
[3] Kenworthy, A.K. & Edidin, M. Distribution of a glycosylphosphatidylinositolanchored protein at the apical surface of MDCK cells examined at a resolution of
<100 Å using imaging fluorescence resonance energy transfer. J. Cell Biol. 142,
69-84 (1998).
[4] Nichols, B.J. GM1-containing lipid rafts are depleted within clathrin-coated pits.
Curr. Biol. 13, 686-90 (2003).
[5] Dewey, T.G., and Hammes, G.G. Calculation of fluorescence resonance energy
transfer on surfaces. Biophys. J. 32, 1023–1035 (1980).
[6] Wolber, P.K., and Hudson, B.S. An analytic solution to the Förster energy transfer
problem in two dimensions. Biophys. J. 28, 197–210 (1979)
[7] Fujiwara, T. et al. Phospholipids undergo hop diffusion in compartmentalized cell
membrane. J. Cell Biol. 157, 1071-1081 (2002).
[8] Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells. Science.
296, 913-6 (2002).
[9] Jovin, T.M. & Arndt-Jovin, D.J. Luminescence digital imaging microscopy. Annu.
Rev. Biophys. Biophys. Chem. 18, 271-308 (1989).
[10] Karpova, T.S. et al. Fluorescence resonance energy transfer from cyan to yellow
fluorescent protein detected by acceptor photobleaching using confocal
microscopy and a single laser. J. Microscopy 209, 56-70 (2003)
[11] Keller, P., Toomre, D., Diaz, E., White, J. & Simons, K. Multicolour imaging of
post-Golgi sorting and trafficking in live cells. Nat. Cell Biol. 3, 140-9 (2001).
[12] Pralle, A., Keller, P., Florin, E.L., Simons, K. & Horber, J.K. Sphingolipidcholesterol rafts diffuse as small entities in the plasma membrane of mammalian
cells. J. Cell Biol. 148, 997-1008 (2000).