Golgi sorting regulates organization and activity of GPI-proteins at … · 2014-05-16 · SUPPLEMENTARY INFORMATION Golgi sorting regulates organization and activity of GPI-proteins

SUPPLEMENTARY INFORMATION

Golgi sorting regulates organization and activity of GPI-proteins at apical

membranes

Simona Paladino1,2#, Stéphanie Lebreton3#, Simona Tivodar1, Fabio Formiggini2, Giulia Ossato4,

Enrico Gratton4, Marc Tramier5, Maïté Coppey-Moisan6, and Chiara Zurzolo1,3*.

1Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli

Federico II, Napoli, Italy; 2CEINGE Biotecnologie Avanzate, Napoli, Italy; 3Unité de Trafic

Membranaire et Pathogénèse, Institut Pasteur, Paris, France; 4Laboratory for Fluorescence Dynamics,

University of California, Irvine, California; 5Institut de génétique et dévelopement de Rennes, UMR

6290; 6Complexes macromoléculaires en cellules vivantes, Institut Jacques Monod, UMR 7592

CNRS, University Paris-Diderot, France.

*Correspondence to Chiara Zurzolo, [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.1495

SUPPLEMENTARY RESULTS


Supplementary Figure 1. Apical DRM-associated PLAP and GFP-FR form cholesterol

independent high molecular weight complexes. (a) MDCK cells stably co-expressing

PLAP/GFP-FR were grown for 4 days on filters. LC-biotin was added to the apical (AP) or the

basolateral (BL) surface of the cells. After immunoprecipitation with specific antibodies,

biotinylated proteins were revealed using HRP-streptavidin. Quantifications represent the mean

value of three independent experiments. (b) MDCK cells stably co-expressing PLAP/GFP-FR were

lysed in buffer containing 1% TX-100 at 4°C and purified by centrifugation to equilibrium on

sucrose density gradients (5-40%). Fractions of 1 ml were collected from the top (fraction 1) to the

bottom (fraction 12). After TCA-precipitation, proteins were run on SDS-PAGE and detected using

specific antibodies. An aliquot of each fraction was spotted on nitrocellulose membranes and GM1

was revealed using HRP-cholera toxin. As previously shown, the band at 43 kDa represents a

partially denatured dimer of GFP11. (c) Purification on velocity gradient of GFP-FR and PLAP at

steady-state. MDCK cells stably co-expressing PLAP/GFP-FR, grown to confluence, were lysed

and run on velocity gradient as described in methods. Fractions were collected from top (Fraction 1)

to bottom (Fraction 9), TCA-precipitated and revealed by western blotting with specific antibodies.

Molecular weight markers are indicated on top of the panels. (d) MDCK cells stably co-expressing

PLAP/GFP-FR, grown to confluence, were cross-linked by using BS3 either at 4°C or 37°C for

different indicated times. After lysis, proteins were TCA-precipitated, separated on SDS-PAGE and

revealed by western blotting using specific antibodies. The molecular weight of the monomeric

form of each protein is indicated, together with the position of a 180 kDa marker. *, **, and ***

indicate, respectively, monomers, dimers and trimers of the different proteins. In brackets:

unspecific bands recognized by the anti-GFP or PLAP antibody since they are present also in non-

transfected MDCK cells (panel g). Note that the cross-linked pattern was similar at both

temperatures, indicating that the proteins are in oligomeric complexes at the cell surface at

physiological temperatures. Because none of the proteins were endocytosed during the short

incubation times at 37°C26, the reduced formation of HMW complexes at 4°C most likely depends


on the reduced mobility of molecules in the plane of the membrane at this temperature. (e) MDCK

cells stably co-expressing PLAP/GFP-FR, grown to confluence, treated with saponin (+SAP) or not

(control), were cross-linked by using BS3 at 37°C for different indicated times. After lysis, proteins

were TCA-precipitated and processed as above. Note that the reduction of the monomers in saponin

treated samples possibly derived from their partial extraction from the membrane. (f) Gels of three

different experiments were quantified, and the results were expressed as a percentage of monomer

vs cross-linkable complexes (from dimer to HMW). Error bars, ± SD. (g) Protein lysates of wild-

type MDCK cells or stably expressing GFP-FR (left) or PLAP (right) were separated by SDS-

PAGE and revealed for western blot using anti-GFP (left) or PLAP (right) antibody.


Supplementary Figure 2. PLAP and GFP-FR can be cross-linked in specific cholesterol

dependent hetero-complexes. (a) MDCK cells stably co-expressing GFP-FR/PLAP untreated

(control) or treated with saponin (+ SAP) were cross-linked, lysed and proteins were

immunoprecipitated with monoclonal anti-PLAP antibody and revealed by western blotting using

an anti-GFP antibody. The molecular weight of the position of a 180 kDa marker is indicated. (b)

Cell lysates from MDCK cells stably expressing GFP-FR were immunoprecipitated with an anti-

PLAP antibody and, after separation on SDS-PAGE, proteins were revealed by western blotting by

using anti-GFP antibody. A control western blotting for GFP (gel on the right) shows that GFP-FR

is expressed by these cells. Note that there is no cross-reaction of the anti-GFP antibody with

PLAP. (c) MDCK cells stably co-expressing PLAP/GFP-FR were treated with BS3, lysed and

purified on velocity gradient. Samples were immunoprecipitated by using an anti-PLAP antibody

and revealed by western blotting using an anti-GP114 antibody (top panel). An equal aliquot of

each fraction was collected and TCA-precipitated before immunoprecipitation and revealed by

western blotting (bottom panel) with a polyclonal anti-GP114 antibody (gift of Dr. A. Le Bivic;

Faculté des Sciences de Luminy, Marseille, France). The molecular weight of the monomeric form

of GP114 is indicated together with the position of a 180 kDa marker. Note that GP114 is not cross-

linked with PLAP.



Supplementary Figure 3. Longer incubation times and higher concentrations of saponin do

not affect GPI-APs homo-clusters, while cholesterol depletion affects co-clustering of both

GPI-APs. MDCK cells stably co-expressing PLAP/GFP-FR, treated with different concentrations

of saponin (SAP) (a) or for different times (b), were cross-linked with BS3. After lysis, proteins

were TCA-precipitated, separated on SDS-PAGE and revealed using specific antibodies. The

molecular weight of the monomeric forms of each protein is indicated, together with the position of

a 180 kDa marker. (c) MDCK cells stably co-expressing PLAP/GFP-FR, either in control

conditions (-) or after saponin treatment (+), were cross-linked with BS3 (+), fixed and stained with

specific antibodies and revealed by Cy3- and Cy5-conjugated secondary antibodies. Serial confocal

sections were collected by using a laser scanning microscope (LSM 510 META, Carl Zeiss

Microimaging, Inc.) equipped with a plan apo 63x oil-immersion (NA 1.4) objective lens. The 3D

reconstruction is shown and areas of co-localization are labelled in white. Bar, 4 µm. (d) Ten

randomly chosen fluorescence images from two different experiments were analysed by using

Matlab software (see Methods). We calculated the regions where pixels of the two fluorophores are

excluded (light grey bars)/co-present (dark grey bars) and expressed as percentage of total area

occupied by each fluorophore. The percentage of co-localization in co-presence regions is shown

(punctate bars). Error bars represent ± SD (t test for co-presence/exclusion analysis, p< 0.00001; t

test for co-localization analysis, p< 0.003). One example for each sample (control, SAP) of the

degree of co-localization is visualized by correlation maps. The rainbow indicator is showed.



Supplementary Figure 4. Validation of N&B analysis for GPI-AP cluster detection. (a) The

graph shows the bleaching rate of mGFP-FR and GFP-FR, which is calculated as ratio between

fluorescence intensity of last and first frame of the N&B time-lapse and is expressed as mean of 4

independent experiments (n>20 cells). Error bars, ± SD. Note that mGFP-FR and GFP-FR have

comparable bleaching rate (7-18%). (b) Scattergram of Brightness vs Intensity of a representative

MDCK cell expressing GFP-FR alone (left panel) or co-expressing GFP-FR and PLAP (right panel)

shows that brightness is independent of the fluorescence intensity. (c) Standard curve of brightness

(B) vs number of units per aggregate (Subunits) was built by plotting the brightness values either of

soluble monomeric, dimeric and trimeric GFP (two or three mGFP moiety fused in tandem, left

panel) or of membrane-linked monomeric and dimeric GFP (one or two mGFP are fused to the GPI-

attachment signal of uPAR; mGFP-GPI and mGFP-mGFP-GPI, respectively, right panel)

transiently expressed in MDCK cells. Increase of B is linearly proportional to number of mGFP

(degree of correlation = 0.99 and 1, respectively), thus we could extrapolate with high accuracy the

number of subunits of GFP-FR aggregates by linear interpolation of the experimental B values (see

also panel g). Error bars, ± SD. The two standard curves overlap indicating that there is no

difference in B values between soluble and membrane bound GFP. (d) Quantification of the

brightness of mGFP, mGFP-mGFP, mGFP-mGFP-mGFP, mGFP-GPI (uPAR) and mGFP-mGFP-

GPI (uPAR) expressed as mean of 3 independent experiments (n>30 cells) is plotted. Error bars, ±

SD. *, p<0.0006; **, p<0.00003. As expected from the standard curves shown in panel c, the B

values for the soluble and membrane GPI-anchored form of the mGFP are absolutely comparable:

the membrane-linked dimer GFP has higher B than monomer and comparable to the soluble dimer

GFP. (e) Distribution analysis of the brightness values experimentally obtained for monomeric,

dimeric and trimeric GFP. The percentage of pixels in groups of observed B values (with a value

interval of 0.5) of a representative experiment is shown together with their occurrence (inset)

expressed as percentage of cells falling in each group (n= 40 cells). (f) The graph shows the class

distribution (monomer to trimer) on the basis of the percentage of pixels of reference molecules


(mGFP, mGFP-mGFP, mGFP-mGFP-mGFP) falling in the range of corresponding B values.

Values are expressed as mean of 5 independent experiments. Error bars, ± SD. *, p<0,0001; **,

p<0.00004. (g) Experimental mean B values of GFP-FR from three experiments (black squares) are

plotted together with the standard curve (grey circles) made with soluble GFP. (h) Distribution of

brightness values of p75-GFP compared to soluble monomeric (mGFP) and dimeric (mGFP-mGFP)

GFP. After 4 days in culture, MDCK cells stably expressing p75-GFP were processed for N&B, as

described before. The percentage of pixels in groups (with a value interval of 0.5) of observed B

values of a representative experiment is shown together with their occurrence (inset) expressed as

percentage of cells falling in each group n=20 cells). (i) Graphical representation of the percentage

of pixels falling in the different classes of B values (from monomer to hexamer) on the basis of the

calibration curve (panel g). Values are expressed as mean of 3 independent experiments. Error bars,

± SD. Note that in agreement with previous biochemical data, p75-GFP is present mainly as dimer

at the plasma membrane of cells grown in the presence of growth factors.


Supplementary Figure 5. GFP-FR brightness is not affected by saponin or mevinolin

treatment in polarized MDCK cells. After 4 days in culture, either in control conditions or upon

saponin (a,b) or mevinolin treatment (c-e), N&B analysis was carried out on MDCK cells

expressing GFP-FR. (a) Brightness and fluorescence intensity maps of polarized MDCK cell

expressing GFP-FR upon saponin treatment are shown. Bar, 0.9 µm. (b) The percentage of pixels in

groups (with a value interval of 0.5) of observed B values of a representative experiment is shown

together with their occurrence (inset) expressed as percentage of cells falling in each group (n=35

cells for both conditions). (c) Quantification of the brightness of GFP-FR from 3 independent

experiments is plotted. Error bars, ± SD. (d) The percentage of pixels in groups (with a value

interval of 0.5) of observed B values of a representative experiment is shown together with their

occurrence (inset) expressed as percentage of cells falling in each group (n=30 cells). (e) Graphical

representation of the percentage of pixels falling in the different classes of B values (from monomer

to hexamer) on the basis of the calibration curve in Supplementary Fig. 4g. Values are expressed as

mean of 3 independent experiments. Error bars, ± SD.



Supplementary Figure 6. Quantification of hetero-FRET between GPI-APs. (a) Comparison of

images obtained either with FLIM (left) or N&B (right). In both cases, GFP-FR was imaged in

polarized MDCK cells expressing only GFP-FR. Pixel size of N&B 70 nm (ROI imaged 256x64).

Pixel size of FLIM 250 nm (ROI imaged 140x140). Bars, 0.55 µm. (b) The graph shows the

relationship between the amounts of interacting donor expressed in percentage (%) with the

acceptor/donor (A/D) intensities ratio. This panel is representative of two experiments that have

been performed the same day with exactly the same laser power and detector gain. The quantity of

interacting donor (%) is independent of acceptor/donor intensities ratio (from 0,77 to 5,64)

indicating that hetero-clusters of GPI-APs is driven by an active mechanism. (c) Cells expressing

different levels of GFP-FR and mCherry-PLAP were analysed. GFP and mCherry intensities are

reported respectively on left and middle panels, while the corresponding lifetime values of GFP-FR

are shown on the right panels. Bar, 9 µm. Histograms show GFP-FR lifetime (ns) and A/D

fluorescence intensities for each imaged cell. The lifetime of GFP-FR is independent from A/D

fluorescence intensities. (d, e) The graphs exhibit the relationship between the amounts of

interacting donor (%) with the donor (d) or acceptor (e) fluorescence intensities (from 14357 to

59158 and from 28432 to 123312, respectively). The quantity of interacting donor (%) is

independent of both donor or acceptor fluorescence intensities. Thus, hetero-clusters of GPI-APs

are density independent that is characteristic of a sub-pixel size domains organization. (f) The graph

represents the percentage of polarized MDCK cells having lower (< 20%) or higher (> 20%) values

of interacting donor (13 vs 87%). (g) The graph represents the percentage of CHO cells having

lower (< 20%) or higher (> 20%) values of interacting donor (30 vs 70%).



Supplementary Figure 7. GPI-AP clusters in CHO cells are cholesterol dependent and

accommodate different species. N&B (a,d) or FLIM analysis (e,f) was carried out in CHO cells

after 2 or 3 days in culture. (a) Quantification of the brightness of GFP-FR from 3 independent

experiments performed in cells expressing GFP-FR, either in control conditions or upon saponin

treatment, or in cells co-expressing GFP-FR and mCherry-PLAP is plotted. Error bars, ± SD. *,

p<0.02; **, p<0,000001. (b) Brightness and fluorescence intensity maps of CHO cells expressing

GFP-FR both in control condition and upon saponin treatment are shown. Bar, 0.9 µm. (c) The

percentage of pixels in groups (with a value interval of 0.5) of observed B values of a representative

experiment is shown together with their occurrence (inset) expressed as percentage of cells falling

in each group (n=25 cells for both conditions). (d) Graphical representation of the percentage of

pixels falling in the different classes of B values (from monomer to hexamer) on the basis of the

calibration curve shown in Supplementary Fig. 4g. Values are expressed as mean of 3 independent

experiments. Error bars, ± SD. *, p<0.002; **, p<0.00005. Note: B measurements are taken from

different areas and in control conditions the brightness of GFP-FR is comparable in CHO and

MDCK cells (respectively without and with microvilli), confirming that it is independent on the

presence of microvilli. (e) Intensity and mean fluorescence lifetime maps of GFP-FR alone or in

presence of mCherry-PLAP in control or cholesterol depleted cells are shown. The lifetime scale is

from 1 ns to 3 ns. Bars, 9 µm. (f) Histograms of GFP-FR lifetime alone (blue bars) or in

combination with mCherry-PLAP (pink bars) in control condition (colored bars) or upon cholesterol

depletion (pale colored bars). Experiments were performed 3 independent times, n>35 cells. Error

bars, ± SD. *, p<0,0001. In post-acquisition only the plasma membrane signal was considered for

GFP-FR lifetime (see methods). (g) After 2 days in culture, CHO cells expressing GFP-FR were

treated with trypsin and then subjected to temperature block in order to analyse the Golgi pool of

proteins. Lysates were purified on velocity gradient (see methods). Molecular weight markers are

indicated on top of the panels.


Supplementary Figure 8. Hetero-clusters of different GPI-APs are formed by independent

homo-clusters and not by monomeric GPI-APs. (a) MDCK cells stably expressing PLAP/GFP-

FR were treated with BS3, lysed and purified on velocity gradients. Samples were

immunoprecipitated using an anti-PLAP antibody and revealed by western blotting using an anti-

GFP antibody (upper panel). An aliquot of immunoprecipitated proteins for each fraction was

revealed by western blotting with anti-PLAP antibody (lower panel). The molecular weight of the

monomeric form of each protein is indicated, together with the position of a 180 kDa marker. (b)

MDCK cells stably expressing PLAP/GFP-FR, grown to confluence, were treated (+) or not (-) with

DTT for 5 min, cross-linked at 37°C for 10 min and then lysed. Lysates were immunoprecipitated


with an anti-PLAP antibody and revealed by western blotting by using an anti-GFP antibody (left

panel). An aliquot of the lysate before immunoprecipitation was run on SDS-PAGE and revealed by

western blotting using an anti-GFP antibody (right panel). Note that after DTT treatment GFP-FR

was present only in the monomeric or partially denaturated dimeric (band at 43 kDa) form and does

not co-immunoprecipitate with PLAP. (c, d) The full, uncut gel images from Fig. 4a (c) and 5c (d)

are shown.



Supplementary Figure 9. Cholesterol addition to non-polarized MDCK cells affects the

surface organization of GPI-APs.

(a) B and I map of polarized MDCK (4 days) or non-polarized (1 day) expressing GFP-FR. (b) The

percentage of pixels in groups (with a value interval of 0.5) of observed B values of a representative

experiment is shown together with their occurrence (inset) expressed as percentage of cells falling

in each group (n=25 cells for both conditions). (c) B and I map of 1 day MDCK cells expressing

GFP-FR both in control condition and upon addition of cholesterol. (d) The percentage of pixels in

groups (with a value interval of 0.5) of observed B values of a representative experiment is shown

together with their occurrence (inset) expressed as percentage of cells falling in each group (n=30

cells for both conditions). (e-g) After 1 day in culture, either in control conditions or upon

cholesterol addition, N&B analysis was carried out on MDCK cells expressing p75-GFP. (e)

Quantification of the brigthness of p75-GFP expressed as mean of 3 independent experiments is

plotted. Error bars, ± SD. (f) The percentage of pixels in groups (with an interval of 0.5) of

observed B values of a representative experiment is shown together with their occurrence (inset)

expressed as percentage of cells falling in each group (n=25 cells for both conditions). (g) Graphical

representation of B values grouped in classes (from monomer to hexamer) on the basis of the

calibration curve shown in Supplementary Fig. 4g. Values are expressed as percentage of total

protein of 3 independent experiments. Error bars, ± SD. (h-k) N&B (h-j) and FLIM (k) analysis

was performed in 1-day MDCK cells pre-treated with cycloheximide in order to consider

exclusively the cell surface pool of proteins, in control conditions or upon cholesterol addition (in

presence of cycloheximide). (h) B and I map of 1 day MDCK expressing GFP-FR in control

condition or upon cholesterol addition. (i) The percentage of pixels in groups (with a value interval

of 0.5) of observed B values of a representative experiment is shown together with their occurrence

(inset) expressed as percentage of cells falling in each group (n=30 cells for both conditions). (j)

Graphical representation of B values grouped in classes (from monomer to hexamer) on the basis of

the calibration curve shown in Supplementary Fig. 4g. Values are expressed as percentage of total


protein of 3 independent experiments. Error bars, ± SD. (k) Intensity and mean fluorescence

lifetime maps of GFP-FR alone or in combination with mCherry-PLAP. The lifetime scale is from

1.2 ns to 2.8 ns. Bars, 0.9 µm and 9 µm in N&B and FLIM images, respectively.


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Golgi sorting regulates organization and activity of GPI-proteins at … · 2014-05-16 · SUPPLEMENTARY INFORMATION Golgi sorting regulates organization and activity of GPI-proteins