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Differential insertion of GPI-anchored GFPs into lipid rafts of live cells

Daniel F. Legler^*,,1, Marie-Agnès Doucey, Pascal Schneider, Laurence Chapatte, Florent C. Bender and Claude Bron

^* Department of Biology, Division of Immunology, University of Konstanz, Konstanz, Germany; and
Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, Epalinges, Switzerland

¹Correspondence: Department of Biology, Division of Immunology, University of Konstanz, Universitätsstrasse 10, Room P1105, Konstanz 78457, Germany. E-mail: daniel.legler{at}uni-konstanz.de

SPECIFIC AIM

To design raft-specific fluorescent markers, we engineered different glycosylphosphatidyl-inositol (GPI) -anchored green fluorescent proteins (GFP-GPI) and studied their membrane localization upon cell transfection and after painting of live cells, which allows insertion of purified GFP-GPI into the plasma membrane.

PRINCIPAL FINDINGS

1. Distinct localization of different GFP-GPIs in lipid rafts
Partitioning of proteins in cholesterol and sphingolipid-enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Certain proteins preferentially localize into lipid rafts—in particular, a group of functionally diverse cell surface proteins anchored in the outer leaflet of microdomains by a complex glycosylphosphatidyl-inositol (GPI) moiety. We used this property to generate an extracellular fluorescent marker for lipid rafts. Two GFP-GPI were constructed by fusing the GPI anchor addition sequence of the decay-accelerating factor (DAF) or the TRAIL receptor 3 (TRAIL-R3) to an enhanced GFP variant. Both GFP constructs were stably transfected in HEK 293 cells and revealed comparable protein expression levels; both GFP-GPI fusion proteins localized at the plasma membrane as evidenced by FACS analysis and confocal laser microscopy. Only marginal intracellular staining was observed, suggesting limited recycling of both membrane-associated proteins. Transfected cells were treated with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves specifically the ectodomains of many GPI-anchored proteins. After this treatment, 91% of surface expressed GFP-GPI(DAF) was released from the cell surface. In contrast, PI-PLC treatment removed only 19% of GFP-GPI(TRAIL-R3) from the plasma membrane of HEK 293 transfectants.

To investigate the localization of GFP-GPI fusion proteins within the plasma membrane, HEK 293 cells stably transfected with GFP-GPI (DAF) or GFP-GPI(TRAIL-R3) were lysed in 1% Triton X-100 and fractionated on a sucrose density gradient. Both GPI-anchored proteins were floated in low density fractions of the gradient that contain the detergent-insoluble lipid rafts (Fig. 1 ). These fractions were enriched in cholesterol and contained the doubly acylated Fyn kinase and the raft-associated ganglioside GM1, but not the epidermal growth factor receptor. A remarkable difference in the lipid raft partitioning was observed: only 25–55% of the GFP anchored via the TRAIL-R3 GPI moiety was found in the cholesterol/sphingolipid-enriched light density fractions, whereas the GFP linked to the DAF GPI anchor exclusively partitioned in the lipid raft fractions (Fig. 1) .

View larger version (75K): [in this window] [in a new window]   Figure 1. Distinct partitioning of GFP-GPI(DAF) and GFP-GPI(TRAIL-R3) in lipid rafts. HEK293 cells stably expressing GFP-GPI(DAF) and GFP-GPI(TRAIL-R3) were lysed in 1% Triton X-100 and subjected to sucrose density gradient centrifugation. Proteins from equal volumes of collected representative fractions were separated by SDS-PAGE and assessed by Western blot analysis using specific antibodies against GFP, EGF receptor, and Fyn. To analyze the distribution of the ganglioside GM1, 5 µL of each fraction was dot blotted onto a nitrocellulose membrane and detected using CTxHRP. Total protein distribution was monitored by Ponceau red staining. Cells were labeled with 14C cholesterol prior to sucrose density gradient centrifugation and integrated counts were measured in each fraction.

2. Cell surface painting of live cells with purified GFP-GPIs
A property of purified GPI-anchored proteins is their ability to reinsert into the plasma membrane upon incubation with a target cell. To exploit this property for fluorescent labeling of lipid rafts, we purified both GPI-anchored GFPs from HEK293 transfected cells. For cell surface painting, the murine EL-4 thymoma cell line and primary human PBL were incubated with purified GPI-anchored GFPs at 37°C. After 1.5–3 h, cells were washed extensively and the plasma membrane insertion of painted proteins analyzed by FACS (Fig. 2 A). The efficiency of membrane insertion of the purified molecules was similar on EL-4 cells and PBL. The expression level of GFP-GPIs on painted lymphocytes and transfected HEK293 cells was comparable.

View larger version (42K): [in this window] [in a new window]   Figure 2. Cell surface painting of lymphocytes with GFP-GPIs. A) EL-4 cells and primary human PBL were incubated for 2 h in the presence (filled) or absence (open profiles) of purified GFP-GPI(DAF) and GFP-GPI(TRAIL-R3), respectively. After extensive washing of the cells, plasma membrane insertion of GPI-linked GFP molecules was directly quantified by FACS analysis. B) Localization of painted GFP-GPIs on EL-4 cells and primary human PBL was monitored by confocal microscopy. Bars, 5 µm.

Faint cell surface staining was observed after 5 min of incubation. Maximal plasma membrane insertion of the GFP-GPIs was reached after 90 min. Incubation of up to 6 h did not increase the efficiency of the surface labeling. Cell surface painting of GFP-GPIs was temperature dependent since no insertion was observed at 4°C and was greatly reduced at room temperature. Monitoring of painted cells by confocal microscopy clearly showed a restricted localization of both GFP-GPIs at the plasma membrane (Fig. 2B ). No intracellular labeling was observed, indicating both proteins were retained at the cell surface and were not internalized. Painted molecules remained stably associated with the cell surface for up to 12 h, followed by a progressive decrease in surface expression upon successive cell division.

Identical results were obtained with different target cells, including primary murine T and B lymphocytes and various cell lines.

3. Role of the GPI anchor and carrier lipids for cell surface painting
To confirm the role of the GPI moiety for an efficient insertion of the recombinant fluorescent protein into the membrane of target cells, purified GFP-GPI (DAF) was subjected to PI-PLC hydrolysis before incubation with EL-4 cells. FACS analysis revealed efficient cell surface painting with intact untreated GFP-GPI (DAF), whereas PI-PLC-cleaved GFP-GPI (DAF) failed to insert into the plasma membrane, confirming the crucial role of the GPI anchor for efficient and stable membrane insertion.

We investigated whether carrier lipids could influence the painting efficiency of GPI-anchored GFP. Purified GFP-GPI(DAF) was mixed with increasing concentrations of cholesterol, dipalmitoyl-phosphatidylethanolamine (DPPE), and sphingomyelin (SM). Alternatively, GFP-GPI (DAF) was mixed with dioleoyl-phosphatidylethanolamine (DOPE), which is not enriched in lipid rafts. Liposomes made of lipids enriched in rafts increased the painting efficiency of GFP-GPI(DAF) in a concentration-dependent manner. Cholesterol was the best carrier, giving a 2-fold increase in painting efficiency as assessed by FACS analysis. Membrane insertion of GFP-GPI(DAF) was enhanced by addition of DPPE or SM. Addition of DOPE had no influence on cell surface painting. These results support the notion that lipids naturally enriched in membrane microdomains facilitate insertion of a GPI-anchored protein into lipid rafts.

4. Exclusive localization of painted GFP-GPI(DAF) in lipid rafts
To further investigate the membrane localization of painted GFP-GPI fusion proteins, lymphocytes were incubated with purified recombinant proteins. The washed, painted cells were lysed in Triton X-100 and subjected to a sucrose density gradient fractionation. Both GFP-GPIs were found in the light density fractions corresponding to lipid rafts. Painted GFP-GPI(DAF) localized entirely in the raft fractions, similar to transfected GFP-GPI(DAF). However, no more than of 55% of GFP-GPI (TRAIL-R3) was found in microdomains. The membrane distribution of endogenous proteins was not affected by the painting procedure.

These experiments demonstrate that GPI-anchored GFPs can be stably inserted into the plasma membrane of a variety of cell types and that GFP-GPI(DAF) can serve as an ideal fluorescent marker for lipid rafts of live cells.

CONCLUSIONS AND SIGNIFICANCE

Compartmentalization of the plasma membrane is of particular interest because it dictates the aggregation and segregation of proteins involved in signal transduction. Upon specific triggering, transmembrane receptors such as TCR, BCR, or TNFR1 translocate to lipid rafts. This raft localization is a prerequisite for efficient receptor-mediated signal transduction. However, the precise mechanism of the translocation to microdomains requires further investigation. Despite accumulating evidence suggesting the existence of lipid rafts in vivo, their size and composition under different physiological conditions remain to be clarified. Due to their preferential localization in lipid rafts, different GPI-anchored GFP fusion proteins have been used as markers to examine the dynamics of rafts and raft-associated proteins. GFP-GPIs have also been used to study the role of lipid rafts in the sorting of lipids and proteins along both secretory and endocytic pathways. However, some of these GPI-anchored proteins are poorly characterized and studies using different GFP-GPIs as lipid raft markers provided contradictory results. The demonstration that the two natural GPI-anchored proteins Thy-1 and PrP are organized in different membrane domains on neuronal cell surfaces highlights the critical importance of selecting the right GPI anchor for the design of a GFP-GPI fluorescent marker of lipid rafts.

We generated an extracellular fluorescent marker for lipid rafts by constructing two GPI-anchored GFPs, which differ only in their GPI signal sequence and confer distinct localization in microdomains. We investigated this unique property of recombinant GFP-GPIs to paint lipid rafts of live cells (Fig. 3 ). Cell surface painting offers several advantages over conventional transfection/infection methods: 1) virtually any cell type can be painted; 2)the painting efficiency is high and reveals homogeneous membrane insertion; 3)painting is rapid and surface expression is immediate; 4) painting is performed under normal culture conditions with no detectable toxicity for the cells; and 5) the introduced protein retains its biological function.

View larger version (21K): [in this window] [in a new window]   Figure 3. Cell surface painting of lipid rafts with GFP-GPIs. After extraction and purification, GPI-anchored proteins form micelles in solution and can be reinserted into the plasma membrane when added to target cells. We exploited this protein transfer, or painting, to insert a highly specific fluorescent maker for lipid rafts into microdomains in live cells.

We have demonstrated that the source of a GPI anchor used to generate a GFP-GPI as a fluorescent marker is critical for its membrane localization. GFP-GPI (DAF), but not GFP-GPI (TRAIL-R3), exclusively partition in lipid rafts. Purified GFP-GPI(DAF) could be stably inserted into lipid rafts of lymphocytes. Thus, painting with GFP-GPI(DAF) will help investigate the dynamic events in lipid rafts, such as formation of the immunological synapse, reorganization of the signaling network at the synapse upon receptor engagement, and processes involved in protein sorting of live cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1338fje;

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