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The Density of GM1-Enriched Lipid Rafts Correlates
Inversely with the Efficiency of Transfection Mediated
by Cationic Liposomes
Tamas Kovacs,1 Andrea Karasz,1 Janos Szollo†si,1,2 Peter Nagy1*
� AbstractAlthough cationic liposome-mediated transfection has become a standard procedure,the mechanistic details of the process are unknown. It has been suggested that endocyticuptake of lipoplexes is efficient, and transfectability is largely determined by later steps.In this article, we stained GM1-enriched membrane microdomains, a subclass of lipidrafts, with subunit B of cholera toxin and correlated transfection efficiency with theirdensity by quantitatively evaluating microscopic images. We found a strong anticorrela-tion between the density of GM1-enriched membrane microdomains and the efficacyof transfection monitored by measuring the expression level of GFP in different celllines transfected by lipofection using two different transfection agents. These findingsimply that GM1-enriched membrane microdomains interfere with the process of lipo-fection. The blocked step must be endocytosis since the accumulation of fluorescentlylabeled plasmids was lower in cells with high content of GM1-enriched membranemicrodomains. Such a correlation was not observed in cells transfected by electropora-tion. By comparing the efficiency of lipofection in several cell lines we found that thosewith a high density of GM1-enriched membrane microdomains were the most resistantto transfection. We conclude that the inhibition of lipofection by GM1-enriched mem-brane microdomains is a general rule, and that endocytosis of lipoplexes can be ratelimiting in cells with high density of GM1-enriched membrane rafts. ' 2009 International
Society for Advancement of Cytometry
� Key termslipid raft; lipoplex; transfection efficiency; CTX-B; GM1 ganglioside
TRANSFECTION of cells with DNA or siRNA has become a routine procedure in
molecular biology. Although viral transfer of genes (transduction) yields a high frac-
tion of transfected cells with high protein expression levels (1), its acceptance in
experimental and clinical applications is hampered by safety concerns (2). Nonviral
gene transfer can be broadly divided into two types: (i) physical methods including
electroporation and microinjection; (ii) chemical approaches using cationic lipo-
somes, dendrimers, or calcium phosphate (3). Although probably there is not a single
molecular biology lab in the world where cationic liposome-mediated transfection is
not used, the mechanism of DNA transfer is unclear, and transfection protocols are
empirical. Currently used second generation liposome formulations contain a mix-
ture of a cationic lipid and a helper lipid (4). Plasmid DNA is large and charged;
therefore, it encounters barriers during the transfection process. Electrostatic interac-
tions between cationic lipids and the negatively charged nucleic acid lead to the
formation of DNA–lipid complexes (lipoplexes) in which DNA is neutralized, con-
densed, and protected from degradation (5). It is widely accepted that lipoplexes are
endocytosed (6), and that productive endocytosis leading to gene expression is gener-
ally achieved by the clathrin-mediated pathway (7), although lipid raft/caveolae-
dependent endocytosis has also been shown to play a role under certain circumstances,
1Department of Biophysics and CellBiology, University of Debrecen,Debrecen 4012, Hungary2Cell Biophysical Workgroup of theHungarian Academy of Sciences,Research Center for MolecularMedicine, University of Debrecen,Debrecen 4012, Hungary
Received 10 April 2009; RevisionReceived 13 May 2009; Accepted 18 May2009
Additional Supporting Information may befound in the online version of this article.
Grant sponsor: Hungarian Scientific Re-search Fund, Grant numbers: OTKA72677, 68763; Grant sponsor: EuropeanCommission; Grant numbers: LSHB-CT-2004-503467, LSHC-CT-2005-018914,MCRTM-CT-2006-0359462.
*Correspondence to: Peter Nagy,Department of Biophysics and CellBiology, University of Debrecen,Nagyerdei krt 98, Debrecen 4012,Hungary
Email: [email protected]
Published online 12 June 2009 in WileyInterScience (www.interscience.wiley.com)
DOI: 10.1002/cyto.a.20756
© 2009 International Society forAdvancement of Cytometry
Original Article
Cytometry Part A � 75A: 650�657, 2009
especially in gene transfer mediated by polyplexes (8,9). Since
endocytosis of lipoplexes is more efficient than later steps of
the transfection process, there are more cells taking up plas-
mid DNA than those expressing it (10). Rate limiting steps of
transfection are thought to include escape of the lipoplex from
endosomes, release of DNA from the lipoplex and nuclear
import (5,11,12). Helper lipids are assumed to play an instru-
mental role in the escape of the DNA from the endosomal
compartment by inducing lipid phase transition (4). In parti-
cular, the superiority of multicomponent lipoplexes has been
shown to be the consequence of their ability to induce rupture
of the endosomal membrane (13).
Lipid rafts are dynamic membrane microdomains with a
peculiar composition characterized by high cholesterol, sphin-
golipid, and ganglioside content (14). Since the entity defined
as raft depends on the method used for its detection, lipid rafts
prove to be difficult to investigate (15). Flow cytometric
(16–18) and complex microscopic techniques (19) have gained
importance in the investigation of lipid rafts due to their
potential for medium to high throughput assays or their ability
to reveal raft heterogeneity. Lipid rafts have been implicated in
organizing transmembrane signaling and membrane trafficking
(20), but their relationship to lipoplex-mediated transfection
has been barely investigated. It has been reported that in HeLa
cells raft-mediated internalization of polyethylenimine poly-
plexes was more efficient than the clathrin-mediated pathway
(8), but productive endocytosis of DNA-lipoplex complexes
followed the clathrin-mediated pathway (9). Since glycosyl-
phosphatidylinositol (GPI)-anchored proteins and ganglioside
GM1 accumulate in lipid rafts, both of them are used as speci-
fic raft markers (21,22). Lipid rafts are heterogeneous with
regard to their composition and function (23,24). Specifically,
GM1-enriched rafts show only partial overlap with microdo-
mains containing GPI-anchored proteins (25).
In this article, we used subunit B of cholera toxin (CTX-
B) to specifically label and quantitate the density of GM1-
enriched membrane microdomains, a subtype of lipid rafts.
We show that a high density of GM1-enriched membrane
microdomains inhibits transfection mediated by cationic lipo-
somes, and conclude that endocytosis is the rate limiting step
in cationic liposome-mediated transfection in cells with a high
density of GM1-enriched membrane microdomains. This
principle can be used to design more powerful transfection
agents rationally.
MATERIALS AND METHODS
Cells
The breast cancer cell line JIMT-1, available from the
German Collection of Microorganisms and Cell Cultures
(www.dsmz.de), was grown in F-12/DMEM (1:1) supplemented
with 20% FCS, 60 units/L insulin and antibiotics (26). The
human breast cancer cell line SKBR-3, the human cervix adeno-
carcinoma cell line HeLa, the human epithelial carcinoma cell
line A431, the mouse fibroblast cell line NIH/3T3 and the Chi-
nese hamster ovary cell line CHO were obtained from the
American Type Culture Collection (Rockville, MD) and grown
according to their specifications. The immortalized human
keratinocyte cell line HaCaTwas obtained from the Department
of Physiology, University of Debrecen and cultured in DMEM
supplemented with 10% FCS and antibiotics. For microscopic
experiments, cells were cultured on Lab-Tek II chambered cov-
erglass (Nalge Nunc International, Rochester, NY).
Transfection and Labeling of Cells, Plasmids
Cells grown on 2-well chambered coverglass were trans-
fected with Lipofectamine2000 (Invitrogen, Carlsbad, CA)
using 1.5 lg DNA/well and a lipid to DNA ratio of 2:1 (v/w).
Transfection with Effectene (Qiagen, Valencia, CA) was carried
out at an Effectene:Enhancer:DNA ratio of 25:8:1 (v/v/w). The
transfection protocols were otherwise according to the manu-
facturers’ specifications. Electroporation was performed by the
nucleofector device of Amaxa (Cologne, Germany) using
solution V and protocol T-20.
The GFP plasmid pmaxGFP was purchased from Amaxa
(Cologne, Germany). The GFP-GPI plasmid was a kind gift
from Jennifer Lippincott-Schwartz (NIH, Bethesda, MD). An
irrelevant plasmid (pSUPER from Oligoengine, Seattle, WA)
was fluorescently labeled by performing nick translation in the
presence of fluorescein-dUTP using the nick translation kit of
Roche (Cat. No. 10,976,776,001) according to the protocol
provided by the manufacturer. GM1-enriched membrane rafts
were labeled by incubating cells in the presence of 8 lg/ml
AlexaFluor647-tagged subunit B of cholera toxin (CTX-B,
Molecular Probes-Invitrogen, Eugene, OR) for 30 min on ice
to prevent internalization of CTX-B. Afterwards, cells were
washed twice in PBS and fixed in 1% formaldehyde. For label-
ing the light chain of clathrin cells were fixed in 3.7% formal-
dehyde for 30 min, stained with Ab-1 (clone CON.1, Dianova,
Hamburg, Germany) in PBS containing 0.1% BSA and 0.1%
TX-100 followed by labeling with an anti-mouse secondary
antibody tagged with AlexaFluor488.
Transferrin Uptake Experiment
Cells were starved in medium199 containing 0.1% FCS
for 1 h, then incubated in the same type of medium contain-
ing 10 lg/ml AlexaFluor488-labeled transferrin (Molecular
Probes-Invitrogen) for 60 min at 378C followed by staining
with CTX-B as described earlier.
Confocal Microscopy and Image Analysis
Image acquisition was performed using an LSM510 confo-
cal laser scanning microscope (Carl Zeiss AG, Gottingen, Ger-
many). GFP was excited with the 488 nm line of an argon ion
laser and its fluorescence was detected between 505 and 530
nm. AlexaFluor647 was excited with the 633 nm line of a red
He-Ne laser, and its emission was measured over 650 nm. Fluo-
rescence images were taken as 1-lm optical sections using a
403 (NA5 1.3) or 633 (NA5 1.4) oil immersion objective.
Image processing was carried out with the DipImage
toolbox (Delft University of Technology, Delft, The Nether-
lands) under Matlab (Mathworks, Natick, MA). Segmentation
of images into membrane and nonmembrane pixels was car-
ried out with the manually seeded watershed algorithm using
ORIGINAL ARTICLE
Cytometry Part A � 75A: 650�657, 2009 651
a custom-written Matlab program as described previously
(27,28). Cells were analyzed on a cell-by-cell basis, and single
cells were identified by the flood-fill operation carried out on
the image segmented by the watershed algorithm. Each dot in
the presented dot plots represents the mean fluorescence
intensity of a single cell, and a dot plot contains data of 500–
1,000 cells. The fluorescence intensities of GFP, GFP-GPI,
clathrin, and transferrin were measured in the whole cell,
whereas that of CTX-B was calculated in the membrane. The
membrane mask around each cell was determined by taking
the boundary around the object, i.e., the cell, thickened by 1
pixel both inwardly and outwardly. The background-corrected
mean fluorescence intensities of single cells calculated for the
whole cell (GFP, GFP-GPI, clathrin, transferrin) or for the
membrane (CTX-B) are presented. Background correction
was carried out by subtracting the mean fluorescence intensity
of a cell-free area from each pixel in the image.
RESULTS
The Efficiency of Lipofection of JIMT-1 Cells Inversely
Correlates with the Density of GM1-Enriched
Membrane Microdomains
Since the lipid raft content of single cells has not been
related to the efficiency of transfection with cationic lipo-
somes, we examined whether the two parameters are corre-
lated. In the first experiments, JIMT-1 cells were transfected
with GFP-GPI plasmid using Lipofectamine2000, and GM1-
enriched membrane rafts were stained by fluorescently labeled
subunit B of cholera toxin (CTX-B) 48 h after transfection.
Since GPI-anchored proteins are known to accumulate in lipid
rafts, the GFP and CTX-B signals were expected to show
strong positive correlation. Surprisingly, the two signals were
strongly anticorrelated in the majority of cells, while a minor
subpopulation showed the expected positive correlation (Fig.
1A). The conclusion of the quantitative analysis is visually
supported by the confocal microscopic image in which most
of the cells are either green (GFP-GPI) or red (CTX-B), and a
couple of them appearing in yellow show colocalization of
GFP-GPI and CTX-B (Supporting Information Fig. S1).
To exclude the possibility that the observed anticorrela-
tion is the consequence of lipid raft heterogeneity (24) or
GFP-GPI-induced changes in lipid raft structure, cells were
transfected with GFP plasmid. Not a single cell displayed the
yellow color characteristic of colocalization between GFP and
CTX-B (Fig. 2), and an even stronger anticorrelation was
revealed by the quantitative analysis of single cells (Fig. 1B).
Cells with a high density of GM1-enriched membrane micro-
domains were never transfected, and cells showing high pro-
duction of the transfected protein were always very dim in the
CTX-B channel. The negative correlation between the density
of GM1-enriched rafts and transfection efficiency was also
revealed by plotting the relationship between mean GFP fluo-
rescence as a function of raft density (CTX-B fluorescence,
Fig. 3A). To rule out the possibility that transfection by cati-
onic liposomes influences the binding of CTX-B we compared
the mean fluorescence intensities of control and GFP-trans-
fected cells and observed no significant difference (Fig. 4). We
tentatively concluded that a high density of GM1-enriched
membrane microdomains somehow interferes with the
process of lipofection.
The Endocytosis of a Fluorescently Labeled Plasmid is
Correlated Inversely with the Density of GM1-Enriched
Membrane Microdomains
Although lipoplex uptake is considered to be more
efficient than later steps of transfection (5,29), the assumption
that a high density of GM1-enriched membrane microdo-
mains interferes with endocytic uptake of DNA seemed
reasonable. Therefore, we transfected JIMT-1 cells with an
irrelevant plasmid (pSUPER) fluorescently labeled by nick
translation in the presence of fluorescein-dUTP, and stained
them with AlexaFluor647-CTX-B 8 h after transfection.
Confocal microscopy revealed a remarkable anticorrelation
between the plasmid content of single cells and their
Figure 1. The density of GM1-enriched membrane microdomains inversely correlates with lipofection efficiency. JIMT-1 cells were trans-
fected with GFP-GPI (A), GFP (B) or an irrelevant fluorescently labeled plasmid (C) using Lipofectamine2000 followed by staining with CTX-
B 2 days after transfection. The fluorescence intensity of the expressed protein (A,B) or that of the plasmid (C) is plotted as a function of
CTX-B fluorescence intensity. Each dot in the figure represents the mean fluorescence intensity of single cells measured in the respective
fluorescence channel of the confocal microscope.
ORIGINAL ARTICLE
652 Lipid Rafts Inhibit Transfection
GM1-enriched raft density (Figs. 1C and 5). We concluded
that a high density of GM1-enriched membrane microdo-
mains does not allow the efficient endocytic uptake of DNA-
liposome complexes.
The Inhibitory Role of GM1-Enriched Membrane
Microdomains is a General Property of Lipofection
To check whether the observed anticorrelation between
GM1-enriched raft density and transfectability by lipoplexes is
limited to the transfection of JIMT-1 cells with Lipofecta-
mine2000 we extended the investigation to a different cell line
and to a different transfection agent. Transfection of SKBR-3
cells with GFP using Lipofectamine2000 showed anticorrela-
tion between GFP intensity and the density of GM1-enriched
membrane microdomains, although the strength of the inverse
relationship was somewhat smaller than in JIMT-1 (Figs. 3B,
6A; Supporting Information Fig. S2). Transfection of JIMT-1
cells with GFP using a different lipid formulation (Effectene)
also resulted in negative correlation between transfection effi-
ciency and the density of GM1-enriched membrane microdo-
mains (Figs. 3B, 6B; Supporting Information Fig. S3). Assum-
ing that GM1-enriched membrane microdomains indeed
interfere with endocytic uptake of the lipoplex GFP expression
is expected to show no dependence on CTX-B staining after
electroporation. Analysis of JIMT-1 cells transfected by elec-
troporation with GFP revealed no correlation between the
density of GM1-enriched membrane microdomains and GFP
expression (Figs 3A, 6C, Supporting Information Fig. S4).
These results present convincing evidence that GM1-enriched
membrane rafts generally inhibit lipofection without influen-
cing the transfectability of cells by electroporation.
The Transfectability of Cell Lines is Predictable from
their Density of GM1-Enriched Membrane Rafts
Some cell lines are empirically known to be easily trans-
fectable, whereas others hardly lend themselves to transfection.
We selected seven cell lines including epithelial and fibroblast
lines, cancerous and noncancerous ones with known differ-
ences in their transfectability. All of them were transfected
with the same Lipofectamine2000-DNA complex and their
mean GFP expression levels were examined as a function of
the density of GM1-enriched rafts 48 h after transfection. The
data reveal a remarkably predictable tendency; cells with a
high density of GM1-enriched membrane microdomains are
difficult to transfect, while cells known and found to be easily
transfectable had low content of GM1-enriched membrane
rafts (Fig. 3C). We conclude that the level of GM1-enriched
membrane microdomains plays a fundamental role in deter-
mining the transfectability of cells with cationic liposomes.
Lack of Correlation Between the Expression Level of
Clathrin, Endocytic Uptake of Transferrin and the
Density of GM1-Enriched Membrane Microdomains
The inhibition of productive endocytosis of lipoplexes in
cells with a high density of GM1-enriched membrane micro-
domains could be explained by the low level or absence of
Figure 2. Staining with CTX-B shows strict anticorrelation with the expression of GFP. JIMT-1 cells were transfected with GFP using Lipo-
fectamine2000 and were stained with CTX-B 2 days after transfection. The fluorescence images of GFP (A), CTX-B (B), the phase contrast
image (C) and their overlay (D) reveal anticorrelation between the expression of GFP and CTX-B fluorescence intensities. Part E shows the
cells in color identified by the manually seeded watershed algorithm, whereas part F shows the membrane mask in red. GFP and CTX-B
fluorescence intensities were evaluated in the cell (E) and membrane (F) masks, respectively. The scale bar corresponds to 50 lm.
ORIGINAL ARTICLE
Cytometry Part A � 75A: 650�657, 2009 653
clathrin expression in these cells. To find evidence for or
against the aforementioned assumption we stained JIMT-1
cells with CTX-B and a monoclonal antibody against the light
chain of clathrin. The image (Fig. 7A) and its quantitative
evaluation (Fig. 4B) show a lack of correlation between the
density of GM1-enriched membrane microdomains and cla-
thrin expression level. Although clathrin expression did not
depend on the density of GM1-enriched lipid rafts, it was still
possible that clathrin-dependent endocytosis was inhibited by
a high density of GM1-enriched lipid microdomains. There-
fore, we incubated cells in the presence of fluorescently labeled
transferrin and stained them with CTX-B afterwards (Fig. 7B).
The uptake of transferrin depended negligibly on the density
of GM1-enriched membrane domains compared to that
observed for the uptake of fluorescent plasmids or the expres-
sion of transfected proteins (Fig. 4B).
DISCUSSION
Modification of gene expression by exogenous nucleic
acids (DNA, siRNA) is a widely used tool in research, and it
gains importance in clinical applications as well. Although
transfection of cells is most often carried out using cationic
liposomes, the efficiency of this approach in some cell types is
not as high as desirable. More importantly, factors influencing
Figure 4. CTX-B binding is independent of lipofection and clathrin expression. (A): JIMT-1 cells were transfected with GFP using Lipofecta-
mine2000, and nontransfected (thin solid line) and transfected cells (thick dashed line) were stained with AlexaFluor647-CTX-B 2 days after
transfection. For comparison nontransfected CHO cells were also stained with AlexaFluor647-CTX-B (thick solid line). CHO and JIMT-1 cells
were imaged with different photomultiplier settings, and the measured intensities of CHO cells were corrected for the higher gain. The his-
tograms show the distribution of the mean CTX-B intensity of single cells measured by confocal microscopy. (B): JIMT-1 cells were stained
with CTX-B and against the light chain of clathrin (thin solid line). In another experiment JIMT-1 cells were incubated in the presence of
fluorescently labeled transferrin for 1 h at 378C followed by staining with CTX-B (thick dashed line). The fluorescence intensity of singlecells was calculated and the range of CTX-B fluorescence intensity was divided into bins. The average clathrin or transferrin fluorescence
intensity in these bins was calculated and plotted against CTX-B intensity.
Figure 3. High density of GM1-enriched membrane microdomains in single cells and cell lines predicts low lipofection efficiency. (A,B):
JIMT-1 cells were transfected with GFP using Lipofectamine2000 (A, filled circle), electroporation (A, open triangle) or Effectene (B, open trian-
gle). SKBR-3 cells were transfected with GFP using Lipofectamine2000 (B, filled circle). Cells were stained with CTX-B 2 days after transfection,
and the cells were analyzed by confocal microscopy. The range of fluorescence intensities in the CTX-B channel was divided into 20 bins, and
the mean GFP fluorescence intensity was calculated separately for each bin. (C): Seven different cell lines were transfected with GFP using
Lipofectamine2000. The same lipid-DNA mixture was used for each cell line. Cells were stained with fluorescent CTX-B 2 days after transfec-
tion, and the fluorescence intensity of GFP and CTX-B were measured by confocal microscopy. The mean GFP intensity of the cell lines is
plotted against their average CTX-B intensities. The CTX-B intensity histograms of two of the cell lines (CHO, JIMT-1) are shown in Figure 4A.
ORIGINAL ARTICLE
654 Lipid Rafts Inhibit Transfection
distinct steps and the overall efficiency of transfection are
largely unknown making the procedure unpredictable or em-
pirical at best. Although gene transfer mediated by viral trans-
duction is more efficient, there are concerns about its safety
(2). Therefore, there is an urgent need for better gene transfer
technologies. A better understanding of the mechanisms of
cationic liposome-mediated transfection can lead to signifi-
cant improvements in the efficiency of this approach.
In this article, we observed a strong negative correlation
between the density of GM1-enriched membrane microdo-
mains and transfectability of cells by two commercial cationic
liposome formulations when the efficiency of transfection was
evaluated either at the level of lipoplex endocytosis or gene
transcription, i.e., protein expression. These findings imply
that a high density of GM1-enriched rafts blocks endocytosis
of the DNA-lipid complex. It has to be noted that caution
should be exercised in the generalization of the observed
inhibitory role of a high density of GM1-enriched rafts on
lipoplex endocytosis, since only two different commercial cati-
onic lipid mixtures were tested. We believe that it is still
Figure 5. Uptake of a fluorescent plasmid inversely correlates with the density of GM1-enriched membrane microdomains. JIMT-1 cells
were transfected using Lipofectamine2000 with an irrelevant plasmid (pSUPER) fluorescently labeled by nick translation, and they were an-
alyzed by confocal microscopy 8 h after transfection. (A) and (B) show the fluorescence of the plasmid and CTX-B, respectively, while (C) is
the phase contrast image. The overlay of the fluorescence and phase contrast images is shown in (D). (E) and (F) show the cell and mem-
brane masks, respectively. The scale bar corresponds to 30 lm.
Figure 6. The inverse correlation between transfection efficiency and the density of GM1-enriched membrane microdomains is a property
of lipofection. SKBR-3 cells were transfected with GFP using Lipofectamine2000 (A). JIMT-1 cells were also transfected with GFP using
Effectene (B) or by electroporation (C). Cells were stained by CTX-B 2 days after transfection and the cells were analyzed by confocal
microscopy. The mean intensity of GFP fluorescence is plotted against CTX-B fluorescence intensity on a cell-by-cell basis.
ORIGINAL ARTICLE
Cytometry Part A � 75A: 650�657, 2009 655
plausible to assume that the endocytosis of DNA complexed
with cationic liposomes is generally inhibited by a high density
of GM1-enriched lipid rafts, since the overall endocytic and
post-endocytic route of different lipoplex formulations is
known to be very similar (6).
The inhibition of cationic liposome-mediated transfec-
tion by GM1-enriched membrane microdomains seems to be
a general rule, since (i) it was observed in several cell lines; (ii)
it was reproduced with two transfection agents; (iii) it was not
observed with electroporation. The latter finding also supports
the conclusion that GM1-enriched membrane microdomains
inhibit the endocytic step of transfection, since in electropora-
tion DNA is taken up by cells through voltage-induced mem-
brane pores bypassing the endocytic machinery (30).
It is widely accepted that endocytosis of the lipoplex is
much more efficient than later steps of transfection, therefore
lipoplex internalization usually does not play a decisive role in
determining the outcome of transfection (5,29). However, the
results presented in the article show that endocytosis is the
limiting step (or one of the limiting steps) of transfection if
the density of GM1-enriched membrane rafts is high. We pro-
pose that the process of transfection is hindered in cells with a
high density of GM1-enriched lipid rafts at the level of endo-
cytosis. These cells take up a substantially lower amount of
lipoplex from which only insufficient quantities are able to get
out of endosomes and into nuclei. The inhibitory role of
GM1-enriched membrane microdomains is manifested at the
single cell level (i.e., within a population of cells) and when
different cell types are compared. Single cells and cell types
with a low density of GM1-enriched membrane microdomains
take up the lipoplex more efficiently, and later steps of the
transfection process limit the efficiency of transfection in
them.
We do not know the underlying principle behind the
inhibitory role of GM1-enriched membrane microdomains in
lipofection. Although it is usually accepted that uptake of
lipoplexes leading to gene expression proceeds via the
clathrin-dependent pathway (7), such a profound inhibition
of the process by a high density of GM1-enriched membrane
microdomains has not been observed. The fact that the
expression level of clathrin was independent of CTX-B stain-
ing and that transferrin endocytosis was only weakly
influenced by the density of GM1-enriched membrane micro-
domains argues that receptor-mediated endocytosis is not
generally inhibited by a high density of GM1-enriched rafts. It
is known that lipid rafts contain a high concentration of gang-
liosides each type of which contains one or more negatively
charged sialic acid. The high density of lipid rafts may change
the surface charge of cells in such a way which hinders the
interaction of the cell surface with lipoplexes which themselves
contain both positive and negative charges. It is worth men-
tioning that HeLa cells seemed to be an exception to the
inhibitory role of GM1-enriched membrane microdomains on
lipofection, since there was no correlation between the density
of GM1-enriched lipid rafts and transfection efficiency in this
cell line (our unpublished observation). The mechanism of
post-endocytic processing of internalized DNA complexes in
HeLa may be different from other cells, since DNA complexed
with polyethylenimine (PEI) polyplexes (branched or linear)
was taken up and expressed by HeLa cells by both the clathrin-
and raft/caveolae-mediated pathways, but the latter was in
general more efficient. In contrast, in other cell types (COS-7,
HUH-7) clathrin-dependent endocytosis was more relevant
for transfection by PEI (8). However, there is some contro-
versy since another study put forward that polyplex-mediated
transfection proceeds via the caveolae-dependent pathway in
general (9). However, lipoplex-mediated uptake of DNA was
found to be mediated by the clathrin-dependent pathway even
in HeLa cells (9). Although the evidence for the distinct post-
endocytic sorting of internalized DNA complexes by HeLa
cells is controversial, it can still be assumed that cell specific
parameters typical of HeLa cells may explain the lack of corre-
lation between transfection efficiency and GM1-enriched raft
density in these cells.
We have considered alternative explanations for the
observed anticorrelation between raft density, i.e., fluorescence
of AlexaFluor647-CTX-B, and transfection efficiency, i.e., the
fluorescence of fluorescein-labeled plasmid or that of GFP.
Fluorescence resonance energy transfer (FRET) between GFP
(or fluorescein) and AlexaFluor647 could account for the
observed anticorrelation, but the large separation between the
spectra of the dyes makes FRET very unlikely to happen. Alter-
natively, transfection could lead to such modifications in lipid
raft structure that would block the binding of CTX-B, i.e.,
instead of our assumption that GM1-enriched lipid rafts
inhibit transfection, the transfection process itself could alter
the binding of CTX-B to cells. However, we consider this unli-
kely since (i) the negative correlation was observed with differ-
ent transfection agents; (ii) expression vectors producing a raft
protein (GFP-GPI) or a cytoplasmic protein (GFP) lead to
similar results; (iii) the uptake of fluorescently labeled plasmid
which did not produce any protein was also inversely
Figure 7. Relationship between the density of GM1-enriched lipid
rafts, clathrin expression, and transferrin uptake. (A): JIMT-1 cells
were stained with CTX-B (red channel) followed by fixation, per-
meabilization and staining with a monoclonal antibody against
the light chain of clathrin (blue channel). The scale bar corre-
sponds to 15 lm. (B): JIMT-1 cells starved in the presence of 0.1%FCS in high iron medium (Medium199) were incubated in the pre-
sence of 10 lg/ml AlexaFluor488-transferrin (blue channel) for 60min at 378C followed by staining with AlexaFluor647-CTX-B (redchannel). The scale bar corresponds to 10 lm.
ORIGINAL ARTICLE
656 Lipid Rafts Inhibit Transfection
correlated with CTX-B intensity; (iv) the CTX-B intensity cor-
related inversely with the observed and known transfectability
of several cell lines; (v) the negative correlation between trans-
fection efficiency and CTX-B binding was not observed after
electroporation; (vi) lipofection did not lead to any change in
the binding of CTX-B. Therefore, we conclude that a high
density of GM1-enriched lipid rafts inhibits transfection
mediated by cationic liposomes.
GFP-GPI is used as a marker of lipid rafts (15,31). The
results presented in this article suggest that care should be
taken when interpreting these results, since GFP-GPI transfec-
tion does not label a subpopulation of cells in which the
density of GM1 gangliosides is very high. In addition to this
high-GM1, low-GFP-GPI subpopulation we found two more
subpopulations among these cells (Fig. 1A). There were cells
which had a low amount of GM1-enriched lipid rafts, there-
fore they expressed a high amount of GFP-GPI, since endocy-
tosis of the lipoplex was not inhibited in them. Although the
third subpopulation expressed a high amount of GM1-
enriched rafts, they got transfected, since the inhibition of
lipoplex endocytosis was not complete. These cells displayed
the expected positive correlation between the two lipid raft
markers.
In conclusion, the results presented in this article identify
high density of GM1-enriched lipid rafts as a strong negative
predictor of efficient transfection mediated by cationic lipo-
somes and suggest that productive endocytosis inhibited by
GM1-enriched membrane microdomains limits the efficiency
of transfection if the density of GM1-enriched lipid rafts is
high. The fact that cell lines with a high density of GM1-
enriched rafts exhibited low transfectability can be used to
select experimental model systems. In the future these princi-
ples can also be used to design better lipid formulations for in
vitro and in vivo gene transfer.
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ORIGINAL ARTICLE
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