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J. Biol. Chem., Vol. 276, Issue 42, 39476-39483, October 19, 2001
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From the
Received for publication, June 1, 2001, and in revised form, August 9, 2001
A human genetic disorder, Tangier disease, has
been linked recently to mutations in ATP-binding cassette protein A1
(ABCA1). In addition to its function in apoprotein
A-I-mediated lipid removal, ABCA1 was also shown to be a
phosphatidylserine (PS) translocase that facilitates PS exofacial
flipping. This PS translocation is crucial for the plasma membrane to
produce protrusions enabling the engulfment of apoptotic cells. In this
report, we show that ABCA1 also plays a role in endocytosis.
Receptor-mediated endocytosis, probed by both transferrin and low
density lipoprotein, is up-regulated by more than 50% in homozygous
Tangier fibroblasts in comparison with controls. Fluid-phase uptake is
increased similarly. We also demonstrate that bulk membrane flow,
including lipid endocytosis and exocytosis, is accelerated greatly in
Tangier cells. Moreover, endocytosis is similarly enhanced in normal
fibroblasts when ABCA1 function is inhibited by glyburide, whereas
glyburide has no effect on endocytosis in Tangier cells. In addition,
we demonstrate a decreased annexin V binding in Tangier fibroblasts as
compared with controls, supporting the notion that PS transmembrane
distribution is indeed defective in the presence of ABCA1 mutations.
Furthermore, adding a PS analog to the exofacial leaflet of the plasma
membrane normalizes endocytosis in Tangier cells. Taken together, these data demonstrate that ABCA1 plays an important role in endocytosis. We
speculate that this is related to the PS translocase function of
ABCA1. A loss of functional ABCA1, as in the case of Tangier cells, enhances membrane inward bending and facilitates endocytosis.
ATP-binding cassette
(ABC)1 protein A1 belongs to
the ABC transporter superfamily, one of the largest and most highly
conserved gene superfamilies (1). This family of transporters consists, minimally, of a highly conserved ABC-ATPase and a much less conserved multimembrane-spanning domain. By hydrolyzing ATP, ABC transporters are
capable of transporting a wide variety of substrates including lipids
across the membrane. The substrate specificity of ABC transporters is
thought to be determined by the nature of the membrane-spanning domains. In humans, several clinical disorders are linked to defects in
ABC transporters (2).
Tangier disease is a rare genetic disorder in humans characterized by
extremely low plasma concentrations of high density lipoprotein
cholesterol and apoprotein A-I (3). The absence of high density
lipoprotein in Tangier patients was attributed to the impairment of
apoprotein A-I-mediated cholesterol efflux (4). Recently, several
mutations in ABCA1 have been linked genetically to the disease (5-8).
This was confirmed further by studies using a knockout mouse model (9,
10). These animals also demonstrate a nearly complete absence of high
density lipoprotein. The mechanisms by which ABCA1 facilitates
apoprotein A-I-mediated lipid efflux, however, remain largely
unknown. ABCA1 may serve as a receptor on the cell surface for apo-AI
as indicated by chemical crosslinking studies (11, 12). Apo-AI and
ABCA1, however, were shown to have rather distinct diffusional
coefficients on the plasma membrane, indicating that direct interaction
between apo-AI and ABCA1 is limited (13). ABCA1 is known to
specifically transport phosphatidylserine (PS) from the internal
leaflet of the plasma membrane to the exofacial leaflet (PS
translocase) (14). This could influence the lipid microenvironment on
the cell surface and may sequentially facilitate apoprotein A-I binding (13). In addition, the plasma membrane, together with endosomal membranes, is the main reservoir of cellular free cholesterol (15).
Endosomal membrane trafficking may be linked directly to
apo-AI-mediated cholesterol and phospholipid efflux (16).
We have hypothesized that impairment of PS translocase caused by
functional mutations in ABCA1 or inhibition of ABCA1 would result in
the alteration in endosomal membrane trafficking. In the present
report, we provide evidence demonstrating that a loss of functional
ABCA1 results in an increase in endocytosis. Both receptor-mediated
endocytosis and fluid-phase uptake are enhanced in Tangier fibroblasts.
Membrane recycling in the endosomal system is also accelerated.
Importantly, this enhanced endocytosis can be duplicated in normal
fibroblasts by pharmacologically inhibiting ABCA1 function.
Furthermore, endocytosis in Tangier cells can be attenuated by lyso-PS,
a phospholipid analog specifically inserted into the exofacial leaflet
of the plasma membrane.
Cell Culture--
Tangier fibroblasts (TD1 and TD2) and
two normal control fibroblasts (N1 and N2) were obtained from Dr.
J. Oram (Washington University, Seattle, WA). These cells were
immortalized by transfection with human papillomaviruses E6 and E7
(17). Another independent Tangier primary fibroblast cell line (TD3)
and two normal control primary fibroblast cell lines (N3 and N4),
characterized by one of the authors (J. G.) were also studied.
The described mutations were as follows: TD1, Arg-527 to
tryptophan (homozygous), and TD2, Gln-537 to arginine (a compound
heterozygote in which the second allele failed to produce detectable
mRNA) (18). TD3 was a compound heterozygote. All cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and penicillin/streptomycin (100 milliunits/ml).
Cells were used before 20 passages. 2-3 days before experiments, cells
were seeded onto 35-mm coverslip-bottom dishes coated with poly-lysine
(MatTek Corp., Ashland, MA).
Materials and Reagents--
LDL was obtained from the sera of
healthy individuals by density centrifugation. DiI-LDL was prepared as
described (19). Human transferrin (Sigma) was column-purified
and Cy3-labeled following manufacturer instructions (Amersham Pharmacia
Biotech). All the fluorescent reagents were checked by competition
experiments with unlabeled materials. Fluorescein dextran (70 kDa),
BODIPY-C5-SM, FM 1-43, and Alexa Fluor 488 conjugated
annexin V were purchased from Molecular Probes (Eugene, OR). Glyburide
was from Sigma, and
1-oleyl-2-hydroxy-sn-glycero-3[phospho-L-serine]
(lyso-phosphatidylserine or lyso-PS) was from Avanti Polarlipids, Inc.
(Alabaster, AL). All live cell experiments were carried out in medium 1 (150 mM NaCl, 5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, and 20 mM HEPES, pH 7.4) plus either BSA or glucose.
Endocytosis Measurements--
Cells were grown 2-3 days on
coverslip dishes before the experiments. For LDL uptake, cells were
changed into Dulbecco's modified Eagle's medium with 10%
lipoprotein-deficient serum overnight to up-regulate LDL receptors. For
all uptake experiments, the cells were preincubated in Dulbecco's
modified Eagle's medium/HEPES plus 2 mg/ml BSA for 10 min at 37 °C
followed by either Cy3-transferrin (Tf) (10 µg/ml) for 10 min or
DiI-LDL (10 µg/ml) for 30 min in the same medium. The cells were then
rinsed three times by PBS and fixed with 4% paraformaldehyde. Surface
receptor binding was performed by incubating cells on ice with Cy3-Tf
or DiI-LDL for 30 min. This was followed by PBS rinses and fixation on
ice with paraformaldehyde. Fluid-phase endocytosis was measured by
incubating cells with fluorescein-dextran (5 mg/ml) for 30 min at
37 °C. The cells then were rinsed with PBS three times and fixed
with 4% paraformaldehyde.
Membrane Recycling--
Liposomes containing
BODIPY-C5-SM were made by mixing BODIPY-C5-SM
and dioleylphosphatidylcholine, both in ethanol, by a molar ratio of
2:3. The mixture was then dried under N2 and
redissolved in ethanol. Liposome stock (20 mM total lipid
concentration) was produced by rapidly injecting lipid mixture into
medium 1 while vortexing followed by dialyzing against PBS overnight at
4 °C to remove the ethanol. Cell surface membrane labeling was
achieved by incubating cells with liposomes in medium 1 (50 µM total lipid concentration) on ice for 30 min. The
cells were then washed three times on ice before chasing at 37 °C
for 10 or 30 min to allow endocytosis to occur. Cells were cooled down
to 4 °C immediately and washed with ice-cold medium 1 containing 5%
fatty acid-free BSA (six times and five min each) to remove remaining
surface label. This was followed by a light fix with paraformaldehyde on ice, and the cells were examined immediately by microscopy.
For membrane recycling, cells were incubated with FM 1-43 (10 µM) in medium 1 plus 2 mg/ml glucose for 15 min at
37 °C to label the endosomal system. Cells were rinsed three times
with room temperature dye-free medium/glucose to remove
surface-associated dye. Cells were then chased on the microscope stage
maintained at 32-34 °C, and a time series of fluorescence images
was acquired at 0, 0.5, 1, 2, 3, 5, 7, 10, 15, and 20 min.
Annexin V Surface Binding--
Both control and Tangier cells
were preincubated on ice for more than 30 min. Cells were then
incubated with Alexa Fluor 488 conjugated annexin (1:5 dilution from
manufacturer stock) in medium 1 for 1 h at 4 °C. After three
washes with cold PBS, cells were lysed with 2% Triton X-100.
The lysates were measured for fluorescence using a spectrofluorometer,
RTC-2000 (Photon Technology International, Inc., Princeton, NJ). The
protein contents in the lysates were determined by the Lowry assay.
Fluorescence Microscopy Measurement--
Most experiments
reported in this paper involved fluorescence intensity measurement
following a procedure described earlier (20, 21). The measurement was
performed on an Olympus IX50 inverted fluorescence microscope equipped
with a cooled 12-bits CCD camera (microMax, Princeton
Instruments). Images were acquired with a 40× (0.75 numerical
aperture) objective to collect fluorescence from the entire cell
thickness. Total fluorescence intensity of each image was measured
using Winview, the program that also drives the CCD camera. After
background correction, the total fluorescence intensity of each image
was then divided by the number of cells (~20-30 cells for
fibroblasts) in the image to give a mean fluorescence intensity/cell (FI/cell). Each data point represented an
average of 5-8 such images (~200 cells). For membrane recycling
experiments, a time series of images was taken from each dish of cells,
and 4-5 dishes were used for each cell line. For each dish,
fluorescence intensity at time 0 was counted as 100% or normalized to
1. Fluorescence intensities at each time point were normalized
according to initial fluorescence intensity
(FIt/FIinitial) to give
relative FI. The data are the average relative FIs at
each time point from 3 or 4 dishes. Curve fitting was a single
exponential decay using SigmaPlot.
ABCA1 Inhibitor, Glyburide, Increases Endocytosis in Human
Fibroblasts--
Because ABCA1 has been shown to influence lipid
distribution in the plasma membrane, we first asked if the inhibition
of ABCA1 would affect endocytosis. Normal human fibroblasts were
preincubated with glyburide for 30 min and then incubated with Tf in
the presence of glyburide for an additional 10 min at 37 °C. Tf
uptake was measured and compared with correspondent untreated cells. As
shown in Fig. 1a, glyburide
treatment resulted in an increase in Tf endocytosis in three
independent control fibroblast cell lines, ranging from 30 to 50%.
This strongly suggested that ABCA1 is indeed involved in membrane
trafficking. We then tested glyburide on three Tangier fibroblast cell
lines. The inhibition of ABCA1 by glyburide, as expected, had no
detectable effect on all the Tangier fibroblasts tested (Fig.
1b). Because Tangier fibroblasts express only nonfunctional
ABCA1, we then used these Tangier cells to study in detail the effect
of impaired ABCA1 function on endocytosis.
Receptor-mediated Endocytosis Is Increased in Tangier
Fibroblasts--
We then compared receptor-mediated endocytosis in
control and Tangier fibroblasts. Tf and LDL are known to bind to
surface receptors and to be internalized through clathrin-coated pits (22). Fibroblasts were incubated with Cy3-Tf for 10 min at 37 °C
before microscopy observation. A typical Tf intracellular distribution in control fibroblasts is shown in Fig.
2a (left). Tf is
seen in punctate endosome structures throughout the cells. There were also bright perinuclear clusters in most of the cells, presumably the
recycling endosomes. Tf uptake in two Tangier fibroblasts (TD1 and TD2)
evidently is greater compared with the two normal (N1 and N2) cell
lines. This was verified by quantitative measurements in three Tangier
fibroblasts and three normal controls as shown in Fig. 2b.
There is an ~50% increase of Tf endocytosis during a 10-min
incubation in Tangier cells in comparison with controls. Increased
endocytosis of Tf is not caused by an altered surface receptor
expression, because Tf surface binding was similar in all six cell
lines (Fig. 2b, light gray bar).
We next measured LDL uptake in these cells. Both control and Tangier
fibroblasts were incubated with DiI-LDL for 30 min at 37 °C and then
fixed for microscopy measurements. Typical DiI-LDL distribution is
shown in Fig. 3a. To correct
for possible differences in surface receptor expression, LDL surface
binding was first measured by incubating cells with DiI-LDL on ice for
30 min (Fig. 3b, black bars). This was used to
normalize final DiI-LDL uptake in each cell line (Fig. 3b).
Similar to Tf, DiI-LDL uptake in all three Tangier cell lines is more
than double that of control cells after a 30-min incubation. Together
with Tf uptake results, we conclude that the receptor-mediated
endocytosis is up-regulated in Tangier cells including both
immortalized and primary Tangier fibroblasts.
Fluid-phase Uptake Is also Increased in Tangier
Fibroblasts--
In addition to receptor-mediated endocytosis,
extracellular nutrients can also be taken up by cells through other
mechanisms such as pinocytosis (23). A fluid-phase marker,
fluorescein-dextran, was used to measure overall endocytosis. The cells
were incubated with dextran for 30 min at 37 °C, and the amount of
uptake was quantified. The two Tangier cell lines accumulated more than
double the amount of dextran during a 30-min period as compared with normal control cells (Fig. 4).
Both Membrane Lipid Endocytosis and Recycling Is Accelerated in
Tangier Fibroblasts--
Another method to examine endocytosis is to
monitor membrane lipid flow. A short chain sphingomyelin fluorescence
analog (BODIPY-C5-SM) was used to assay membrane
endocytosis (24). Cells were incubated on ice with liposomes containing
BODIPY-C5-SM to allow the sphingomyelin analog to insert
into the cell surface. The cells then were rinsed with dye-free medium
and chased at 37 °C for 10 or 30 min to allow endocytosis to occur.
By this time, a population of endosomes containing
BODIPY-C5-SM became visible (Fig.
5a). The majority of the
labeling was still on the cell surface, and the overall degree of
labeling was similar in Tangier and control cells. The cells were then
cooled down on ice once again, and the remaining surface
BODIPY-C5-SM was removed by back exchange with fatty
acid-free BSA. The only BODIPY-C5-SM remaining with cells
at this point was within the endosomes. Intracellular
BODIPY-C5-SM was then quantified as shown in Fig.
5b. BODIPY-C5-SM endocytosis was more than
doubled in Tangier fibroblasts in comparison with normal controls at
both 10 and 30 min. This is again in agreement with results described
earlier.
Membrane recycling was measured with a fluorescence lipophilic dye, FM
1-43. This dye partitions between membrane and aqueous phase and is
fluorescent only in the membrane (a 105 increase in quantum
yield) (25). Cells were incubated with FM 1-43 for 15 min at 37 °C
to label endosomal compartments. The cells then were rinsed several
times with medium to wash off the dye on the cell surface. The only dye
left associated with cells at this time was in the endosomal
compartments. The cells were then chased in a dye-free medium. During
the chase, lipids in the endosomal compartments move back to the cell
surface (membrane recycling), and FM 1-43 loses its fluorescence upon
reaching the surface by rapidly dissociating from membrane. The rate of
cell-associated fluorescence decay, therefore, is an indicator of
membrane exocytosis (26). When this experiment was performed with
control cells, membrane lipids recycle back to the cell surface with
t1/2 ~ 9 min (Fig.
6), similar to the rate observed by
others (26). In Tangier cells, however, the fluorescence decay is
faster with t1/2 ~ 5 min. This indicates that the
membrane flow from endosomal compartments back to the cell surface is
accelerated in Tangier fibroblasts. Together with our observations with
BODIPY-C5-SM described above, we conclude that the rate of
membrane recycling in Tangier cells is about twice that of normal
control fibroblasts.
Exogenous Lyso-PS Attenuates Endocytosis in Tangier Fibroblasts But
Not in Normal Cells--
ABCA1 has shown to be a PS translocase that
transports PS from the internal leaflet to the exofacial leaflet of the
membrane. The loss of functional ABCA1, such as in Tangier fibroblasts, could lead to an alteration of PS distribution across the bilayers of
the plasma membrane. PS distribution between bilayers is thought to be
maintained minimally by two flippases (27); an aminophospholipid flippase flops PS inward, and ABCA1 flips outward. At steady state, most PS is on the internal leaflet of the plasma membrane with a small
fraction in the exofacial leaflet. With impaired ABCA1 function and a
normal aminophospholipid flippase, however, Tangier fibroblasts may
have less PS in the exofacial leaflet. This might be expected to have
an impact on membrane bending, which consequently could influence
endocytosis. To verify that Tangier cells indeed have an altered PS
distribution, Alexa Fluor 488 conjugated annexin V was used to
quantitate the amount of PS in the outer leaflets. Annexin V is
known to have a high affinity for PS and is widely used to detect
apoptotic cells where the membrane asymmetry is lost (50). Alexa 488 annexin V surface binding (1 h at 0 °C) was extremely low in
fibroblasts, and we failed to detect any significant signal by
fluorescent microscopy, indicating a predominant inner leaflet PS
distribution in nonapoptotic cells. When we, however, measured annexin
V binding by fluorospectrometer using lysates from a large number of
cells, we consistently could detect decreased annexin V binding in
Tangier fibroblasts (Fig. 7). This supports the notion that PS transmembrane distribution is indeed defective in the presence of ABCA1 mutations.
To determine whether enhanced endocytosis in Tangier cells is caused by
an alteration in PS distribution, we used a lyso-PS that shares the
same head group as PS but lacks one of the fatty acid chains. Lyso-PS,
unlike PS, is not the substrate of aminophospholipid flippase (28).
This enabled us to specifically supplement PS to the exofacial leaflet
and possibly to "restore" Tangier cells to "normal" to some
extent. Both immortalized and primary Tangier as well as normal
fibroblasts were preincubated with lyso-PS (10 µM) for 20 min and then incubated with Tf in the presence of lyso-PS for 10 min at
37 °C. As shown in Fig. 8, Tf
endocytosis was decreased by 30% in the two Tangier fibroblast cell
lines treated with lyso-PS, whereas lyso-PS at this concentration had
no significant effect on normal cells. The number of Tf receptors on
the cell surface was not altered significantly by lyso-PS treatment
(data not shown). This suggests that enhanced endocytosis in Tangier
cells could be the direct consequence of an alteration in PS
distribution caused by the loss of functional ABCA1.
The studies presented here demonstrate that the loss of ABCA1
function in Tangier fibroblasts is associated with enhanced endocytosis. Although the three Tangier cell lines employed here have
different mutations in ABCA1, all share a common phenotype: the total
absence of apo-AI-mediated lipid efflux (17, 29). This was also
observed in cells lacking ABCA1 or its equivalent such as in the
ABCA1 Using three primary or immortalized Tangier cell lines, we obtained
several lines of evidence indicating that ABCA1 plays a role in
membrane trafficking. First, we demonstrated that receptor-mediated endocytosis, specifically the uptake of Tf and LDL, was increased significantly in Tangier fibroblasts. We observed similar increases of
fluid-phase uptake and endosomal membrane recycling in Tangier cells.
Importantly, we demonstrated that the Tangier phenotype of enhanced
endocytosis could be reproduced in normal control fibroblasts by a
specific ABCA1 inhibitor, glyburide (31). As anticipated, this
inhibitor had no effect on endocytosis in the three Tangier
fibroblasts. Interestingly, glyburide has also been shown to have a
potent inhibitory effect on cholesterol and lipid efflux in normal
cells, reproducing what is observed in Tangier cells (32). These data
support our conclusion that the loss of functional ABCA1 in Tangier
cells is indeed responsible for the observed alteration in endocytosis.
It is not entirely clear at present how ABCA1 might regulate
endocytosis. Signaling processes such as activation of phospholipases D
and C were shown to be partially impaired in Tangier cells (33). This
could have an impact on membrane trafficking. Activation of
phospholipase D or C, however, was shown to increase endocytosis (34,
35). It is therefore unlikely that the defects in the phospholipase D
or C signaling pathway could explain our observations of enhanced
endocytosis in Tangier cells.
Alternatively, as suggested by the "rescue effect" of lyso-PS in
Tangier fibroblasts, ABCA1 is necessary for the maintenance of
cross-leaflet PS distribution of the plasma membrane. Lipids in the
plasma membrane are distributed asymmetrically (36), with
phosphatidylcholine and SM mainly in the exofacial leaflet and
phosphatidylethanolamine and PS in the internal leaflet of the
membrane. This asymmetry is maintained actively by flippases and
translocases that utilize ATP (37). Such a system provides cells with a
stable yet highly responsive and dynamic membrane. Within a lipid
bilayer, for example, any unidirectional lipid flipping or flopping
between two leaflets would cause an alteration of relative surface area
of the leaflets and therefore a change in membrane curvature (27). This
would facilitate either inward (invagination) or outward membrane
bending (evagination) (38). Membrane bending then initiates
vesiculation leading to either endocytosis or blebbing.
Although some invaginations on the cell surface are assisted by protein
coatings such as clathrin-coated pits or caveolae, a large number of
vesiculation events occur without apparent coating. This is evident by
the fact that fluid-phase uptake, a measure of overall vesiculation
events, is rather insensitive to the inhibition of clathrin-coated pit
endocytosis (39). This implies that vesiculation events could occur
simply by the modifications of membrane leaflets, possibly through a
dynamic adjustment of relative surface areas in a lipid bilayer.
Hydrolysis of sphingomyelin (an exofacial leaflet lipid on the plasma
membrane), for example, results in extensive vesiculations in the
absence of visible coatings and is independent of ATP (40). This has
been attributed to a decrease of surface area in the exofacial leaflet
of the membrane, which in turn forces inward membrane bending (40).
Similarly, enrichment of the internal leaflet with exogenous PS is
known to produce an increase in endocytosis (41) that was
dose-dependent and relying on PS being translocated into
the internal leaflet of the plasma membrane by an aminophospholipid
flippase (42). A lyso-PS, similar to the one employed in our study, had
an inhibitory effect on endocytosis by remaining on the exofacial
leaflet of the membrane (28). Interestingly, both receptor-mediated and fluid-phase endocytosis was affected in this case, similar to our
observations. When lyso-PS was added in a concentration that did not
have any significant impact on the endocytosis of cells that have
functional ABCA1, we found that endocytosis in Tangier cells was
attenuated significantly. We cannot rule out the possibility that
Tangier cells may have other metabolic defects causing enhanced endocytosis. The facts that Tangier cells have less PS on exofacial leaflet of the membrane and lyso-PS can differentially affect Tangier
and normal cells, however, point to the importance of PS in Tangier
phenotypes including endocytosis.
If indeed ABCA1 functions as PS flippase, it would be responsible for
supplying extra PS to the exofacial leaflet of the membrane while
depleting PS from internal leaflet. Both these movements favor an
outward membrane bending (43). This in fact is consistent with the
observation that ABCA1 or its homologue is required for the engulfment
of apoptotic cells (31, 44), a process characterized by membrane
protrusion initiated by an outward membrane bending (45). A loss of
functional ABCA1 would lead to relatively more PS in the inner leaflet
of the membrane. This relative excess inner leaflet area not only
suppresses membrane protrusion (engulfment), but also favors membrane
inward bending, which in turn facilitates endocytosis. Our
interpretation is also in line with the recent finding that another ABC
transporter, Drs2p, influences the formation of clathrin-coated
vesicles from the trans-Golgi network in yeast (46).
This ABC transporter was shown to be an aminophospholipid flippase
(47) that translocates PS or phosphatidylethanolamine from luminal
leaflet to cytoplasmic leaflet on the trans-Golgi network.
This may imply that although the budding of clathrin-coated vesicles
depends on protein coats, changes in membrane bending energy caused by
defects in lipid flippase (Drs2P) or translocase (ABCA1) could add an
additional driving force to inhibit or promote initial invagination.
The most striking phenotype of Tangier disease is the total absence of
lipid efflux from these cells. Recently, a retroendocytosis model has
been proposed as a possible mechanism for lipid efflux (16). Lipid
acceptors, either apo-AI or high density lipoprotein, may constantly
traffic through endosomes and then recycle back to the cell surface.
Possibly within endosomal organelles, the acceptors acquire lipids.
Tangier fibroblasts have an enhanced endocytosis yet totally lack lipid
efflux. This apparent paradox may be resolved by the fact that the
binding of apo-AI is correlated positively with the expression of a
functional ABCA1 (12, 13). Without functional ABCA1, Tangier cells
would not be able to bind to apo-AI efficiently or to shed lipid.
Apo-AI cell association and binding in these fibroblasts is extremely
low, and in these studies we failed to detect any significant binding
or association (data not shown).
An outstanding question regarding ABCA1 is how a PS translocase
activity possibly facilitates lipid efflux. Using cell lines with or
without ABCA1 expression, Fielding et al. (32) recently demonstrated that to acquire cholesterol apo-AI must first be lipidated
with phospholipids, especially phosphatidylcholine. Lipidated apo-AI
can then efficiently acquire cholesterol independent of ABCA1
expression. This is in line with recent evidence that ABCA1-mediated
cholesterol efflux does not depend on "rafts," a membrane
microdomain rich in cholesterol and sphingomyelin but depleted in
phosphatidylcholine (48). It seems unlikely that PS can interact with
cholesterol directly. In contrast, the relationship between PS and
phosphatidylcholine may be important and remains to be explored.
In summary, we report that ABCA1 plays an important role in endosomal
membrane trafficking. These data support recent studies indicating that
ABCA1 functions as a PS translocase (14) and are in line with reports
that ABCA1-green fluorescent protein is mainly localized to the
plasma membrane (14, 49). Further work is required to understand
whether ABCA1 also functions in other vesicular transport processes.
We are grateful to Drs. Jack Oram (University
of Washington, Seattle, WA) for providing immortalized Tangier and
control fibroblasts. We also thank Drs. Ross Milne, Zemin Yao, Heidi
McBride, and Gerard Vassiliou for critical comments.
*
This work was supported by a Canadian Institutes of Health
Research (CIHR) Group Grant in Atherosclerosis (44360) (to
R. M.) and CIHR/Wyeth-Ayerst Chair in Cardiovascular Disease (to
R. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence may be addressed. Tel.: 613-761-5256;
Fax: 613-761-5281; E-mail: rmcpherson@ottawaheart.ca or
xzha{at}ottawaheart.ca.
Published, JBC Papers in Press, August 14, 2001, DOI 10.1074/jbc.M105067200
The abbreviations used are:
ABC, ATP-binding cassette;
PS, phosphatidylserine;
TD, Tangier fibroblasts;
N, normal control fibroblast;
LDL, low density lipoprotein;
DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate;
BODIPY, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;
lyso-PS, 1-oleyl-2-hydroxy-sn-glycero-3[phospho-L-serine];
BSA, bovine serum albumin;
Tf, transferrin;
FI, fluorescence intensity;
SM, sphingomyelin.
Endocytosis Is Enhanced in Tangier Fibroblasts
POSSIBLE ROLE OF ATP-BINDING CASSETTE PROTEIN A1 IN ENDOSOMAL
VESICULAR TRANSPORT*
§,§
Lipoprotein and Atherosclerosis Group,
University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario
K1Y 4W7, Canada and the ¶ Division of Cardiology,
McGill University Health Center, Montreal, Quebec H3A 1A1,
Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
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Fig. 1.
ABCA1 inhibitor, glyburide, increases Tf
endocytosis in normal fibroblasts but not in Tangier cells. Both
control (a) and Tangier (b) fibroblasts were
preincubated with or without glyburide (100 µM) for 30 min at 37 °C and then incubated with Cy3-Tf (+ glyburide)
for 10 min. Tf uptake in glyburide-treated cells was normalized to that
of nontreated correspondent cells and presented as relative Tf uptake:
(FI/cell) + g/(FI/cell) 
g. There is a significant increase in Tf endocytosis after
glyburide treatment in control (a) (*, p < 0.001) but not in Tangier fibroblasts (b).
View larger version (48K):
[in a new window]
Fig. 2.
Transferrin endocytosis is up-regulated in
Tangier fibroblast. Both control and Tangier fibroblasts were
incubated with Cy3-Tf for 10 min at 37 °C, and the cells were then
fixed for fluorescence measurement. Intracellular Tf distributions in
two Tangier (TD1 and TD2) and two control
(N1 and N2) cell lines are shown in a.
Tf uptake is shown in b. The uptake of Tf by three Tangier
(TD1, TD2, and TD3) and three control
(N1, N2, and N3) fibroblasts was
quantified by fluorescence microscopy following an established method
(20, 21) (black bars in b). Tf surface bindings
are presented as light gray bars. Each data point represents
an average of FI/cell from 5-8 fields of cells. Error
bars represent the standard deviations among the fields.
View larger version (36K):
[in a new window]
Fig. 3.
LDL uptake is increased in Tangier
fibroblasts. Both control and Tangier fibroblasts were incubated
with DiI-LDL for 30 min at 37 °C. Fluorescence microscopic images of
DiI-LDL uptake are shown in a. Surface receptor expression
was measured by incubating cells on ice with DiI-LDL for 30 min. The
quantitative measurements of DiI-LDL uptake are indicated in
b. The amount of DiI-LDL uptake (FI/cell) is
presented as a light gray bar, and surface binding is
presented as a black bar.
View larger version (9K):
[in a new window]
Fig. 4.
Dextran uptake is up-regulated in Tangier
fibroblasts. Both normal and Tangier fibroblasts were incubated
with fluorescein-dextran (F-dextran) (5 mg/ml) for 30 min at
37 °C. The cells then were fixed and quantified for
FI/cell.
View larger version (59K):
[in a new window]
Fig. 5.
Membrane lipid endocytosis in increased in
Tangier fibroblasts. Both normal and Tangier fibroblasts
(TD1) were labeled on ice with liposomes containing
BODIPY-C5-SM for 30 min, rinsed with ice-cold medium
free of lipid analog, and then warmed to 37 °C for a 10- or 30-min
chase. The live cells were then imaged with fluorescence microscopy.
BODIPY-C5-SM is seen on the plasma membrane and in the
punctate dots, the endosomal compartments (a). The cells
then were cooled on ice and washed with 5% BSA to remove
surface-remaining BODIPY-C5-SM. This resulted in
BODIPY-C5-SM only in the intracellular compartments,
representing the amount of membrane lipid endocytosed during the chase
at 37 °C. The amount of cell-associated BODIPY-C5-SM was
then measured, and the results are shown in b.
View larger version (14K):
[in a new window]
Fig. 6.
Lipid exit from endosomal compartments is
accelerated in Tangier cells. Both control and Tangier
(TD1) cells were labeled with 10 µM FM 1-43 for 15 min at 37 °C and rinsed several times with PBS/glucose at
37 °C. The cells then were moved to a microscope stage maintained at
32-34 °C. Fluorescence images were taken at 0, 0.5, 1, 2, 3, 5, 7, 10, 15, and 20 min. FI at each time point (FIt)
was divided by initial FI (FIinitial) to
normalize cell-associated fluorescence:
FIt/FIinitial. The data
are averages from 3-4 dishes at each time point. Curve fitting was by
a single exponential decay.
View larger version (12K):
[in a new window]
Fig. 7.
Annexin V surface binding is reduced in
Tangier fibroblasts. Both primary control (N3) and
primary Tangier (TD3) fibroblasts were incubated with Alexa
488 conjugated annexin V for 1 h at 0 °C and then lysed with
2% Triton X-100. Cell-associated fluorescence (FI)
was measured and then ratioed to cell protein (mg/ml). A representative
binding from three measurements is shown here, and error
bars are the standard deviations (p < 0.001).
View larger version (16K):
[in a new window]
Fig. 8.
Lyso-phosphatidylserine attenuates Tf
endocytosis in Tangier fibroblasts but not in control cells.
Control (N1, N2, N3, and
N4) and Tangier (TD1 and TD3)
fibroblasts were preincubated with or without lyso-PS (10 µM) for 20 min at 37 °C and then incubated with Cy3-Tf
(+ lyso-PS) for an additional 10 min. Tf uptake in
lyso-PS-treated cells was normalized to nontreated correspondent cells
as described in the Fig. 1 legend. *, p < 0.001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ mouse (9, 14). In addition, overexpression of ABCA1
correlates with an increase in apo-AI-mediated lipid efflux (12, 30).
All these data strongly support an absence of functional ABCA1 in
Tangier cell lines. We therefore used Tangier cells to probe the role
of this transporter in endocytosis.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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