Rapid Nonvesicular Transport of Sterol between the Plasma
Membrane Domains of Polarized Hepatic Cells*
Daniel
Wüstner
§,
Andreas
Herrmann,
Mingming
Hao, and
Frederick R.
Maxfield
From the Department of Biochemistry, Weill Medical
College of Cornell University, New York, New York 10021 and
Humboldt-Universität zu Berlin,
Mathematisch-Naturwissenschaftliche Fakultät I, Institut
für Biologie/Biophysik, Invalidenstrasse 43, D-10115 Berlin, Germany
Received for publication, March 18, 2002, and in revised form, May 10, 2002
 |
ABSTRACT |
We studied the transport of the fluorescent
cholesterol analog dehydroergosterol (DHE) in polarized HepG2 human
hepatoma cells. DHE delivered via methyl-
-cyclodextrin was delivered
to both the apical and basolateral membranes and became concentrated in the apical membrane within 1 min. Intracellular DHE was targeted mainly
to vesicles of the subapical compartment or apical recycling compartment (SAC/ARC), where it colocalized with fluorescent
transferrin and fluorescent analogs of phosphatidylcholine and
sphingomyelin. In contrast, transport of DHE from the plasma membrane
to the trans-Golgi network was found to be very low. Vesicles
containing DHE traversed the cells in both directions, but vesicular
export of DHE from the SAC/ARC to the plasma membrane domains was low. Disruption of the microtubule cytoskeleton disturbed vesicular transport of DHE but not its enrichment in the apical (canalicular) membrane. Transport of DHE to the canalicular membrane after
photobleaching was very rapid (t1/2 = 1.6 min) and was largely ATP-independent in contrast to enrichment of DHE in the
SAC/ARC. Release of DHE from the canalicular membrane was also
ATP-independent but slower than the enrichment of sterol in the biliary
canaliculus (t1/2 = 5.4 min). Canalicular DHE could
completely redistribute to the basolateral plasma membrane but could
not transfer from one cell to the other cell of an HepG2 couplet. We
conclude that sterol shuttles rapidly among the plasma membrane domains
and other membrane organelles and that this nonvesicular pathway
includes fast transbilayer migration.
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INTRODUCTION |
One of the hallmarks of polarized epithelial cells is the
formation of apical and basolateral plasma membrane domains with distinct lipid and protein compositions. These plasma membrane domains
are separated by tight junctions that prevent diffusion of lipids and
proteins in the exoplasmic but not in the cytoplasmic leaflet of the
membrane bilayer (1, 2). Sorting mechanisms in the biosynthetic,
transcytotic, and endocytic recycling pathways ensure the transport of
lipids and proteins to the correct plasma membrane domain (3-5). In
several types of polarized cells cholesterol has been found to be
enriched in the apical membrane along with (glyco)sphingolipids (6).
Moreover, establishment of the polarized state in epithelial cells
depends on cholesterol (7-9). Cholesterol depletion of polarized cells
has been shown to specifically perturb the apical transport of membrane
proteins, including various glycosylphosphatidylinositol-anchored proteins (10). This has been attributed to disruption of cholesterol and (glyco)sphingolipid-rich membrane domains in the trans-Golgi network (TGN)1 (for a review
see Refs. 11 and 12).
Despite recognition of the importance of cholesterol in the properties
of polarized cells, the transport pathways of cholesterol itself remain
obscure. This is largely due to the lack of appropriate cholesterol
analogs that would allow one to follow intracellular transport of
sterols but which also have physical properties closely resembling
those of cholesterol. Dehydroergosterol (DHE) is a natural yeast sterol
that does not have a bulky reporter group. Although differences in the
desorption rate of DHE compared with cholesterol from lipid monolayers
have been reported, DHE shows very similar physicochemical behavior to
cholesterol in respect to its lateral and transverse organization in
model membranes (13-16). DHE can be used by Caenorhabditis
elegans as its only sterol source (17). In pulse-chase
studies we demonstrated recently that DHE has an intracellular
distribution very similar to cholesterol in CHO cells, with the
endocytic recycling compartment as a major sterol pool (18). Thus, DHE
can be used to determine the transport pathways of sterol in polarized
cells and to study the involvement of cholesterol transport in apically
directed membrane trafficking.
Hepatocytes are specifically adapted to secrete cholesterol on their
apical (canalicular) membrane into the bile. Biliary secretion of
cholesterol and bile salts at the canalicular membrane of hepatocytes
along with phosphatidylcholine (PC) is the only pathway of sterol
clearance from the body (19). Thus, these cells have to
transport cholesterol continuously to their apical membrane, and
studying cholesterol dynamics in polarized hepatic cells should set the
stage for understanding sterol transport in epithelial cells in
general. Several lines of evidence suggest that cholesterol destined
for biliary secretion is not synthesized de novo in
hepatocytes but is transported to the liver in a process named reverse
cholesterol transport (20). During reverse cholesterol transport high
density lipoproteins (HDL) carrying cholesterol and cholesteryl esters bind to the basolateral membrane of hepatocytes and deliver their content of free cholesterol to the bile probably independent of uptake
and transport of the lipoprotein particle (20, 21). The intracellular
trafficking pathways of cholesterol destined for biliary secretion in
hepatocytes are unknown. Here we used a well differentiated hepatoma
cell line, HepG2, to study the intracellular transport of DHE by
fluorescence microscopy. HepG2 cells form an apical vacuole between
adjacent cells, which resembles the biliary canaliculus (BC) of
hepatocytes in many respects (22). They have been used previously to
study the polarized transport of fluorescent sphingolipids as well as
the canalicular enrichment of fluorescent PC (23-25). Because the
couplets of polarized HepG2 cells are oriented with the
apical-basolateral axis lying horizontally, they are well suited for
microscopic observation of trafficking between apical and
basolateral membranes.
We incorporated DHE initially into the plasma membrane of HepG2 cells
and studied its intracellular itineraries and apically directed
transport. We found colocalization of DHE with markers of the endocytic
pathway and to a small extent with the TGN. Some vesicles containing
DHE shuttled bidirectionally between both plasma membrane domains.
However, no pronounced vesicle traffic of DHE to either plasma membrane
domain could be observed. Enrichment of DHE in intracellular organelles
was ATP-dependent, but transport of the sterol between the
plasma membrane domains was largely ATP-independent. By using
quantitative fluorescence microscopy and image analysis, including
fluorescence recovery after photobleaching (FRAP), we show that the
transport of DHE to the BC from other membrane components is faster
than the observed vesicle traffic and is not slowed by ATP depletion of
cells. DHE also redistributed from the canalicular to the basolateral
membrane after ATP depletion. DHE could not traverse the BC from one
cell to the other in an HepG2 couplet nor shuttle between neighboring
cells. These results indicate that vesicular transport of sterols
occurs, but nonvesicular pathways appear to predominate in determining
sterol distribution in polarized HepG2 cells.
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EXPERIMENTAL PROCEDURES |
Chemicals
Red fluorescent PC
2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine (-BODIPY-PC),
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine (BODIPY-Cer),
N-[6-[(7-nitro-2-1,3-benzooxadiazol-4-yl)amino]hexanoyl]-sphingosine (C6-NBD-Cer),
2-[6-[(7-nitro-2-1,3-benzooxadiazol-4-y)amino]hexanoyl-1-hexadecanoyl]-sn-glycero-3-phosphatidylcholine, N-[6-[(7-nitro-2-1,3-benzo-oxadiazol-4-y)amino]-hexanoyl]-sphingosyl]-phosphocholine (C6-NBD-SM), rhodamine-labeled dextran (70 kDa), Alexa-488,
Alexa-546, and Alexa-488 phalloidin were purchased from Molecular
Probes. Medium 1 contained 150 mM NaCl, 5 mM
KCl, 1 mM CaCl2, 1 mM
MgCl2, 5 mM glucose, and 20 mM
HEPES (pH 7.4). Medium 2 was identical to medium 1 except that it
contained no glucose but 5 mM sodium azide and 50 mM 2-deoxyglucose for energy depletion of cells (see below). Release medium is Medium 1 supplemented with 25.5 mM citric acid, 24.5 mM sodium citrate, 100 mM deferoxamine mesylate, was adjusted to pH 5.2, and
contained 280 mM sucrose instead of glucose (see above).
Rhodamine-labeled dextran was dissolved in phosphate-buffered saline
and repeatedly dialyzed before use to remove unconjugated dye. Fetal
calf serum and Dulbecco's modified Eagle's medium were from
Invitrogen. All other chemicals were from Sigma. Transferrin (Tf) was
iron-loaded as described previously (26). Succinimidyl ester of
Alexa-488 and Alexa-546 were then conjugated to the iron-loaded Tf
following the manufacturer's instructions. Texas Red-asialoorosomucoid was a generous gift from Dr. John Murray (Albert Einstein College of
Medicine, New York).
Cell Culture
HepG2 cells were grown in Dulbecco's modified Eagle's medium
with 4.5 g/liter glucose, supplemented with 10% heat-inactivated fetal
calf serum and antibiotics. Cells were routinely passaged in plastic
tissue culture dishes. For experiments, cells were plated onto glass
coverslips coated with poly-D-lysine and used after
reaching the highest degree of polarization as described previously
(25).
Preparation of Lipid Labeling Solutions
Fluorescent Lipid Analogs--
Fluorescent phospholipids and
ceramide were stored at 
20 °C in chloroform/methanol (1:1, v/v).
Analogs were transferred to a glass tube and dried under argon. For
labeling of the cells with -BODIPY-PC and C6-NBD-SM, a
previously described protocol was used (25, 27).
DHE-Cyclodextrin Complexes--
A stock solution of DHE (5 mM) was made in ethanol and stored under argon. For
labeling cells with DHE, the analog was loaded on
methyl--cyclodextrin (MCD) as described previously (18). Briefly,
from the stock solution of DHE 750 µl were transferred to a glass
tube, and ethanol was evaporated under argon, and a solution of 30 mM MCD dissolved in Medium 1 with 0.1% (w/v) BSA was
added to get a MCD/DHE-ratio of 1:8 (mol/mol) (28). The solution was
carefully resuspended, vortexed, and bath-sonicated. After
centrifugation at 20,000 × g for 20 min, the
supernatant DHE/MCD solution was carefully collected and stored at
4 °C under argon. The properties of the labeling solution and
reproducibility of the loading procedure was routinely checked by
measuring its fluorescence excitation and emission spectra using a Spex
Fluorolog spectrofluorometer (Spex Industries, Inc., Edison, NJ).
Intracellular Transport of DHE Derived from
DHE/MCD
HepG2 cells were routinely labeled with DHE/MCD for 1 min at
37 °C except for studying time course of labeling (see below). Cells
were washed and further incubated at 37 °C for the indicated time
points (see Fig. 1).
Colocalization Experiments--
For triple labeling cells with
DHE/MCD, -BODIPY-PC and Alexa488-Tf cells were incubated for 10 min at 37 °C with 5 µg/ml Alexa488-Tf, washed with Medium 1, labeled for 1 min with DHE/MCD, washed, and incubated for 2 min at
37 °C with -BODIPY-PC. Cells were washed and subsequently
incubated at 37 °C for 30 min. Cells were briefly
back-exchanged with BSA (2 times for 5 min, 5% BSA w/v on ice),
washed, and fixed with 2% paraformaldehyde (PFA) for 10 min on ice.
For triple labeling cells with DHE/MCD, -BODIPY-PC, and
C6-NBD-Cer, the same protocol was used but without
preincubation with Alexa488-Tf. After fixation with PFA, cells were
washed and incubated with 10 µM C6-NBD-Cer
for 5 min at 37 °C. For colabeling with BODIPY-Cer, cells were
labeled first with DHE/MCD as described above, washed, and chased
for 30 min at 37 °C with 10 µM BODIPY-Cer present
during the last 10 min of incubation. Colocalization studies of DHE
with Texas Red-ASOR were performed as described previously for studying
transport of fluorescent PC in HepG2 cells (25).
Energy Depletion and Uptake of Transferrin--
To deplete ATP,
cells were incubated for 30 min at 37 °C in Medium 2 containing 5 mM sodium azide and 50 mM 2-deoxyglucose. To
quantify clathrin-dependent endocytosis, cells were
incubated for 10 min with 5 µg/ml Alexa488-Tf, washed, and chased for
30 min at 37 °C. Cells were chilled with ice-cold Medium 1 or 2 and incubated in release medium for 10 min at 2 °C to remove
surface-bound Tf by a mild acid wash. Those cells were washed with
Medium 1 or 2 to adjust the pH to 7.4, and uptake of Alexa-Tf was
quantified on the microscope by measuring fluorescence of Alexa-Tf as
intensity per cell (see "Results"). In those experiments designed
to determine the energy dependence of transport processes, all
incubation steps except labeling with DHE/MCD and rhodamine-dextran
were performed in Medium 2.
Incubation with Drugs to Disrupt the Cytoskeleton--
Cells
were incubated with 33 µM nocodazole to disrupt
microtubules for 30 min at 37 °C, prior to labeling with DHE/MCD
as described above. Nocodazole was present during a subsequent chase for 30 min at 37 °C. Visualization of the microtubule cytoskeleton in control and drug-treated cells was performed according to Ref. 25.
Quantification of Cholesterol and Amount of Incorporated
DHE--
Cells growing in 6-well cell culture plates were labeled with
DHE/MCD as described above or kept non-labeled, washed extensively, and incubated for 5 min at 37 °C in trypsin (0.05% v/v). Cells were
washed twice in Medium 1 by centrifugation at 400 × g.
Lipids of the cell pellet were extracted in hexane/isopropyl
alcohol 3:2 (v/v) as described for cholesterol (29). Solvent was
evaporated under argon, and lipids were resolubilized in either
reaction buffer (Molecular Probes) for quantification of cellular
sterols or in ethanol to measure DHE. DHE was quantified by measuring fluorescence intensity in ethanol at 
ex = 326 nm and
em = 370 nm on a Spex Fluorolog spectrofluorometer (Spex
Industries, Inc., Edison, NJ). Sterols were quantified using a
Quantification Kit from Molecular Probes, which is based on hydrolysis
of cholesteryl esters to cholesterol and oxidation of cholesterol by
cholesterol oxidase (30).
Time-lapse Fluorescence Microscopy and Fluorescence Recovery
after Photobleaching (FRAP)
Time Course of Labeling Cells with DHE and Time-lapse
Fluorescence Microscopy of DHE--
HepG2 cells were labeled with 2.5 mg/ml rhodamine-dextran for 1 h or with -BODIPY-PC for 1 min at
37 °C. They were washed and labeled with DHE/MCD for the
indicated time points at 37 °C (see Fig. 1 and above). Cells were
washed and either immediately placed on a temperature-controlled
microscope stage maintained at 35 ± 1 °C of a wide field
microscope or incubated for 30 min at 37 °C prior to imaging,
respectively. BC were identified by rhodamine-dextran or -BODIPY-PC
fluorescence in the red. For studying vesicle-based transport from the
SAC/ARC in cells prelabeled with -BODIPY-PC, DHE in the plasma
membrane was selectively extracted by incubation for 5 min with Medium
1 containing 5 mM MCD + 10 mM
cholesterol-loaded MCD. Cells were washed, and images of DHE fluorescence were acquired every 1 min for a total time of 20 min.
FRAP of DHE--
HepG2 cells were labeled with 2.5 mg/ml
rhodamine-dextran for 1 h (canalicular FRAP) or with -BODIPY-PC
for 1 min at 37 °C (basolateral FRAP) and subsequently with
DHE/MCD as described above. Cells were washed with pre-warmed
buffer, and immediately the coverslip dish was placed on a
temperature-controlled microscope stage maintained at 35 ± 1 °C of a wide field microscope. BC were identified by
rhodamine-dextran fluorescence, and basolateral plasma membrane was
indicated by -BODIPY-PC fluorescence in the red channel right after
placing the dish on the microscope stage. After acquiring a
fluorescence image of rhodamine-dextran or -BODIPY-PC, as well as an
image of DHE (see below), the field aperture of the condenser was
closed to ~13.3 µm diameter while placing the region of interest
(BC or basolateral membrane) in the red channel into the center of the
field. Prelabeling of the cells with rhodamine-dextran or
-BODIPY-PC, respectively, did not influence the FRAP kinetics measured for DHE. By switching the filter set to DHE excitation (see
below) and opening the fluorescence shutter for 1 min, the fluorescence
of DHE was selectively bleached in the BC area or in the basolateral
membrane. To study the transport of the sterol analogs to the bleached
region the shutter was closed, the field aperture opened, and a
fluorescence image of DHE was acquired after the indicated time points
using a cooled CCD camera. Bleaching of an entire cell was complete
after 1 min and did not result in any recovery. For the fluorescence
loss in photobleaching (FLIP) experiment, the DHE in the basolateral
membrane was bleached repeatedly with 3-min intervals between the
bleaches to allow diffusion of DHE. Every bleach in the basolateral
region resulted in a fluorescence loss of DHE in the couplet forming a
BC of about 28%.
FRAP of -BODIPY-PC--
Cells were labeled for 2 min with
-BODIPY-PC at 37 °C, washed, and placed on the microscope
stage as described above for DHE. BC were first identified in the red
channel and placed in the center of the field using a neutral density
filter of 33% in order to prevent bleaching of the fluorophores. For
bleaching of the BC and acquisition of images which was performed as
described above for DHE, no neutral density filters were used.
Fluorescence Microscopy and Image Analysis
Wide field fluorescence microscopy and digital image acquisition
were carried out using a Leica DMIRB microscope with a 63×, 1.4 NA oil
immersion objective (Leica Lasertechnik GmbH, Wetzlar, Germany)
equipped with a Princeton Instruments cooled CCD camera driven by
Image-1/MetaMorph Imaging System software. -BODIPY-PC, Texas
Red-ASOR, and rhodamine-dextran were imaged using a standard rhodamine
filter set (535-nm (50-nm bandpass) excitation filter, 565-nm long pass
dichromatic filter, and 610-nm (75-nm) bandpass) emission filter),
whereas Alexa 488-Tf and NBD-lipids were imaged using a standard
fluorescein filter set (470-nm, (20-nm bandpass) excitation filter,
510-nm long pass dichromatic filter, and 537-nm (23-nm) bandpass)
emission filter). DHE was imaged using a specially designed filter cube
obtained from Chroma Technology Corp. (Brattleboro, VT) with 335-nm
(20-nm bandpass) excitation filter, 365-nm long pass dichromatic
filter, and 405-nm (40-nm bandpass) emission filter as described
previously (31). All other components of the microscope were adapted
for UV imaging as described previously (18). Image analysis was carried
out using the Image-1/MetaMorph Imaging System software (Universal
Imaging Inc.). Determination and subtraction of crossover of
fluorescence between the channels were performed as described (30,
32).
Colocalization and Time-lapse Experiments--
BC were first
identified in the red channel by fluorescence of -BODIPY-PC focusing
in the equatorial plane of the BC. In the case of studying
colocalization with Texas Red-ASOR, a focal position 1 µm above the
largest diameter of the cells was chosen as start point for the
z-stack. Three serial focal plane images 0.5 µm apart above as well
as below this position were acquired subsequently for all channels
(i.e. DHE; -BODIPY-PC or Texas Red-ASOR; and Alexa-Tf,
C6-NBD-Cer, or C6-NBD-SM). The
wavelength-dependent variation in the focal position was
determined independently by acquiring serial focal plane images of 0.1- and 0.5-µm TetraSpec fluorescent beads (Molecular Probes) mounted in
gelvatol on a glass coverslip. No wavelength-dependent
variation in the focal position was found between the red and green
channels. However the corresponding focal plane for DHE was 1 µm
below that of the red and green channel. Thus, a DHE image 1 µm below
that of the other fluorescent markers was chosen to compare the
distribution of the fluorescent probes. For all acquired stacks a
Nearest Neighbor deconvolution algorithm implemented in MetaMorph
software (Universal Imaging Inc.) was applied in order to remove
out-of-focus fluorescence. This was of particular importance for DHE to
improve the spatial resolution. The sum projection of individual stacks
from single corresponding planes of each channel was generated using
MetaMorph software. In time-lapse experiments vesicle positions were
tracked manually by using the method of Barak and Webb (33), and
vesicle velocity was calculated from the distance moved between
successive images. Release of DHE from the SAC/ARC was quantified by
measuring DHE fluorescence in an 8 × 8 pixel box placed in the
center of the SAC/ARC and BC, respectively (Fig. 5).
Fluorescence resonance energy transfer (FRET) has been reported to take
place between DHE (putative donor) and NBD analogs (putative acceptors)
if they reside in the same compartment (34). To test whether FRET
between DHE and NBD analogs might interfere with our colocalization
studies, fluorescence of NBD analogs in double-labeled cells was
bleached as described previously (25). An image of DHE fluorescence was
acquired before and after bleaching NBD fluorescence in those cells.
Fluorescence of DHE was quantified for both images in regions defined
by the fluorescence of the Golgi (for C6-NBD-Cer) or the
SAC/ARC (for C6-NBD-SM) from the corresponding image of the
acceptor (see above) (FRET by sensitized acceptor fluorescence
(35)).
FRAP Experiments--
For each image of the time series, a
background correction was routinely performed by subtracting the
intensity of regions without cells from the whole image. To quantify
fluorescence intensity, regions of interest (ROI) were traced manually
for the BC according to their geometry in the first image of the time
series (i.e. in the prebleach image) and for two cells
forming a BC. For basolateral FRAP an image of -BODIPY-PC with
closed field aperture was chosen for defining the ROI. The integrated
intensity was measured for the defined ROI and for the whole cells. The
ratio, R, of fluorescence of ROI and of whole cells was then
calculated and normalized as described previously (25). Fluorescence
loss of DHE in the canalicular membrane due to basolateral FRAP was
calculated by normalizing all fluorescence ratios of a time series to
the fluorescence ratio in the first image after bleaching. The data of
9 to 10 or 5 measurements for DHE and fluorescent PC, respectively,
were averaged for presentation purposes, whereas all individual
recovery kinetics were fitted to a mono-exponential function using
Sigma Plot 4.0 (SPSS Inc., Chicago).
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RESULTS |
Labeling with DHE/MCD Results in Bright and Exclusive
Staining of the Plasma Membrane Domains of HepG2 Cells--
To follow
the transport of cholesterol analogs from the plasma membrane to
intracellular compartments requires (i) sufficient labeling of cells
with those analogs and (ii) initial staining exclusively of the plasma
membrane. First, the time course of labeling HepG2 cells with DHE was
studied. Cells were labeled for 10-60 s at 37 °C, washed, and
placed on the microscope stage. DHE rapidly inserts into the plasma
membrane of HepG2 cells incubated with the labeling solution containing
DHE loaded on MCD (DHE/MCD) (Fig.
1A). After a 10-s pulse, faint
staining of the cells was found, and after 20 s DHE was seen in
both the basolateral and the canalicular membrane (arrows).
After 60 s of labeling, cellular fluorescence intensity of DHE was
very bright and could not be increased by longer pulse times (not
shown). Thus, in subsequent experiments cells were pulse-labeled for 1 min at 37 °C. Note that images in Fig. 1A have the same
intensity scaling to allow visual comparison of the fluorescence
intensity of DHE after different times of labeling. The total cellular
fluorescence intensity of DHE is more than 5-fold above cellular
autofluorescence in the UV region of the spectrum after a 1-min pulse
at 37 °C. The amount of incorporated DHE corresponded to 10 mol %
of sterols in the cells. Under our labeling conditions about 0.5% of
DHE from DHE/MCD became incorporated into the plasma membrane of
HepG2 cells. Incubating cells with DHE/MCD did not alter the total
sterol content of HepG2 cells which was 20 ± 4 nmol per
106 cells. This indicates that DHE supplied to cells via
MCD replaces cholesterol in the plasma membrane without altering
cellular sterol homeostasis. We tested uptake of Tf and found no
difference between DHE-labeled and non-labeled cells, indicating that
clathrin-coated pit internalization, which is altered by cholesterol
depletion (36, 37), is not affected by the labeling procedure.
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Fig. 1.
Transport of DHE after pulse labeling of the
plasma membrane with DHE/MCD.
A, HepG2 cells were labeled with rhodamine-dextran (70 kDa)
for 1 h at 37 °C, washed, and labeled with DHE/MCD for
10 s, 20 s, or 1 min at 37 °C. All images in the
upper panel are of the same intensity scaling. The
lower panel shows rhodamine-dextran fluorescent BC
(arrow) corresponding to the upper panel for DHE.
B, cells were labeled with DHE/MCD for 1 min at 37 °C,
washed, and incubated for 0, 7, or 30 min at 37 °C. Bright labeling
of the basolateral and canalicular membrane (thick arrows)
but no intracellular staining was observed after pulse labeling.
Already after 7 min of incubation DHE-labeled vesicles (thin
arrows) and a perinuclear enrichment of DHE in non-polarized cells
(thin arrowheads) were found. Incubation for 30 min at
37 °C resulted in a subapical enrichment of DHE in polarized cells
(thick arrowheads). Bar, 20 µm.
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In Fig. 1B the time course of uptake of DHE from the plasma
membrane is shown. In contrast, to Fig. 1A, the contrast of
images was adjusted to distinguish plasma membrane labeling of DHE from its intracellular fluorescence in labeled compartments. Labeling for 1 min at 37 °C resulted in bright, homogeneous staining of the
plasma membrane of HepG2 cells with DHE (Fig. 1B, 0 min chase). After a 7-min chase, diffuse cytoplasmic staining as
well as vesicles carrying DHE (thin arrows) became apparent
(Fig. 1B). After 30 min of chase at 37 °C, additional
accumulation of DHE in the perinuclear (thin arrowheads) or
subapical region (thick arrowheads) in nonpolarized and
polarized cells was found, respectively. At later time points, as the
labeling in the perinuclear and subapical regions became brighter,
distinct membrane structures (i.e. vesicles and tubules) could be seen. The DHE distribution did not change significantly after
30 min of chase. The DHE-labeled vesicles and the apparently organelle-associated intracellular DHE distribution at steady state
indicated that a vesicular pathway might transport DHE between intracellular organelles in HepG2 cells.
DHE Colocalizes with Markers of the Endocytic Recycling System in
HepG2 Cells--
To identify the organelles along the intracellular
itinerary of DHE, cells were colabeled with DHE/MCD, the fluorescent
PC analog -BODIPY-PC (fluorescence emission in red), and Alexa488-Tf (fluorescence in green), chased for 30 min at 37 °C, fixed, and observed by fluorescence microscopy. The trafficking of -BODIPY-PC in HepG2 cells has been described previously (25). In polarized couplets of HepG2 cells, DHE as well as -BODIPY-PC were enriched in
the canalicular membrane (thick arrows), and they
colocalized in vesicles in the subapical region of the cells
(thick arrowheads) and in vesicles scattered throughout the
cytoplasm (thin arrowheads) (Fig.
2, A and B). These
vesicles were often triple-labeled with both lipid probes and with
transferrin (Tf) (Fig. 2C). In addition, vesicles labeled
with DHE and -BODIPY-PC but not with Alexa-Tf were found (thin
arrows). Colocalization in vesicles in the subapical region is
seen more clearly at higher magnification (thick arrowheads) (Fig. 2, D-F). Because the observed vesicles contain
markers of the recycling pathway (i.e. fluorescent Tf), they
probably belong to a subapical compartment or apical recycling
compartment (SAC/ARC) as described in HepG2 cells as well as in the
hepatocytic cell line WIF-B (24, 25, 38, 39). Some vesicles and tubules labeled only with DHE were found in the subapical region of the cells
(thin arrowheads in Fig. 2D).
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Fig. 2.
Colocalization of DHE with markers of
the endocytic system. A-L, cells were labeled with 5 µg/ml Alexa-Tf, DHE/MCD, and with -BODIPY-PC as described under
"Experimental Procedures." Cells were washed and subsequently
incubated at 37 °C for 30 min, washed, and after a brief
back-exchange with ice-cold BSA (2 times for 5 min, 5% BSA w/v on
ice), fixed with PFA. Fixed cells were imaged as described under
"Experimental Procedures." DHE (A, D,
G, and J) as well as -BODIPY-PC (B,
E, H, and K) became enriched in the
canalicular membrane (thick arrows). All three probes were
found in the subapical region (thick arrowheads)
(A-C). Additionally, vesicles containing all probes
(A-C, thin arrowheads) or only DHE and
-BODIPY-PC (A-C, thin arrow) but not Alexa-Tf
(C, F, I, and L) were found
throughout the cytoplasm. The zoomed regions of A-C show
that DHE colocalizes with -BODIPY-PC and Alexa-Tf (thick
arrowheads) in vesicles underneath the BC in polarized cells
(D-F) and in a perinuclear compartment in
nonpolarized cells (J-L), respectively. Vesicles and
tubular structures labeled only with DHE were also found (thin
arrowheads). M-O, DHE (M) colocalizes with
C6-NBD-SM (N) in the canalicular membrane
(central ring) and in adjacent vesicles (thick
arrowheads); however, also vesicles only labeled with DHE but not
with C6-NBD-SM were found (thin arrowhead).
O, overlay with DHE in green and
C6-NBD-SM in red, colocalizing regions appear
orange. Bar, 20 µm.
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Colocalization of DHE with -BODIPY-PC and Alexa488-Tf was also found
in non-polarized HepG2 cells (Fig. 2, G-L). Those cells are
defined by the absence of an apical membrane (i.e. no BC) and the perinuclear localization of recycling endosomes similar to that
found in CHO cells (17, 23). In non-polarized HepG2 cells overlap of
all markers in the perinuclear region was found (Fig. 2,
G-I, arrows). Triple-labeled vesicles were
detected in this area (Fig. 2, J-L, thick
arrowheads). The degree of colocalization of DHE with the
recycling markers seemed to be higher in non-polarized cells than in
polarized cells. These results show that DHE partially associates with
the recycling pathway in both polarized and non-polarized HepG2 cells.
Previously, it was shown (23) that fluorescent sphingolipids are
transcytosed and delivered to the BC via a subapical compartment. As
shown in Fig. 2, M-O, DHE colocalizes in the BC as well as in subapically enriched vesicles (thick arrowheads) with
C6-NBD-SM. In addition, vesicles labeled only with DHE
(thin arrowheads) were found. These results show that some
DHE derived from the plasma membrane is delivered to the SAC/ARC, which
contains markers of the recycling as well as the transcytotic pathway.
The TGN Is Not a Major Pool of Intracellular DHE--
One site of
intracellular cholesterol accumulation has been proposed to be the TGN
(for a review see Refs. 12 and 40). We examined whether DHE derived
from the plasma membrane is transported to the TGN. First cells were
colabeled with DHE and BODIPY-Cer, which is a vital stain for the TGN
(Fig. 3, A-I). DHE
colocalized to some extent in tubules with BODIPY-Cer detected in the
green channel in polarized cells (Fig. 3,
A-C, arrowheads). However, regions with
enrichment of BODIPY-Cer, identified by its red emission due
to formation of excimers (41), largely lacked DHE (not shown). This
suggests that DHE is not highly enriched in the TGN. In nonpolarized HepG2 cells DHE was mostly found in the perinuclear region. Some DHE
did overlap in tubules or dots containing BODIPY-Cer in the rim
surrounding the perinuclear area (thin arrowheads). A very similar fluorescence pattern was observed in cells that are in the
process of forming a BC (semi-polarized cells). Those HepG2 cells are
characterized by the diaphragm-like structure on the apical pole of the
adjacent cells still segregating the two canalicular membranes (Fig. 3,
G-I, arrows). In those semi-polarized cells enrichment of DHE in the two regions resembling the recycling compartment (thick arrowheads) and the TGN (thin
arrowheads) was found, but both compartments did not yet have a
subapical location like in fully polarized cells (see Fig. 3,
A-C). In an independent experiment colocalization of DHE
and -BODIPY-PC with C6-NBD-Cer, another vital stain for
the TGN (42), was investigated. The TGN, labeled with
C6-NBD-Cer, was not a major site for accumulation of DHE or
-BODIPY-PC (Fig. 3, J-O). The nearest neighbor
correction for out of focus fluorescence imaging protocol outlined
under "Experimental Procedures" allowed us to clearly distinguish
Golgi tubules or stacks from vesicles stained with DHE and
-BODIPY-PC. Typically, DHE and -BODIPY-PC were excluded from
C6-NBD-Cer positive Golgi tubules (see outlined
tubules in Fig. 3, J-L and M-O for polarized and nonpolarized cells, respectively). However, part of the
DHE labeling seemed to overlap with C6-NBD-Cer in the
regions adjacent to the outlined tubules (Fig. 3, J-O).
This suggests that some of the sterol is also delivered to the TGN.
FRET has been reported to occur between DHE and NBD analogs if they
reside in the same compartment in the cell (34), and this could have reduced DHE fluorescence in the TGN. We tested this possibility by
bleaching the putative acceptor, C6-NBD-Cer (35). No
fluorescence increase of DHE could be detected after bleaching of
C6-NBD-Cer, so the possibility that part of DHE overlapping
with C6-NBD-Cer is quenched in the TGN can be excluded. For
both ceramide analogs we found no increased colocalization in cells
labeled with DHE and chased for 1.5 h at 37 °C (not shown).
This indicates that DHE residing in the SAC/ARC or the plasma membrane
does not redistribute significantly to the TGN after longer incubation.
Taken together, the results indicate that DHE is targeted to some
extent to the TGN, but they also show that this organelle is not a
major pool for DHE derived from the plasma membrane in HepG2 cells.
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Fig. 3.
Transport of DHE to the TGN in polarized and
in nonpolarized HepG2 cells. Cells were labeled with DHE/MCD,
washed, and incubated for 20 min at 37 °C. Cells were labeled with
BODIPY-Cer for 10 min at 37 °C, washed, and imaged. DHE
(A, D, and G) labeled the BC
(arrow) and colocalized with BODIPY-Cer (B,
E, and H) in tubules (thin arrowheads)
underneath the BC in polarized cells BC (A-C) and of a
perinuclear rim or dot in non-polarized (D-F)
and semi-polarized cells (G-I), respectively. DHE
additionally stained a clearly segregated region in the perinuclear
area of non-polarized (D-F) and semi-polarized
cells (G-I) (thick arrowheads).
C, F, and I, overlay with DHE in
green and BODIPY-Cer in red. Colocalizing regions
appear orange. J-O, HepG2 cells were labeled with
DHE/MCD and with -BODIPY-PC as described under "Experimental
Procedures." Cells were washed and subsequently incubated at 37 °C
for 30 min, washed, and after a brief back-exchange (2 × 5 min,
5% BSA w/v on ice), fixed with PFA. Fixed cells were incubated for 5 min at 37 °C with C6-NBD-Cer, washed, and imaged as
described under "Experimental Procedures." DHE (J and
M) as well as -BODIPY-PC (K and N)
became enriched in the canalicular membrane (arrows), in a
subapical region in polarized cells (J-L), and a
perinuclear region in nonpolarized cells (M-O),
respectively. Only partial overlap is found in the central part of
those regions whereas most of C6-NBD-Cer (L and
O) is in tubules not containing DHE or -BODIPY-PC (see
outlined areas). Bar, 20 µm.
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Bidirectional Movement of DHE Carrying Vesicles and the Involvement
of the Cytoskeleton in Transport of DHE--
DHE derived from the
plasma membrane becomes enriched in vesicles of the SAC/ARC as shown by
its colocalization with fluorescent Tf, PC, and sphingolipids (Fig. 2).
The intracellular route and time course of vesicle-based transport of
DHE was next investigated. In time-lapse experiments, we found movement
of some vesicles containing DHE (i) from the basolateral region toward
the BC, (ii) from the apical pole directed toward the basolateral
membrane, and (iii) from the BC toward the SAC/ARC. However, in all
acquired video sequences (n = 15) the number of
vesicles released from the SAC/ARC pool was found to be very low. A
typical time sequence demonstrating all types of movement of
DHE-carrying vesicles is shown in Fig.
4A. In the first 4 min of the
sequence two DHE labeled vesicles (green arrowheads) moved
from the vesicular pool resembling the SAC/ARC (arrow)
toward the basolateral membrane. Between 2 and 4 min after starting the
sequence, a vesicle moved from the BC toward the SAC/ARC pool
(red arrowheads). This suggests apical endocytosis of DHE.
Between 9 and 14 min after starting the time sequence another vesicle
moved from the basolateral region toward the apical pole of the cell.
Longer acquisition was not possible because of artifacts introduced by
photobleaching after about 14 min of recording. Thus, we were unable to
record a complete vesicle path from one plasma membrane domain to the
other. The velocity of the vesicle movements was found to be as fast as
0.18 µm/s as measured by distance moved between two captured images (data from 4 video sequences recorded for 12-14 min). The measured vesicle velocity is similar to that found previously for fluorescent PC
and is in line with those observed for motor driven movement along
microtubule tracks (25, 43). Further evidence that DHE vesicles move on
microtubule tracks was obtained from experiments with drugs disrupting
the microtubule cytoskeleton. Incubating cells with nocodazole resulted
in scattering of the SAC/ARC (24, 25), so DHE-containing vesicles
became dispersed throughout the cytoplasm (Fig. 4C). In
contrast, labeling of the basolateral or canalicular membrane with DHE
was not affected by nocodazole treatment (Fig. 4, B and
C). This indicates that vesicle-based transport of DHE but
not its delivery to either plasma membrane domain in polarized HepG2
cells requires an intact microtubule cytoskeleton.
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Fig. 4.
Visualization of vesicle-based transport of
DHE in polarized HepG2 cells. A, cells were labeled for
1 min at 37 °C with DHE/MCD, washed, incubated for 30 min at
37 °C, and placed on a temperature-controlled microscope stage of a
wide-field microscope maintained at 36 ± 1 °C. Images were
acquired every minute using a cooled CCD camera with 2 × 2 binning. Two vesicles (green arrowheads) moved from the
vesicle pool resembling the SAC/ARC (arrowhead) toward the
basolateral membrane (0, 2, 3, and
4). Those vesicles moved together and are therefore
indicated by the same color. Another vesicle moving from the BC
(arrow) to the SAC/ARC shown after 2 min (red
arrowheads) was found. Moreover, a vesicle moving from the
basolateral plasma membrane toward the apical pole of the cell was
found between image 9 and 12 min after starting
the time lapse (blue arrowheads). The nucleus of the imaged
cell with tracked vesicle paths is located below the
arrow pointing to the BC. The bright spot in the
adjacent nonpolarized cell located left to the observed cell resembles
a perinuclear recycling compartment. The observed vesicle pathways are
summarized in a schematic in the lower right panel.
In this image tracked vesicle paths are shown and colored according to
the color of the arrowheads for outlined vesicles (see
above). Arrows indicate the direction of observed vesicle
movement. Cells were incubated with 33 µM nocodazole
(B and C) for 30 min at 37 °C, washed, and
labeled with DHE/MCD for 1 min at 37 °C. Cells were washed and
either immediately imaged (B) or incubated for 30 min at
37 °C in the presence of the drug (C). In cells treated
with nocodazole to disrupt microtubules, DHE labeled the canalicular
membrane (arrows) at 0 and 30 min chase as found in control
cells (see Fig. 1). However, vesicles were scattered throughout the
cytoplasm after 30 min of chase (arrowheads) but not
enriched in the subapical or perinuclear region in polarized or
nonpolarized cells, respectively. Bar, 20 µm.
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Contribution of Vesicular Transport of DHE to Sterol Exchange
between the Plasma Membrane Domains Is Low--
To obtain quantitative
information about the role of vesicular traffic from the SAC/ARC in
transport of DHE between the plasma membrane domains, cells were
colabeled with -BODIPY-PC and DHE/MCD and chased for 30 min at
37 °C. Plasma membrane-associated DHE was removed by incubating
cells with medium containing 5 mM MCD and 10 mM cholesterol-loaded MCD (Fig.
5A). The intracellular vesicle
pool, which is noted as the SAC/ARC adjacent to the BC (outlined by a circle), remained well labeled by
DHE (thick arrowheads). Interestingly, the BC identified by
the corresponding fluorescence image of -BODIPY-PC (1st
panel) does not contain DHE after this extraction procedure
(arrows). During the time-lapse sequence some DHE rapidly
redistributes to the BC as indicated by the faint staining in this area
seen already after 2 min of chase at 37 °C. Vesicles adjacent to the
SAC/ARC were occasionally observed (thin arrowheads), but
they did not move vectorially toward the BC or basolateral plasma
membrane. Little redistribution of DHE to the basolateral or
canalicular membrane could be observed. Quantification of these results
is shown in Fig. 5B. Only about 10% of initial DHE
fluorescence is released during the chase. The measured half-time of
this process is 2.9 min, and no further release could be observed
during longer incubations. Some DHE was transported to the BC with a
half-time of 1.7 min raising its initial fluorescence by about 10%.
These results indicate that vesicles of the SAC/ARC containing DHE do
not contribute to a large extent to transport of DHE between the
basolateral and canalicular membrane.
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Fig. 5.
Release of DHE from the SAC/ARC and transport
to the canalicular membrane studied by time-lapse fluorescence
microscopy. Cells were colabeled with -BODIPY-PC and DHE/MCD
and chased for 30 min at 37 °C. Plasma membrane-associated DHE was
removed by incubating cells with Medium 2 containing 5 mM
MCD and 10 mM cholesterol-loaded MCD for 5 min at
37 °C. Cells were washed and placed on a temperature-controlled
microscope stage of a wide-field microscope maintained at 35 ± 1 °C. A, the incubation in extraction medium resulted in
complete removal of DHE from the basolateral membrane and from the BC.
The latter is indicated by a circle between the brightly
labeled SAC/ARC (thick arrowheads) as judged from
fluorescence of the corresponding image of -BODIPY-PC (not shown)
(see corresponding phase contrast image for cell borders and position
of BC (arrows)). Some labeled vesicles could be seen after 2 min of incubation after starting the time lapse (thin
arrowhead); however, no cross-redistribution of DHE from the
SAC/ARC toward the BC or basolateral membrane could be observed.
B, quantification of fluorescence of DHE in the SAC/ARC
(black circles) and the BC (white circles)
normalized to the fluorescence in the first image (i.e. 0 min) after starting the time lapse. The solid lines indicate
a monoexponential fit to the data. Data represent mean ± S.E. of
4 measurements. Bar, 20 µm.
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Transport of DHE to the Canalicular Membrane in ATP-depleted HepG2
Cells--
Vesicular traffic of proteins and lipids are
energy-requiring processes (44, 45). To study the relevance of
endocytosis and vesicular traffic in DHE distribution and transport,
cells were energy-depleted by incubation in azide and 2-deoxyglucose for 30 min at 37 °C. Under these conditions, uptake of Alexa-Tf was
completely blocked, indicating inhibition of
clathrin-dependent endocytosis (compare Fig.
6, C and F). Uptake
of Alexa-Tf in ATP-depleted cells was 0.7 ± 0.7% that in control
cells (mean ± S.E. of three fields with about 6-10 cells per
field for each condition). Resupplying glucose to ATP-depleted cells
not treated with a mild acid wash to remove surface-bound Tf resulted
in endocytosis of Alexa-Tf and its delivery to an endocytic recycling
compartment as found in control cells (not shown). Energy depletion
blocked the vesicular intracellular enrichment of DHE (Fig.
6D) as well as of -BODIPY-PC (Fig. 6C) as
found after 30 min at 37 °C in control cells in the subapical
(thick arrowheads) and perinuclear region (thin
arrowheads) of the cells (Fig. 6, A and B).
In contrast, ATP depletion did not affect the enrichment of both lipids
in the BC (arrows) or the diffuse staining of the cytoplasm
by both analogs (Fig. 6, D and E). Note that the
images shown in Fig. 6 were not deconvolved like those in Fig. 2 which
are also sum projections of individual planes (see above and
"Experimental Procedures"). This explains the slight difference in
intracellular distribution of DHE in Fig. 6A compared with
Fig. 2A. In control cells as well as in ATP-depleted cells
fluorescence of DHE in the canalicular membrane quantified as fraction
of total cellular DHE fluorescence increased by about 15% during the
first 1 min after removing the DHE/MCD-labeling solution and
remained stable for at least 20 min. This shows that DHE continues to
be transported to the BC during the chase in both conditions, and this
transport is very rapid.
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Fig. 6.
ATP depletion blocks vesicular uptake of DHE
and -BODIPY-PC but not the canalicular
enrichment of both lipid probes. HepG2 cells were washed and
incubated for 30 min at 37 °C in Medium 1 (A-C, control
cells) or in ATP-depletion medium (D-F,
ATP-depleted cells). Cells were colabeled with DHE/MCD (A
and D) and -BODIPY-PC at 37 °C (B and
E). After incubation for 30 min at 37 °C in control
cells, subapical (thick arrowheads) and perinuclear
(thin arrowheads) enrichment of DHE and -BODIPY-PC as
well as brightly stained BC were found (A and B)
(arrows). In ATP-depleted cells only diffuse intracellular
fluorescence but no vesicular uptake was found for DHE (D)
and -BODIPY-PC (E). However, BC were labeled with both
probes (arrows). As a control for ATP depletion, uptake of
Alexa-Tf was studied in a separate experiment in which cells were
labeled for 10 min at 37 °C with Alexa-Tf (C and
F). ATP depletion resulted in complete inhibition of uptake
of Alexa-Tf (F), which was in control cells accumulated in a
recycling compartment (arrowheads, C).
Bar, 20 µm.
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FRAP Reveals Rapid Transport of DHE and -BODIPY-PC
to the Canalicular Membrane--
To obtain kinetic information about
transport of DHE to the BC, a FRAP technique was employed. BC were
bleached after pulse labeling at 37 °C, and the fluorescence
recovery was measured on a temperature-controlled microscope stage. As
shown in Fig. 7A, DHE
fluorescence in the BC (arrows) is strongly reduced after the bleach, but the BC fluorescence recovers substantially within 5 min. No vesicular transport of DHE to the canalicular membrane was
observed during the measurement, suggesting that most DHE is rapidly
transported to the BC by a nonvesicular pathway. The kinetics of
recovery of DHE fluorescence in the BC were quantified to provide a
rate and a fraction of recovery (Q) as shown in Fig. 7B and Table I. The measured
half-time for recovery was 1.6 min for control cells (Fig.
7B, black circles). The fraction of recovery, Q = 0.77, suggests that there is a portion of DHE that
cannot be delivered to the BC in the time course of the experiment.
Remarkably, in ATP-depleted cells fluorescence recovery was even
slightly faster than in control cells (t1/2 = 1.2 min), whereas the recovery fraction was reduced (Q = 0.55) (Fig. 7B, open circles). Because vesicular
traffic of DHE was blocked after ATP depletion (Fig. 6), these results
indicate that nonvesicular transport accounts for most of the
canalicular transport of DHE.
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Fig. 7.
Transport of DHE to the canalicular membrane
studied by FRAP. Cells were labeled with rhodamine-dextran (70 kDa) for 1 h at 37 °C, washed, and labeled with DHE/MCD for
1 min at 37 °C. After washing cells were placed on a
temperature-controlled microscope stage of a wide-field microscope
maintained at 35 ± 1 °C. A, after acquiring an
image of cells containing a BC (arrows), the BC region was
bleached by closing the field aperture to about 13 µm in the object
plane as described under "Experimental Procedures." Subsequently
the field aperture was opened, and images were acquired at the
indicated times. B, normalized fluorescence recovery for DHE
in control (black circles) or ATP-depleted cells
(white circles) and for -BODIPY-PC (triangles)
in control cells. A mono-exponential fit for enrichment of DHE
(solid lines) and -BODIPY-PC (dashed line) in
the BC are shown. Data represent mean ± S.E. of 9-10 (DHE) or 5 measurements (-BODIPY-PC). Bar, 20 µm.
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Table I
Quantification of transport of DHE and -BODIPY-PC as studied by FRAP
Cells were labeled, and FRAP of DHE and -BODIPY-PC was performed for
the BC or the basolateral plasma membrane as described under
"Experimental Procedures." Images of DHE and -BODIPY-PC were
acquired prior and after the bleach at the indicated time points (see
Figs. 7 and 8). Fluorescence of DHE and -BODIPY-PC in the ROI (BC or
basolateral membrane) and in the BC-forming cells was quantified after
background subtraction of the images. Recovery curves normalized to the
prebleach image were fitted with a mono-exponential function using
Sigma Plot 4.0 (SPSS Inc., Chicago).
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The kinetics of transport of -BODIPY-PC to the
canalicular membrane have been measured previously by quantifying
fluorescence of -BODIPY-PC in the BC immediately after pulse
labeling of the basolateral plasma membrane with this analog. A
half-time for this process at 3.5 min was determined (25). Fig.
7B shows the kinetics of recovery of -BODIPY-PC in the BC
using the FRAP method (triangles). A half-time for recovery
of 3.0 min was obtained for -BODIPY-PC. The half-time for enrichment
of this PC analog in the BC closely resembles that previously measured
with an independent method (25). The fraction of fluorescence recovery
of -BODIPY-PC (Q = 1.0) points to some differences
in canalicular transport of sterols versus a fluorescent PC
analog. The result is in line with a nonvesicular transport of
fluorescent PC to the canalicular membrane as demonstrated previously
(25).
ATP-independent release of DHE from the Canalicular Membrane and
Redistribution to the Basolateral Domain--
The surprisingly fast
and ATP-independent recovery of DHE fluorescence in the BC raised the
question whether this rapid sterol enrichment is a property unique to
the canalicular membrane. We next studied the kinetics of transport of
DHE from the BC to the basolateral plasma membrane. Cells were labeled
with DHE/MCD and -BODIPY-PC. After acquiring an image of DHE,
fluorescence of DHE in the basolateral membrane was selectively
bleached, and fluorescence recovery in the basolateral as well
fluorescence loss in the canalicular membrane were measured. As shown
in Fig. 8, A and B,
DHE rapidly recovers in the bleached basolateral area. ATP depletion
did not affect the extent of fluorescence recovery, which was about
100% (Table I). However, the kinetics of fluorescence recovery in the
basolateral region is faster in ATP-depleted cells (Fig.
8B, white circles) (t1/2 = 1.4 min) compared with control cells (Fig. 8B,
black circles) (t1/2 = 2.8 min). Release
of DHE from the BC due to the basolateral bleach was rather low during the time course of the experiment (Fig. 8, A and
C). This suggests replenishment of DHE from regions outside
the bleach spot in the basolateral membrane. ATP depletion slightly
accelerated release of DHE from the BC (t1/2 = 4.1 min) compared with control cells (t1/2 = 5.4 min).
The results indicate that vesicular transport of DHE is not a major
factor in delivery of this sterol to the basolateral membrane.
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Fig. 8.
Release of DHE from the canalicular membrane
and transport to the basolateral domain studied by FRAP. Cells
were labeled with -BODIPY-PC for 1 min at 37 °C, washed, and
labeled with DHE/MCD for 1 min at 37 °C. After washing cells were
placed on a temperature-controlled microscope stage of a wide-field
microscope maintained at 35 ± 1 °C. A, after
acquiring an image of cells containing a BC (arrows), the
basolateral region (outlined circle) was bleached by closing
the field aperture as described under "Experimental Procedures."
Subsequently the field aperture was opened, and images were acquired at
the indicated times. B, normalized fluorescence recovery for
DHE in control (black circles) or ATP-depleted cells
(white circles) in the basolateral plasma membrane.
C, normalized fluorescence of DHE in the BC during the
basolateral recovery in control (black circles) or
ATP-depleted cells (white circles). A mono-exponential fit
for DHE fluorescence (solid lines) in control or
ATP-depleted cells is shown. Data represent mean ± S.E. of 8-13
measurements. Bar, 10 µm.
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DHE in the Canalicular Membrane Can Completely Exchange
with DHE in the Basolateral Plasma Membrane of the Same Cell--
The
slower release of DHE from the BC compared with its
recovery in the basolateral membrane might have been explained by replenishment of DHE from the second unbleached cell of a couplet, while being continuously released from the canalicular membrane. Alternatively, DHE could be released slowly from the BC and then transported to the basolateral membrane. To distinguish between these
possibilities, we performed a FLIP experiment. As shown in Fig.
9, DHE fluorescence throughout one cell
was destroyed due to repeated bleaching, whereas it appeared stable in
the second cell forming the BC (thin arrows). Interestingly,
DHE fluorescence could be destroyed in the canalicular membrane of the
bleached, but not the unbleached, cell as indicated by the brightly
labeled half-BC in images after bleaching (Fig. 9, thick
arrows). The nonpolarized cell adjacent to that forming the
couplet was also completely bleached because it was also illuminated by
the bleach beam (see outlined circle). However, no
fluorescence loss of DHE in neighboring unbleached cells was observed,
indicating that DHE does not diffuse rapidly from one cell to the next.
The experiments demonstrate that DHE does not cross the BC from one
cell to the other of an HepG2 couplet, nor is it released into the BC
lumen. However, DHE associated with the canalicular membrane can
completely redistribute to the basolateral membrane. The slow loss of
DHE from the BC observed following a single bleach of the basolateral membrane (see above and Fig. 8) is therefore not due to continuous sterol transport from one cell of a couplet to the other. We conclude that DHE associates preferentially with the canalicular membrane but
that it can be transported from this membrane to the basolateral membrane by a nonvesicular mechanism.
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Fig. 9.
Dynamic exchange of DHE between the
canalicular and basolateral membrane studied by FLIP. Cells were
labeled with -BODIPY-PC for 1 min at 37 °C, washed, and labeled
with DHE/MCD for 1 min at 37 °C. After washing cells were placed
on a temperature-controlled microscope stage of a wide-field microscope
maintained at 35 ± 1 °C. After acquiring an image of cells
containing a BC (arrows) the basolateral region was bleached
as described under "Experimental Procedures." The field aperture
was opened, an image of DHE fluorescence was acquired, and DHE
fluorescence was allowed to recover in the bleached region for 3 min.
The field aperture was closed and cells were bleached again. This cycle
was repeated until no further fluorescence loss in the bleached cell
could be observed. Fluorescence loss including the canalicular membrane
was observed for the bleached cell but not for the neighboring cell of
the couplet (thin arrow). Fluorescence of DHE in the BC
(thick arrow) decreased only half, i.e. in the
bleached but not the non-bleached cell. Neighboring cells also forming
a BC (arrowhead) appeared bright during the whole experiment
(see text for further explanations). Bar, 20 µm.
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DISCUSSION |
Cholesterol is crucial for establishment and
maintenance of the polarized state in epithelial cells. However, the
intracellular transport pathways of cholesterol in polarized cells are
not well understood. We have studied the intracellular itineraries and dynamics of a cholesterol analog, DHE, in polarized HepG2 cells. We
find that DHE transport occurs by a combination of several pathways.
Some DHE is found associated with organelles and is seen to move
through the cytosol in ways that are consistent with vesicular traffic.
However, the majority of sterol transport into and out of the apical
and basolateral plasma membranes occurs by an ATP-independent,
nonvesicular process. The results of this study are summarized in a
proposed model of sterol transport in polarized HepG2 cells as shown in
Fig. 10.
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Fig. 10.
Model of the transport pathways of sterol
between the plasma membrane domains of polarized HepG2 cells. DHE
incorporated into the exoplasmic leaflet of the plasma membrane of
HepG2 cells can be transported in vesicles, summarized as early
endosomes (EE), to the SAC/ARC and from there to the
opposite plasma membrane domain (1). Additionally, DHE
traverses the plasma membrane by rapid transbilayer migration,
indicated by the zoomed region of both plasma membrane domains, and
traffics to the opposite membrane domain by lateral diffusion along the
inner plasma membrane leaflet (2) or through the cytosol
bound to a cytosolic protein carrier (3). All transport
pathways (arrows) are bi-directional. Tight junctions
(TJ) might prevent lateral diffusion between the membrane
domains in the exoplasmic plasma membrane leaflet. Sterol-enriched
compartments are black (i.e. the basolateral and
canalicular membrane and the SAC/ARC), and those with low or
intermediate sterol content judged by poor or absent labeling with DHE
are shown in gray (i.e. the Golgi or TGN and
other endocytic compartments named here as EE and the
nucleus/endoplasmic reticulum membrane system (Nu)). Sterol
transport is not possible from one cell of a couplet to the other by
crossing either the inter-membrane space along the basolateral membrane
or by crossing the biliary canaliculus by release from the canalicular
membrane into the BC lumen (crossed arrows). See text for
further explanation.
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We demonstrate that DHE is transported to the SAC/ARC as shown by its
colocalization with -BODIPY-PC, with the recycling marker Alexa-Tf
and especially with C6-NBD-SM. Interestingly, vesicular structures were found in the region of the SAC/ARC
in HepG2 cells that were labeled only with DHE but not with
-BODIPY-PC, Alexa-Tf, or C6-NBD-SM (Fig. 2). This
points to the heterogeneous nature of the organelles in the
SAC/ARC region as suggested earlier (46-48). The cholesterol
content of late endosomes and lysosomes was reported to be 6-10% of
total cellular cholesterol (49). In order to test whether DHE is
transported also to these compartments, HepG2 cells were colabeled with
DHE and Texas Red-ASOR, a marker of late endosomes and lysosomes as
described under "Experimental Procedures." Notably, no
colocalization of DHE with Texas Red-ASOR was found, suggesting that
the amount of DHE in late endosomes/lysosomes is too low to be detected
(not shown).
The fluorescence of DHE in the SAC/ARC reached a steady state after
about 30 min of chase from the plasma membrane at 37 °C (Fig. 1).
Note that cholesterol derived from the plasma membrane gets esterified
at a rate of 0.25% per min in hepatoma cells (50). Moreover, a lag
time due to sterol transport from the plasma membrane to intracellular
compartments prior to the onset of sterol esterification has been
demonstrated (51). If DHE is esterified with the same kinetics as
cholesterol, maximally 7.5% of cholesterol or DHE derived from the
plasma membrane should become esterified during the time course of our experiments.
It has been proposed that the Golgi complex is a major
cholesterol-containing organelle, playing an important role in
polarized sorting of membrane components in the biosynthetic pathway
(40, 52). Cholesterol and (glyco)sphingolipids have been proposed to
form ordered microdomains or rafts in the TGN (for a review see Ref.
12). By using DHE as a marker of cholesterol in the plasma membrane, we
found that most vesicular DHE resides in vesicles belonging to the
SAC/ARC and that only a small fraction is found in the TGN at steady
state. This suggests that the sterol content of the TGN is lower than
that of the plasma membrane or of recycling endosomes, as we found
previously in CHO cells (18, 31). We cannot rule out the presence of
high levels of sterol in small regions of the TGN nor did we
investigate the fate of newly synthesized sterol. It might be that
de novo synthesized cholesterol accumulates in the TGN
creating the lipid environment in this compartment that is required for
polarized sorting. However, although several studies (48, 53, 54) have
shown that the recycling compartment is enriched in cholesterol, the
published data on cholesterol content of the Golgi apparatus is
contradictory and depends on the method used for purification of Golgi
membranes (18, 55-57). It has been reported that de novo
synthesized cholesterol reaches the plasma membrane with a half-time of
about 10 min and that this transport only partially involves the Golgi
complex (58, 59). According to our results, synthesized cholesterol
exported to the plasma mem
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ABSTRACT
INTRODUCTION
