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J Biol Chem, Vol. 275, Issue 20, 15279-15286, May 19, 2000
From the Lipids and other membrane constituents recycle
between the plasma membrane and intracellular endocytic compartments.
In CHO cells, approximately half of the internalized
C6-NBD-SM, a fluorescent lipid analogue widely used
as a membrane maker, recycles via the endocytic recycling compartment
with a t1/2 of ~12 min (Mayor, S., Presley,
J. F., and Maxfield, F. R. (1993) J. Cell
Biol. 121, 1257-1269). Surprisingly, the rest returns to the
plasma membrane very quickly. A detailed kinetic study presented in
this paper indicates that after a brief internalization pulse, 42-62%
of the internalized C6-NBD-SM returns to the plasma membrane with a t1/2 of 1-2 min. Similar results
are obtained using HEp2 and nonpolarized Madin-Darby canine kidney
cells. Using FM dyes of different hydrophobicity, we show that rapid
recycling involves passage through an endocytic organelle that was
subsequently identified as the sorting endosome by co-localization with
internalized transferrin and low density lipoprotein. These results
imply that the membrane internalization rate is much higher than
previously estimated, with a t1/2 as short as 5-10
min. Rapid internalization and recycling would facilitate processes
such as nutrient uptake and cholesterol efflux.
Endocytic recycling is essential for regulation of surface
expression of proteins and for the uptake of nutrients. After
internalization, endocytosed molecules are delivered rapidly to sorting
endosomes (1, 2), which consist of vesicles with tubular extensions that are involved in transport of recycling material (e.g.
transferrin receptor) out of the sorting endosomes. A major recycling
pathway involves subsequent passage through the endocytic recycling
compartment (ERC),1 which in
CHO cells is a collection of tubules concentrated near the centriole,
from which molecules recycle back to the plasma membrane
(t1/2 ~9-12 min) (3-5).
Fluorescent lipid probes are very well suited for kinetic studies of
endocytic recycling. Very bright signals can be obtained, and after
internalization pulses, efficient desorption of certain lipid analogs
from the plasma membrane allows accurate measurements of recycling with
minimal interference from probe molecules left in the plasma membrane
(6). After nonselective internalization, a fluorescent lipid analog,
C6-NBD-sphingomyelin (C6-NBD-SM), exits sorting
endosomes, enters the ERC, and then returns to the plasma membrane with
kinetics indistinguishable from transferrin (Tf) in CHO cells (4).
After a 10-min internalization pulse with C6-NBD-SM, the
efflux kinetics from CHO cells suggested the existence of a second,
faster recycling pathway, in addition to the pathway through the ERC
with a t1/2 of ~12 min (7). Taking full advantage
of the properties of lipid analogs, we have now characterized rapid
kinetics of membrane recycling in various cell types, and we found that
nearly half of the internalized membrane recycles with a
t1/2 of about 1.5 min. This surprisingly rapid
recycling requires internalization of the lipids with a
t1/2 of 5-10 min in order to maintain membrane
balance. These results imply that in most cells the exchange of
membrane between the plasma membrane and the endosomes is much more
extensive than formerly estimated. Rapid recycling was previously
thought to be unique to specialized cells, and lipid analogs
(e.g. C6-NBD-SM) have been used to study rapid
recycling in cells such as erythroblasts, showing a recycling
t1/2 of 2 min (8). Our findings, however, indicate
that rapid recycling exists in many cell types. This has important
consequences for processes such as nutrient uptake, cholesterol efflux,
and regulation of surface expression of receptors.
We confirmed the rapid recycling using another series of fluorescent
lipophilic dyes, the FM dyes (Fig. 1),
having a dicationic head group that prevents flip-flop across the
bilayers (9). The partition of the FM dyes between the aqueous phase
and the lipid is determined by the tail length, and the quantum yield increases up to 2 orders of magnitude when FM dyes are transferred from
water into membranes (9). Taking advantage of these properties, we
could determine whether rapid recycling involved fusion with a larger
endosomal structure (e.g. a sorting endosome), followed by
budding of recycling membrane (see Fig. 7). When the amphiphilic FM
dyes enter an endocytic organelle, they distribute between membrane and
aqueous volume according to their partition coefficients. As a
consequence, a higher fraction of the less hydrophobic probes would be
left behind when recycling membrane buds out from a parent organelle
such as a sorting endosome. On the other hand, if rapid recycling
involves direct return of vesicles that pinch from the plasma membrane,
there should be no difference in recycling efficiency of various
probes, because all internalized contents are released upon fusion.
Using three FM dyes of different hydrophobicity, we show that the rapid
recycling pathway involved an organelle that was subsequently identified as the sorting endosome by double labeling with LDL.
Materials--
C6-NBD-SM, FM 2-10, FM 1-43, and FM
1-84 were purchased from Molecular Probes, Inc. (Eugene, OR). The Cy5
labeling kit was obtained from Amersham Pharmacia Biotech. Human Tf was
from Sigma. Iron-loaded Tf was passed through a Sephacryl S-300 gel
filtration system as described previously (5). Cy5 was then conjugated to iron-loaded Tf following the manufacturer's instructions. Unbound dye was removed first by passage through a sizing column and then by
overnight dialysis in phosphate-buffered saline. DiI-LDL (LDL labeled
with 3,3'-dioctadecylindocarbocyanine) was a gift from Dr. Ira Tabas
(Columbia University, New York). All tissue culture supplies were from
Life Technologies, Inc. Dioleoylphosphatidylcholine was from Avanti
Polar lipids, Inc. (Birmingham, AL). All other chemicals were
from Sigma.
Cells--
TRVb-1 is a modified CHO cell line that lacks
endogenous Tf receptor and expresses the human Tf receptor (10). MDCK
type II and HEp2 cells were from ATCC (Manassas, VA). All cells were grown at 37 °C in a 5% CO2 humidified incubator. TRVb-1
cells were grown in bicarbonate-buffered Ham's F-12 medium
supplemented with 5% fetal bovine serum, 200 mg/ml Geneticin as a
selection for the transfected Tf receptors, 100 units/ml penicillin,
100 µg/ml streptomycin, and 2 g/liter glucose; MDCK and HEp2 cells were grown in bicarbonate-buffered Dulbecco's modified Eagle's medium
supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and
100 µg/ml streptomycin. Cells to be labeled with DiI-LDL were transferred to a similar Ham's F-12 medium but with 5%
lipoprotein-deficient serum in place of fetal bovine serum to
up-regulate the cells' LDL receptors (11). Cells for microscopy were
plated 24 h before the experiments in 35-mm plastic tissue culture
dishes with a 7-mm hole in the bottom covered by
poly-D-lysine-coated coverslips (12).
Biochemical Study of C6-NBD-SM Efflux
Kinetics--
To create the C6-NBD-SM labeling solution, a
mixture (2:3 mol/mol) of C6-NBD-SM and
dioleoylphosphatidylcholine (both stocks in ethanol) was dried down
under argon and redissolved in ethanol (20 mM total lipid
concentration). This ethanol solution was injected into medium 1 (150 mM NaCl, 5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, and 20 mM Hepes, pH 7.4) while vortexing and dialyzed at 4 °C overnight against excess phosphate-buffered saline to remove ethanol present in the solution (initially <8%). It was then diluted in medium 1 (M1) to a final total lipid concentration of 50 µM to make the labeling solution.
Cells were plated in 28-cm2 tissue culture dishes 24 h
before the experiment. Kinetic measurements were made as described
previously (7) with a few exceptions. In brief, cells were incubated in M1-glucose (medium 1 with 2 g/liter glucose) for 5 min and pulsed with
C6-NBD-SM labeling solution for 2-10 min at 37 °C. The
cells were then immediately washed with ice-cold M1-glucose and
incubated with back-exchange medium (5% fatty acid-free bovine serum
albumin in M1-glucose) on ice for 1 h, during which six washes of
ice cold back-exchange medium with 10-min intervals were applied. After
the back-exchange process, cells were washed with 37 °C M1-glucose,
and 6 ml of 37 °C chase medium (1% fatty acid-free bovine serum
albumin in M1-glucose) was added to the cell dish. This was defined as
chase time 0. Cells were chased in the chase medium at 37 °C for up
to 60 min. At each time point, 600 µl of chase medium was taken from
the cell dish, and 600 µl of fresh prewarmed chase medium was added
back to the dish. At the end of the 60-min chase, the remaining chase
medium was removed, and cells were incubated in 2.5 mM EDTA
on ice for 15 min. Cells were then harvested with a cell scraper to
determine cell-associated fluorescence.
Chase medium aliquots taken at each time point, and the remaining chase
medium, EDTA wash, and harvested cells were all extracted with butanol.
Fluorescence of butanol solutions was measured with a
spectrofluorometer (Fluorolog 2, Spex Industries Inc., Edison, NJ).
Excitation wavelength was 465 nm, and fluorescence was quantified by
integrating peak area from 518 to 558 nm. Unlabeled chase medium was
used for background correction.
To obtain the kinetic curves in Figs. 2
and 3, the following calculations were
made: (a) total lipid = cell-associated fluorescence + fluorescence remaining in chase medium + fluorescence in chase aliquots; (b) fraction of total lipid in the chase medium at
each time point (f) = (10 × (lipid in chase
aliquot) + lipid removed in previous chase aliquots)/total lipid;
(c) fraction of lipid that was cell-associated
(l) = 1
The fraction of lipid that was cell-associated at each chase time
point, l, was fit to a double exponential decay,
l = a·e
To test back-exchange efficiency, two methods were used. The overall
strategy was to measure the amount of surface-bound fluorescence at the
end of the back-exchange procedure, without interference from
exocytosis. This was accomplished by either keeping cells on ice at all
times, thereby preventing endocytosis and exocytosis, or fixing cells
to prevent exocytosis. In the first assay for measuring back-exchange
efficiency, cells were labeled on ice for 30 min to ensure
incorporation of C6-NBD-SM into the plasma membrane and
back-exchanged on ice for 1 h, saving all of the washes. Cells
were then incubated in EDTA and harvested, as described above, to
determine cell-associated fluorescence. To test whether labeling at
37 °C had any effect on the incorporation of C6-NBD-SM that could have altered the back-exchange efficiency, we pulsed the
cells at 37 °C for 2 min, fixed them with 2% paraformaldehyde, and
then applied the normal back-exchange procedure. At the end of this
procedure, 37 °C chase medium was added to the fixed cells, and
aliquots of chase medium were taken for the first 10 min afterwards.
To measure the release rate of C6-NBD-SM by the chase
medium, cells were labeled on ice for 30 min, washed with ice-cold
M1-glucose, and incubated in chase medium at 37 °C. Aliquots of
chase medium were taken at times from 10 s to 5 min. Fluorescence
from these aliquots and cell-associated fluorescence were obtained to
calculate fractions of C6-NBD-SM removed at different chase times.
Kinetic Measurements of FM Dye Trafficking Using Wide Field
Fluorescence Microscopy--
Cells were labeled with one of the FM
dyes (FM 2-10 2 mM, FM 1-43 15 µM, FM 1-84
2 µM) for either 30 s or 15 min at 37 °C. These
concentrations were chosen to give similar initial intensities. The
cells were then washed with ice-cold M1-glucose, kept on ice for 15 min
in M1-glucose, and rinsed repeatedly with ice-cold M1-glucose. Since
uninternalized FM dye molecules readily dissociate from the plasma
membrane, a back-exchange acceptor was not necessary for these dyes.
The release rates of the FM dyes from the plasma membrane were
determined to be on the order of a few seconds at 24 °C (13), and we
verified that essentially all dye molecules were released from the
membrane by our rinse procedure. The same washes were applied to all
three FM dyes. Immediately prior to image acquisition, 37 °C
M1-glucose was added to the cells. To see whether a temperature shift
at the beginning of the efflux experiments had any effect on the rapid
recycling process, we also performed measurements using FM 2-10 and FM
1-43 without the temperature change. Cells were labeled with either FM
2-10 or FM 1-43 for 30 s at 37 °C, rinsed several times with
37 °C M1-glucose, and taken to the microscope for efflux
measurements. The cell dish was kept on a temperature-controlled
microscope stage maintained at 33-34 °C throughout acquisition.
Fluorescence microscopy was carried out using a Leica DMIRB microscope
(Leica Mikroscopie und Systeme GmbH, Germany) equipped with a cooled CCD camera (Frame Transfer Pentamax camera with a 512 × 512 back-thinned EEV chip, model no. 512EFTB; Princeton Instruments) driven
by Image-1/MetaMorph Imaging System software (Universal Imaging Corp., West Chester, PA). Images were acquired using × 25 oil immersion objective (0.75 NA) to include a large number of cells in one field and
to acquire fluorescence from entire cell thickness. All FM dyes were
imaged using a standard fluorescein filter set. The first image of each
chase experiment was acquired within 40 s after warming up the cells.
Unlabeled cells were used to examine autofluorescence. Under the
acquisition conditions used in kinetic experiments, autofluorescence was negligible. Image background was corrected as follows. Two or three
regions were selected from cell-free areas in a field, and an average
intensity from those regions was taken as the background value for that
field. This background value was then subtracted from every pixel in
the field. An integrated fluorescence power reading of each image was
recorded after background correction. Normalized fluorescence power was
determined by dividing the power in each image by that of the first
reading at the beginning of the chase.
Double Label Experiments by Confocal Microscopy--
Cells grown
in coverslip bottom dishes were incubated with M1-glucose at 37 °C
before labeling. For double label experiments with DiI-LDL and FM dyes,
LDL-receptor up-regulated cells were first pulsed with 6 µg/ml
DiI-LDL for 4 min at 37 °C to label endosomes. For double label
experiments with Cy5-Tf and FM dyes, cells were first pulsed with 20 µg/ml Cy5-Tf for 2 min at 37 °C. In both cases, cells were then
washed and pulsed with one of the FM dyes for 30 s at 37 °C. To
be consistent with kinetic experiments, cells were kept on ice for 15 min in M1-glucose while washes were applied. Confocal images were taken
either immediately after warming the cells up to 37 °C for DiI-LDL
labeled cells or after a 15-min chase at 37 °C for Cy5-Tf-labeled
cells. Live cells were kept on a temperature-controlled microscope
stage maintained at 33-34 °C throughout acquisition.
Confocal microscopy was performed using an Axiovert 100M inverted
microscope equipped with an LSM 510 laser-scanning unit and a 63 × 1.4 NA plan Apochromat objective (Carl Zeiss, Inc.). Since FM dyes
have broad emission spectra, filter sets were selected carefully to
maximize true signal while minimizing crossover of signal from one
channel to the other. Samples double-labeled with DiI-LDL and FM dyes
were excited with a 25-milliwatt argon laser emitting at 488 nm for FM
and a 1.0-milliwatt helium/neon laser emitting at 543 nm for DiI. A
585-615-nm band pass filter was used for collecting DiI emissions, and
a 650-nm long pass filter was used for FM emissions. Samples
double-labeled with Cy5-Tf and FM dyes were excited with the argon
laser emitting at 488 nm for FM and a helium/neon laser emitting at 633 nm for Cy5. A 530-600-nm band pass filter was used for collecting FM
emissions, and a 650-nm long pass filter was used for Cy5 emissions.
The two channels were scanned alternately in a line-by-line fashion, having only one laser line and one detector channel on at each time.
Fraction of cross-over was measured to be less than 10% using
single-labeled samples of each probe. Images were corrected for
background (as described above for FM dyes) and crossover (14).
Partition Coefficient Measurements of the FM
Dyes--
Dioleoylphosphatidylcholine was purchased as a chloroform
solution. It was dried first under argon and then in vacuo
overnight and dissolved in M1, forming multilamellar vesicles (final
lipid concentration was 3.5 mM). This suspension was
sonicated for 30 min to break up large multilamellar vesicles. The
solution cleared after sonication by forming unilamellar vesicles. The
method for measuring partition coefficients was adapted from Huang and
Haugland (15). The solution containing unilamellar vesicles was diluted in M1 to make solutions containing different concentrations of liposomes. For each experiment, fluorescence resulting from titration of liposomes against a constant dye concentration was measured with a
spectrofluorometer. Two concentrations of each dye were used for two
parallel runs: FM 2-10 (0.2 mM, 0.15 mM), FM
1-43 (6 µM, 4 µM), and FM 1-84 (4 µM, 2 µM). The excitation wavelength for FM
2-10, FM 1-43, and FM 1-84 were 465, 468, and 468 nm, respectively, and the emission wavelengths were 608, 593, and 590 nm, respectively. Inner filter effect was corrected for the case of FM 2-10, where the
correction was meaningful, using the equation,
Equations used to obtain partition coefficients were described by Huang
and Haugland (15). The partition coefficient of a membrane probe,
Kp, was defined as follows,
C6-NBD-SM Efflux Kinetics
Back-exchange Efficiency and Rate of Exchange by Chase
Medium--
The experiments reported here required highly efficient
removal of fluorescent lipids from the plasma membrane by
back-exchange. To determine the back-exchange efficiency, two methods
were employed. When cells were labeled and back-exchanged on ice, less
than 2% of total fluorescence was found to be cell-associated at the
end of the back-exchange. This was consistent with previous findings (7, 17). We also tested whether labeling at 37 °C had any effect
that might give a different back-exchange efficiency. We fixed cells
after incubation with C6-NBD-SM at 37 °C, back-exchanged on ice, and then monitored the release of C6-NBD-SM from
cell surface into prewarmed chase medium. Since the fixed cells were incapable of undergoing exocytosis, what was released into the chase
medium was presumably membrane-bound probes left at the end of the
back-exchange procedure. Aliquots taken from the chase medium did not
show fluorescence above background. These results indicated that the
release of C6-NBD-SM measured in our kinetic studies after
back-exchange was due to exit of recycled C6-NBD-SM from
cells, rather than release of uninternalized probes from the cell surface.
The validity of our C6-NBD-SM efflux measurements also
depended on the release rate of C6-NBD-SM from the cell
surface into the chase medium. Our control experiments showed that 50%
of recycled C6-NBD-SM reaching the plasma membrane was
removed by the chase medium within 40 s, and more than 90% was
removed within 2 min. This desorption from the membrane is faster than
the fastest recycling process we measure, but the exocytic rate
constants measured with C6-NBD-SM may be slightly
underestimated, since we ignored the rate of extraction from the plasma
membrane. Because the release process was kinetically complex, we
decided that it would only introduce more errors if we were to correct
the rate of rapid recycling with a calculated release rate.
Existence of a Second Component in C6-NBD-SM
Recycling--
The exit kinetics of C6-NBD-SM were
determined from the amount of C6-NBD-SM recycled back to
the plasma membrane and released into the chase medium. With a pulse
time shorter than 10 min, there was very little hydrolysis of
C6-NBD-SM to C6-NBD-Cer (7), which could
subsequently be transported to the cell surface via nonvesicular
transport. Plots of fraction of cell-associated fluorescence versus chase time are given in Fig. 2. The fraction of lipid
that was cell-associated at each chase time point (open
circles) was fit with a double exponential decay
l = a·e
A double exponential decay fit to data points obtained from 2-min
pulse, 60-min chase experiments of C6-NBD-SM gave a rapid recycling component with a half-time of 1-2 min and a slow recycling component with a half-time of ~12 min (Fig. 2A, Table
I). The rapid recycling component was
found to account for 42% of the recycling population. As the length of
pulse time increased to 5 and 10 min, the relative percentage of rapid
recycling component decreased to 28 and 23%, respectively (Fig. 2,
B and C; Table I). The best fit rate constant for
each component, however, remained approximately constant regardless of
the pulse length.
To see whether a similar rapid recycling pathway existed in other cell
types, we measured C6-NBD-SM efflux kinetics in HEp2 human
carcinoma cells (Fig. 3A, Table I) and nonpolarized MDCK cells (Fig. 3B, Table I). Like CHO cells, these two cell
lines contained a fast recycling component with a
t1/2 of ~1 min. When clathrin-mediated endocytosis
was blocked in CHO cells by preincubation in K+-depleted
buffer (18), both the slow and fast recycling populations were still
detectable (Fig. 3C, Table I). In this case, Tf
internalization was reduced by 74%, and C6-NBD-SM
internalization was reduced by 43%.
Efflux Kinetics of the FM Dyes--
To determine whether an
endocytic organelle compartment was involved in the rapid recycling
pathway, FM dyes of different hydrophobicity were used. The three FM
dyes used in this study have very similar chemical structures, with the
only difference being the number of carbon atoms in their tails (Fig.
1). This difference has the largest impact on each dye's degree of
hydrophobicity, quantified by its partition coefficient.
Using the method described under "Experimental Procedures," the
partition coefficients for FM 2-10, FM 1-43, and FM 1-84 were calculated to be 2.35 × 104, 2.78 × 105, and 7.78 × 105, respectively. The
partition coefficients are useful in representing relative differences
in hydrophobicity among the three FM dyes. The relative differences in
partition coefficients are consistent with differences in release times
measured in cultured neurons (13).
The dye dissociation times from plasma membrane for FM 2-10, FM 1-43,
and FM 1-84 have been measured in synaptic boutons to be 0.7, 3, and
6 s at 24 °C, respectively (13). Because recycled FM dyes
could readily be released into aqueous medium in which they are
nonfluorescent, we used continuous live cell imaging to study efflux
kinetics of the FM dyes. This enabled us to directly measure
cell-associated fluorescence by wide field microscopy on a
temperature-controlled microscope stage.
Fig. 4 shows efflux kinetics of the FM
dyes after a 30-s pulse. Open circles represent
data points from experiments in which a chilling procedure was applied
to remove the membrane-bound probes, and results from experiments
lacking this process are represented by solid
triangles. The two methods gave similar results. FM 1-43
and FM 1-84 showed a biphasic kinetic profile similar to that of
C6-NBD-SM (Fig. 4, B and C). Data
points from these hydrophobic FM dyes fit well to a double exponential
decay, giving a half-time of ~2 min for the fast component and ~11
min for the slow component (Table I). The FM 2-10 efflux curve (Fig.
4A) lacked the rapid efflux component that was present in
the first portion of FM 1-43 and FM 1-84 curves, and the FM 2-10
efflux was well fit by a single exponential decay with a half-time of 12 min (Table I). These results indicated that unlike the FM 1-43 and
FM 1-84 dyes, very little of the more hydrophilic FM 2-10 was
recycled through the rapid pathway. Since some of the FM dyes partition
into the aqueous phase in endosomes, there were residual dye molecules
at the end of a 60-min chase, as would be expected for aqueous solutes
in endosomes.
We also measured efflux kinetics of FM dyes after a 15-min pulse. The
longer pulse time caused a shift in the relative percentages of the two
recycling components (i.e. the slow recycling pathway became
dominant under these conditions). This enabled us to examine whether
the slow recycling pathway behaved similarly with all three FM dyes.
Essentially identical kinetic curves were obtained from all three FM
dyes, which could be fit fairly well with a single exponential decay
having a half-time of 10-12 min.
A summary of all kinetic parameters is presented in Table I. The data
show that all probes share a common recycling kinetic process with a
half-time of 8-14 min depending on the cell type, and hydrophobic
probes have a second, more rapid recycling pathway with a half-time of
1-2 min. The rapid recycling population becomes less detectable as the
pulse length increases, as expected as a consequence of equilibration
of the fast process during the loading pulse.
Co-localization of FM Dyes with DiI-LDL in Sorting Endosomes and
with Cy5-Tf in the ERC--
Double label experiments were performed to
see if FM dyes were entering the same endosomes as LDL and Tf. To
visualize the locations of FM dyes at the beginning of chase
experiments after a 30-s pulse, double label experiments using DiI-LDL
and FM dyes were performed. Cells were first incubated briefly with
DiI-LDL to label endosomes and then pulsed with one of the FM dyes.
Cells were pulsed with the two probes separately to prevent FM dyes from binding to DiI-LDL molecules on the cell surface or in solution and subsequently co-internalizing with DiI-LDL. When imaged by confocal
microscopy, endosomes in CHO cells appear as punctate dots located
under the plasma membrane. Fig. 5,
A-C, shows a cell double-labeled with DiI-LDL and FM 2-10,
and Fig. 5, D-F, shows a cell double-labeled with DiI-LDL
and FM 1-84. In these single optical section confocal microscopy
images, co-localization of FM dyes (encoded green) with
DiI-LDL (encoded red) was indicated by the yellow
and orange dots in C and F,
suggesting that a significant fraction of FM dye molecules indeed went
into endosomes labeled by DiI-LDL. The difference in the trafficking
kinetics of the two probes might have contributed to the fact that the
FM dyes did not co-localize entirely with DiI-LDL. At the time these
live cell images were taken (1-2 min after pulsing), some FM molecules had already trafficked to a pericentriolar ERC, while a portion of
sorting endosomes containing DiI-LDL had become fusion-incapable (19).
Membrane tubules bud off from sorting endosomes and deliver recycling
membrane constituents to a pericentriolar region in CHO cells. The ERC,
consisting of a collection of these tubules, appears as a bright
juxtanuclear spot when imaged by fluorescence microscopy. To see
whether FM dyes recycle through the same compartments as other
recycling molecules (e.g. Tf), double label experiments of
FM dyes with Cy5-Tf were performed. Both probes started to accumulate
in the perinuclear region as soon as 5 min after pulsing and remained
extensively co-localized in the ERC for at least 15 min. Fig.
6, A-D, shows a cell
double-labeled with Cy5-Tf and FM 2-10; and Fig. 6, E-H,
shows a cell double-labeled with Cy5-Tf and FM 1-84. Based on the
efflux kinetics, the fraction of the two dyes remaining is expected to
be different after a 15-min chase, but the purpose of this experiment
is to see if some of the dye remaining in the cells is in the ERC.
Passage through the ERC would indicate that dyes leaving the cell with
a t1/2 of about 12 min are following the recycling
itinerary that has been described previously for transferrin. As shown
in Fig. 6, both dyes have substantial overlap with Cy5-Tf in the ERC.
The FM dyes also showed a few punctate dots away from the
pericentriolar region, which presumably were compartments in the
lysosomally targeted pathway (e.g. late endosomes). Of the
two dyes, more of the FM 2-10 is in these punctate structures, which
is consistent with its higher degree of retention in cells due to its
greater partitioning into the aqueous phase in endosomes. A fraction of FM 2-10 could be transported from the ERC back to sorting endosomes via an intracellular route (20), and this may be partially responsible for the high retention of FM 2-10 by cells.
"Direct" Versus "Indirect" Model--
Two hypothetical
models for rapid recycling are presented in Fig.
7. In the direct return model
(A), vesicles bud off from the plasma membrane and
subsequently fuse back with the plasma membrane, such that there is no
mixing of membrane or contents with other organelles. If this model is
correct, the fraction of all FM dyes passing through the rapid
recycling pathway should be the same, because any FM dye in such a
vesicle would stay trapped until released when vesicles fuse back with
the plasma membrane.
Alternatively, the indirect return model (B) proposes that
the internalized molecules traffic through a larger compartment where
they redistribute between membrane and aqueous volume according to
their partition coefficients. The redistribution process results in a
higher recycling efficiency for the more hydrophobic probes. This
occurs because the more hydrophobic molecules tend to distribute in the
tubular portion of a tubulovesicular compartment where the
membrane/volume ratio is higher. The more hydrophilic molecules, on the
other hand, are distributed more in the luminal volume of such a
compartment and recycle with lower efficiency. This model predicts a
difference in recycling efficiency among different FM dyes through the
rapid recycling pathway. As shown in Fig. 4, the more hydrophobic
probes, FM 1-43 and FM 1-84, are much more efficiently recycled
during the first 10 min of chase, as compared with the more hydrophilic
probe, FM 2-10. This result supports the "indirect return model,"
confirming the involvement of an endocytic compartment larger than the
primary endocytic vesicles in the rapid recycling pathway.
Identification of the Endocytic Compartment by Confocal
Microscopy--
The endocytic compartment was identified as sorting
endosomes by double label microscopy using DiI-LDL as an endocytic
marker. Fig. 5 shows that the FM dyes and DiI-LDL were internalized
into the same endosomes. From the short incubation time (4 min), we know that nearly all of the DiI-LDL is in sorting endosomes at this
time in CHO cells (19). The slow component of FM recycling was
identified based on co-localization with Cy5-Tf, which labeled the ERC
under the conditions used (Fig. 6). A schematic diagram of the two
recycling pathways in CHO cells is presented in Fig. 8.
Membrane Turnover Rate--
The fact that approximately half of
the internalized membrane recycles rapidly makes it necessary to
reconsider the internalization rate of lipid analogs measured
previously (21). As shown in Fig. 8 and in the kinetic model below,
what was taken as the internalization rate previously was, in fact, a
rate describing a combination of internalization and rapid recycling
processes. PM, SE, and RC (Scheme
1) represent the probe concentration in
the plasma membrane, sorting endosomes, and ERC, respectively. Rate
constants k1-k4 are
included in Fig. 8.
A simple kinetic modeling of the rate constants leads to a relationship
between the internalization rate k1 and
k1', the net internalization rate constant after
the rapid recycling has come to steady state.
Biological Role of Rapid Recycling--
It has been recognized
previously that specialized cells may recycle molecules rapidly to
carry out specific functions. In reticulocytes, a high rate of iron
uptake is maintained by recycling Tf receptors with a half-time of
~90 s (24). The kinetics of endocytosis in renal proximal tubule
showed that membrane recycling near the apical surface takes place with
a t1/2 of 1.5 min, and this process is used to
recover small molecules such as vitamins from the urine (25). We now
suggest that many cell types are capable of rapid recycling, and this
process may become dominant in some specialized cells.
The rapid recycling pathway also provides an explanation for a
discrepancy between the uptake kinetics of iron and that of transferrin. In HepG2 hepatoma cells (26) or in phorbol 12-myristate 13-acetate-treated K562 cells (27), the rate of iron uptake is almost
twice as fast as that measured for transferrin. We propose that rapid
recycling of transferrin through acidic sorting endosomes could account
for the additional iron delivered to the cells, since a rapid recycling
would have caused an underestimation of the rate of transferrin uptake.
Rapid membrane turnover may also be essential for uptake of folate,
which requires passage of folate bound to its
glycosylphosphatidylinositol-anchored receptor through an acidic
compartment (28). The folate receptor is delivered to sorting endosomes
(29). The rapid recycling that we observe could also dramatically
increase the rate of cholesterol efflux, which is mediated by high
density lipoprotein endocytosis and recycling (30, 31). Rapid recycling
might be interpreted as direct efflux at the plasma membrane in most
experimental designs.
A kinetic model of Tf recycling and transcytosis in polarized MDCK
cells was described by Sheff et al. (32). The slow recycling component had a half-time of ~12 min for exit to the basolateral plasma membrane, and the fast component had a half-time of ~6 min,
which was interpreted to be direct recycling from early (sorting) endosomes. However, in light of our finding that rapid recycling from
sorting endosomes has a t1/2 of 1.4 min, it seems
likely that the exit with a t1/2 of 6 min is from
another endosomal compartment. We suggest that the 6- and 12-min
half-times described by Sheff et al. may reflect exit from
some of the various Tf-containing endosomal compartments found in
polarized MDCK cells (33) rather than the rapid recycling from sorting
endosomes described in this paper.
In summary, we have detected and characterized a rapid recycling
pathway in various nonpolarized mammalian cells using fluorescent lipid
analogs. This suggests that rapid recycling is a general property of
most cells rather than a specialized adaptation. With an exocytic
half-time of 1-2 min, rapid recycling involves trafficking through
sorting endosomes and accounts for about 33-60% of the recycling
population. The observation of a high fraction of rapid recycling
implies a much higher membrane turnover rate than previously estimated.
We are grateful to Drs. Sushmita Mukherjee
and Timothy E. McGraw for useful discussions. We thank Drs. Sushmita
Mukherjee and William G. Mallet for critical reading of the manuscript.
*
This work was supported by National Institutes of Health
Grant DK 27083.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 should be addressed. Dept. of
Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6405; Fax: 212-746-8875; E-mail: frmaxfie@med.cornell.edu.
The abbreviations used are:
ERC, endocytic
recycling compartment;
C6-NBD-SM, N-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)
sphingosyl phosphocholine;
C6-NBD-Cer, N-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)
ceramide;
DiI, dialkylindocarbocyanine;
LDL, low density lipoprotein;
MDCK, Madin-Darby canine kidney;
Tf, transferrin;
M1, medium 1.
Characterization of Rapid Membrane Internalization and
Recycling*
§ and¶
Department of Biochemistry, Weill Medical
College of Cornell University, New York, New York 10021 and the
§ Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York 14853
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (8K):
[in a new window]
Fig. 1.
Chemical structures of FM 2-10, FM 1-43,
and FM 1-84.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
f.
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[in a new window]
Fig. 2.
Exit of C6-NBD-SM in CHO
cells. Cells were labeled with C6-NBD-SM for various
pulse lengths, back-exchanged on ice for 1 h, and chased in
37 °C chase medium for 1 h. Aliquots of chase medium were taken
at different time points, and the percentage of cell-associated
fluorescence was calculated for each time point (see "Experimental
Procedures"). A computer fitting program was used to give the
parameters that defined the best fit to the data points. Data points
are presented by open circles, and double
exponential decay fits are plotted as solid
lines. The dotted line in A
shows the best fit to a single exponential decay. Kinetic parameters
obtained from the fitting program are listed in Table I. Data points
for all kinetic curves in this paper were derived from an average of
triplicate experiments.
View larger version (17K):
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Fig. 3.
Exit of C6-NBD-SM in other cell
lines. Efflux of C6-NBD-SM was measured in HEp2 cells
(A), nonpolarized MDCK cells (B), and CHO cells
where clathrin-mediated endocytosis was blocked (C). Double
and single exponential decay fits are plotted as solid and
dotted lines, respectively. See the legend to
Fig. 2 for experimental procedures. Kinetic parameters from Fig. 3 are
listed in Table I.
bt + c·edt + e by SigmaPlot Scientific
Graphing Software (Jandel Scientific, San Rafael, CA).
where Aex and Aem
are absorption readings at excitation and emission wavelength,
respectively (16).
(Eq. 1)
where (Pm/M) and
(Pf/W) refer to molar ratios of
membrane-bound probe (Pm) to membrane lipids (M) and of fluid phase probe (Pf) to
water (W), respectively.
(Eq. 2)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
bt + c·edt + e. Theoretical
fits were then plotted as solid lines in Fig. 2.
If C6-NBD-SM efflux occurred by a single component,
first-order kinetic process, a single exponential decay would be
expected to fit the data points. However, this was not the case. To
illustrate this point, a single exponential best fit was added to Fig.
2A. Represented by a dotted line, the
single exponential decay could not account for the rapid decrease of
fluorescence during the first 10 min of chase time. It could, however,
provide a good fit for the slower process dominating the second half of
the chase time. A double exponential decay, on the other hand, very
well accounted for all of the data.
Summary of kinetic parameters of the recycling pathways in CHO cells
bt + c·edt + e. Numbers are
mean ± S.D. Half-time values (min) are provided in parentheses.
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Fig. 4.
Efflux kinetics of FM dyes after 30-s pulse
periods. Cells were labeled with one of the FM dyes for 30 s
at 37 °C, either rinsed in 37 °C M1-glucose (solid
triangles) or rinsed with ice-cold M1-glucose, kept on ice
while further washes of M1-glucose were applied to ensure complete
removal of surface-bound dye molecules, warmed up with 37 °C
M1-glucose (open circles), and imaged on a
37 °C temperature-equilibrated microscope stage for 1 h. Efflux
kinetic curves of FM 2-10 (A), FM 1-43 (B), or
FM 1-84 (C) following a 30-s pulse were obtained by
quantification of wide field microscopy images. Fluorescence power is
normalized to that of the first reading at the beginning of the chase.
FM 2-10 data points were fit with a single exponential decay with a
half-time of 12 min. Data points from FM 1-43 and FM 1-84 fit well to
a double exponential decay, which generated a half-time of ~2 min for
the fast component and ~11 min for the slow component. Kinetic
parameters from Fig. 4 are listed in Table I.
View larger version (36K):
[in a new window]
Fig. 5.
Colocalization of FM dyes with DiI-LDL.
Cells were labeled with DiI-LDL for 4 min at 37 °C and subsequently
with FM 2-10 (B) or FM 1-84 (E) at 37 °C for
30 s in the absence of the labeled LDL. Cells were then washed
with ice-cold medium 1 to remove surface-bound dye molecules. Cells
were imaged 1-2 min after being warmed up by adding 37 °C
M1-glucose and kept on a 37 °C temperature-equilibrated microscope
stage during image acquisition. A and D show
DiI-LDL staining, B shows FM 2-10 staining, E
shows FM 1-84 staining, and C and F are overlays
of the double staining. The images are single plane confocal microscopy
images. The full width at half-height for the optical sections is 0.8 µm. Bar, 10 µm.
View larger version (43K):
[in a new window]
Fig. 6.
Colocalization of FM dyes with Cy5-Tf.
Cells were labeled with Cy5-Tf for 2 min at 37 °C and subsequently
with one of the FM dyes at 37 °C for 30 s in the absence of the
labeled protein. Cells were then washed with ice cold M1-glucose to
remove surface-bound dye molecules. Cells were chased for 15 min at
37 °C before imaging on a temperature-controlled confocal microscope
stage. A and E are single-plane DIC images.
B and F show Cy5-Tf staining, C shows
FM 2-10 staining, G shows FM 1-84 staining, and
D and H are overlays of the double staining.
Fluorescence images are projections of confocal microscopy images. The
full width at half-height for the optical sections is 0.8 µm.
Bar, 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (10K):
[in a new window]
Fig. 7.
Schematic diagrams of two models of the rapid
recycling pathway. The "direct return model" is shown at the
top, and the "indirect return model" is shown at the
bottom. In A, internalized vesicles fuse back
with the plasma membrane without going through sorting endosomes.
Therefore, the number of molecules recycled back to the plasma membrane
should be exactly the same as the number internalized, independent of
partition between the membrane and the aqueous content. In contrast, in
B, internalized vesicles fuse with sorting endosomes, and
recycling membrane subsequently buds from sorting endosomes to return
to the plasma membrane. The fusion and budding process results in a
difference in the percentage of molecules recycled back through rapid
recycling, and the difference is related to the partition coefficient
of each molecule. FM dyes are represented by filled
circles.
View larger version (22K):
[in a new window]
Fig. 8.
Schematic diagram of recycling pathways in
CHO cells. Various lipid molecules and ligand-receptor complexes
are internalized from the plasma membrane, traffic through endocytic
compartments, and return to the plasma membrane via two distinct
recycling pathways. Examples of ligand-receptor complexes given here
are Tf receptor (TR) bound to iron-loaded Tf
( 
Tf) and LDL bound to its receptor (LR).
Lipid molecules are represented in this diagram by the FM dyes (
).
Vesicles carrying internalized molecules and complexes fuse with
sorting endosomes located near the cell periphery. The acidic
environment in sorting endosomes causes dissociation of iron () from
Tf and dissociation of LDL from LR. The FM dyes redistribute between
the membrane and luminal volume of sorting endosomes according to their
partition coefficients. Membrane containing TR-Tf, LR, and FM molecules
will subsequently bud from sorting endosomes and go into one of two
recycling pathways. Lipids and membrane proteins return directly to the
plasma membrane after exiting sorting endosomes with a half-time of
1-2 min via the rapid recycling pathway discussed in this paper.
Alternatively, they go into the ERC and eventually recycle back to the
plasma membrane with a half-time of ~12 min. Sorting endosomes will
mature into late endosomes after some time. Rate constants
k1-k4 correspond to
those mentioned in the kinetic model used under "Discussion."
a, Refs. 4, 22, and 23; b, Refs. 12, 19, and 34;
c, Ref. 4.
View larger version (13K):
[in a new window]
Scheme 1.
k1' was measured to be 0.035-0.046
min
(Eq. 3)
1 (21). If we take k2 as
0.35-0.7 min1 and k3 as 0.35 min1 (4, 22, 23), the true internalization rate,
k1, is calculated to be 0.069-0.138
min1. This result, in turn, suggests that the half-time
for membrane turnover could be as short as 5-10 min, as opposed to
15-20 min (21). Therefore, the observation of a high fraction of rapid recycling reveals the plasma membrane system as being extremely dynamic
with a very rapid turnover rate.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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R. M. Gage, E. A. Matveeva, S. W. Whiteheart, and M. von Zastrow Type I PDZ Ligands Are Sufficient to Promote Rapid Recycling of G Protein-coupled Receptors Independent of Binding to N-Ethylmaleimide-sensitive Factor J. Biol. Chem., February 4, 2005; 280(5): 3305 - 3313. [Abstract] [Full Text] [PDF]
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A. Pagano, P. Crottet, C. Prescianotto-Baschong, and M. Spiess In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5 Mol. Biol. Cell, November 1, 2004; 15(11): 4990 - 5000. [Abstract] [Full Text] [PDF]
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F. Coumailleau, V. Das, A. Alcover, G. Raposo, S. Vandormael-Pournin, S. Le Bras, P. Baldacci, A. Dautry-Varsat, C. Babinet, and M. Cohen-Tannoudji Over-Expression of Rififylin, a New RING Finger and FYVE-like Domain-containing Protein, Inhibits Recycling from the Endocytic Recycling Compartment Mol. Biol. Cell, October 1, 2004; 15(10): 4444 - 4456. [Abstract] [Full Text] [PDF]
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A. Choudhury, D. K. Sharma, D. L. Marks, and R. E. Pagano Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling Mol. Biol. Cell, October 1, 2004; 15(10): 4500 - 4511. [Abstract] [Full Text] [PDF]
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N. Kalin, J. Fernandes, S. Hrafnsdottir, and G. van Meer Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells J. Biol. Chem., August 6, 2004; 279(32): 33228 - 33236. [Abstract] [Full Text] [PDF]
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 J. L. Fisher, I. Levitan, and S. S. Margulies Plasma Membrane Surface Increases with Tonic Stretch of Alveolar Epithelial Cells Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 200 - 208. [Abstract] [Full Text] [PDF]
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K. Hueffer, L. M. Palermo, and C. R. Parrish Parvovirus Infection of Cells by Using Variants of the Feline Transferrin Receptor Altering Clathrin-Mediated Endocytosis, Membrane Domain Localization, and Capsid-Binding Domains J. Virol., June 1, 2004; 78(11): 5601 - 5611. [Abstract] [Full Text] [PDF]
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N. Naslavsky, M. Boehm, P. S. Backlund Jr., and S. Caplan Rabenosyn-5 and EHD1 Interact and Sequentially Regulate Protein Recycling to the Plasma Membrane Mol. Biol. Cell, May 1, 2004; 15(5): 2410 - 2422. [Abstract] [Full Text] [PDF]
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S. C. Frasch, P. M. Henson, K. Nagaosa, M. B. Fessler, N. Borregaard, and D. L. Bratton Phospholipid Flip-Flop and Phospholipid Scramblase 1 (PLSCR1) Co-localize to Uropod Rafts in Formylated Met-Leu-Phe-stimulated Neutrophils J. Biol. Chem., April 23, 2004; 279(17): 17625 - 17633. [Abstract] [Full Text] [PDF]
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M. Hao, S. Mukherjee, Y. Sun, and F. R. Maxfield Effects of Cholesterol Depletion and Increased Lipid Unsaturation on the Properties of Endocytic Membranes J. Biol. Chem., April 2, 2004; 279(14): 14171 - 14178. [Abstract] [Full Text] [PDF]
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 D. Wustner, M. Mondal, A. Huang, and F. R. Maxfield Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells J. Lipid Res., March 1, 2004; 45(3): 427 - 437. [Abstract] [Full Text] [PDF]
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H. Rosen, V. Glukhman, T. Feldmann, E. Fridman, and D. Lichtstein Cardiac Steroids Induce Changes in Recycling of the Plasma Membrane in Human NT2 Cells Mol. Biol. Cell, March 1, 2004; 15(3): 1044 - 1054. [Abstract] [Full Text] [PDF]
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S. X. Lin, W. G. Mallet, A. Y. Huang, and F. R. Maxfield Endocytosed Cation-Independent Mannose 6-Phosphate Receptor Traffics via the Endocytic Recycling Compartment en Route to the trans-Golgi Network and a Subpopulation of Late Endosomes Mol. Biol. Cell, February 1, 2004; 15(2): 721 - 733. [Abstract] [Full Text] [PDF]
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 Z.-L. Xiao, P. Biancani, and J. Behar Role of PGE2 on gallbladder muscle cytoprotection of guinea pigs Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G82 - G88. [Abstract] [Full Text] [PDF]
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A. Remacle, G. Murphy, and C. Roghi Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface J. Cell Sci., October 1, 2003; 116(19): 3905 - 3916. [Abstract] [Full Text] [PDF]
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R. Mohammad-Panah, R. Harrison, S. Dhani, C. Ackerley, L.-J. Huan, Y. Wang, and C. E. Bear The Chloride Channel ClC-4 Contributes to Endosomal Acidification and Trafficking J. Biol. Chem., August 1, 2003; 278(31): 29267 - 29277. [Abstract] [Full Text] [PDF]
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M. K. Loder and H. E. Melikian The Dopamine Transporter Constitutively Internalizes and Recycles in a Protein Kinase C-regulated Manner in Stably Transfected PC12 Cell Lines J. Biol. Chem., June 6, 2003; 278(24): 22168 - 22174. [Abstract] [Full Text] [PDF]
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Y. Sun, M. Hao, Y. Luo, C.-p. Liang, D. L. Silver, C. Cheng, F. R. Maxfield, and A. R. Tall Stearoyl-CoA Desaturase Inhibits ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux and Modulates Membrane Domain Structure J. Biol. Chem., February 14, 2003; 278(8): 5813 - 5820. [Abstract] [Full Text] [PDF]
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E. M. van Dam, T. ten Broeke, K. Jansen, P. Spijkers, and W. Stoorvogel Endocytosed Transferrin Receptors Recycle via Distinct Dynamin and Phosphatidylinositol 3-Kinase-dependent Pathways J. Biol. Chem., December 6, 2002; 277(50): 48876 - 48883. [Abstract] [Full Text] [PDF]
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A. H. van der Luit, M. Budde, P. Ruurs, M. Verheij, and W. J. van Blitterswijk Alkyl-lysophospholipid Accumulates in Lipid Rafts and Induces Apoptosis via Raft-dependent Endocytosis and Inhibition of Phosphatidylcholine Synthesis J. Biol. Chem., October 11, 2002; 277(42): 39541 - 39547. [Abstract] [Full Text] [PDF]
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 S. Roy, B. Wyse, and J. F. Hancock H-Ras Signaling and K-Ras Signaling Are Differentially Dependent on Endocytosis Mol. Cell. Biol., July 15, 2002; 22(14): 5128 - 5140. [Abstract] [Full Text] [PDF]
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 D. Sheff, L. Pelletier, C. B. O'Connell, G. Warren, and I. Mellman Transferrin receptor recycling in the absence of perinuclear recycling endosomes J. Cell Biol., March 4, 2002; 156(5): 797 - 804. [Abstract] [Full Text] [PDF]
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 M. Hao, S. Mukherjee, and F. R. Maxfield Cholesterol depletion induces large scale domain segregation in living cell membranes PNAS, November 6, 2001; 98(23): 13072 - 13077. [Abstract] [Full Text] [PDF]
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 F. Schroeder, A. M. Gallegos, B. P. Atshaves, S. M. Storey, A. L. McIntosh, A. D. Petrescu, H. Huang, O. Starodub, H. Chao, H. Yang, et al. Recent Advances in Membrane Microdomains: Rafts, Caveolae, and Intracellular Cholesterol Trafficking Experimental Biology and Medicine, November 1, 2001; 226(10): 873 - 890. [Abstract] [Full Text] [PDF]
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M. A. Lampson, J. Schmoranzer, A. Zeigerer, S. M. Simon, and T. E. McGraw Insulin-regulated Release from the Endosomal Recycling Compartment Is Regulated by Budding of Specialized Vesicles Mol. Biol. Cell, November 1, 2001; 12(11): 3489 - 3501. [Abstract] [Full Text] [PDF]
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X. Zha, J. Genest Jr., and R. McPherson Endocytosis Is Enhanced in Tangier Fibroblasts. POSSIBLE ROLE OF ATP-BINDING CASSETTE PROTEIN A1 IN ENDOSOMAL VESICULAR TRANSPORT J. Biol. Chem., October 12, 2001; 276(42): 39476 - 39483. [Abstract] [Full Text] [PDF]
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L Citores, D Khnykin, V Sorensen, J Wesche, O Klingenberg, A Wiedlocha, and S Olsnes Modulation of intracellular transport of acidic fibroblast growth factor by mutations in the cytoplasmic receptor domain J. Cell Sci., January 5, 2001; 114(9): 1677 - 1689. [Abstract] [PDF]
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B. P. Ceresa, M. Lotscher, and S. L. Schmid Receptor and Membrane Recycling Can Occur with Unaltered Efficiency Despite Dramatic Rab5(Q79L)-induced Changes in Endosome Geometry J. Biol. Chem., March 23, 2001; 276(13): 9649 - 9654. [Abstract] [Full Text] [PDF]
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G. Kilic, R. B. Doctor, and J. G. Fitz Insulin Stimulates Membrane Conductance in a Liver Cell Line. EVIDENCE FOR INSERTION OF ION CHANNELS THROUGH A PHOSPHOINOSITIDE 3-KINASE-DEPENDENT MECHANISM J. Biol. Chem., July 13, 2001; 276(29): 26762 - 26768. [Abstract] [Full Text] [PDF]
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R. M. Gage, K.-A. Kim, T. T. Cao, and M. von Zastrow A Transplantable Sorting Signal That Is Sufficient to Mediate Rapid Recycling of G Protein-coupled Receptors J. Biol. Chem., November 21, 2001; 276(48): 44712 - 44720. [Abstract] [Full Text] [PDF]
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M. Hao, S. X. Lin, O. J. Karylowski, D. Wustner, T. E. McGraw, and F. R. Maxfield Vesicular and Non-vesicular Sterol Transport in Living Cells. THE ENDOCYTIC RECYCLING COMPARTMENT IS A MAJOR STEROL STORAGE ORGANELLE J. Biol. Chem., January 4, 2002; 277(1): 609 - 617. [Abstract] [Full Text] [PDF]
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D. Sheff, L. Pelletier, C. B. O'Connell, G. Warren, and I. Mellman Transferrin receptor recycling in the absence of perinuclear recycling endosomes J. Cell Biol., March 4, 2002; 156(5): 797 - 804. [Abstract] [Full Text] [PDF]
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S. X. Lin, G. G. Gundersen, and F. R. Maxfield Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin Mol. Biol. Cell, January 1, 2002; 13(1): 96 - 109. [Abstract] [Full Text] [PDF]
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