From the MRC Virology Unit, Church Street, Glasgow
G11 5JR, Scotland, United Kingdom, § MRC Technology,
Crewe Road South, Edinburgh EH4 2SP, Scotland, United Kingdom,
¶ MTM Laboratories AG, Im Neuenheimer Feld 583, D-69120
Heidelberg, Germany, and the
Cell Biology and Biophysics
Programme, EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Received for publication, November 5, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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Neutral lipid is stored in spherical organelles
called lipid droplets that are bounded by a coat of proteins. The
protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we
demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this
system to identify targeting elements, discontinuous segments within
the coding region were required for directing ADRP to lipid droplets.
GFP-tagged protein was employed also to examine the behavior of lipid
droplets in live cells. Time lapse microscopy demonstrated that in
HuH-7 cells, which are derived from a human hepatoma, a small number of
lipid droplets could move rapidly, indicating transient association
with intracellular transport pathways. Most lipid droplets did not show
such movement but oscillated within a confined area; these droplets
were in close association with the endoplasmic reticulum membrane and
moved in concert with the endoplasmic reticulum. Fluorescence recovery
analysis of GFP-tagged ADRP in live cells revealed that surface
proteins do not rapidly diffuse between lipid droplets, even in
conditions where they are closely packed. This system provides new
insights into the properties of lipid droplets and their interaction
with cellular processes.
In mammalian cells, lipid droplets serve as storage organelles,
consisting primarily of cholesterol ester and triacylglyerols (reviewed in Ref. 1). Although long considered to be inert structures, there is now increasing evidence that they play active and
diverse roles in the life cycle of cells. Recently, they have been
implicated in maintenance of intracellular cholesterol balance and
transport of lipids through association with caveolin proteins (2).
Moreover, the lipid stored in droplets is utilized for specialized
purposes in certain cell types. For example, steroid hormones in
steroidogenic cells are produced from cholesterol ester stored in lipid
droplets (3); mammary epithelial cells release milk fat globules from
their apical surface that are directly derived from aggregated lipid
droplets (4); lipid droplets appear to be the primary source of fatty
acids that are converted into triacylglycerols and incorporated into
very low density lipoprotein in hepatocytes (5). There is also a
correlation between particular human diseases and accumulation of lipid
droplets that suggests they are markers of pathological changes. Such
diseases include atheroma, steatosis, obesity, and some cancers (6, 7).
More recently, it has been suggested that aberrant targeting of the Nir2 protein to lipid droplets may induce changes to lipid transport that could correlate with retinal degeneration seen in
Drosophila mutants (8). Indeed, lipid droplets may have
important functions in viral and parasitic infections (9-13).
The surface of lipid droplets has a proteinaceous layer that is thought
to prevent fusion with any adjacent lipophilic surface. However,
analysis of the properties of surface proteins has received only
limited attention. The most widely characterized lipid
droplet-associated proteins are the perilipins, a family of
polypeptides generated by alternative splicing from a single copy gene
(14), and adipocyte differentiation-related protein
(ADRP,1 also termed
adipophilin) (6, 15-17). Expression of the perilipins appears
restricted to adipocytes and steroidogenic cells (Refs. 18 and 19,
reviewed in Ref. 20) whereas ADRP is detected in a broad range of
different tissues (15). Examination of a variety of tissue culture
cells also has revealed ADRP as a ubiquitous component of lipid
droplets (6, 15). Moreover, enhanced expression of ADRP is a useful
marker for pathologies that are characterized by increased
accumulations of lipid droplets (6, 21-24). Hence, analysis of ADRP
would provide valuable insight into the nature of proteins that
associate with lipid droplets. In addition, ADRP offers potential for
describing the characteristics of lipid droplets.
In this study, we have fused green and yellow fluorescent proteins
(EGFP and EYFP) to the human and mouse ADRP coding sequences and
examined the ability of the fusion proteins to associate with lipid
droplets in tissue culture cells. This system was employed to identify
sequences in ADRP that are required for localization to lipid droplets.
ADRP tagged with fluorescent proteins also was used to analyze the
behavior of lipid droplets in live cells and their interaction with
other intracellular compartments.
Generation of Human and Mouse ADRP cDNA--
ADRP
gene-specific primers (primers 1 and 2, Table I) were designed from the
human ADRP (hADRP) mRNA sequence (GenBankTM accession
number BC005127). In conjunction with the Access RT-PCR System
(Promega, United Kingdom), these primers generated hADRP cDNA from
total RNA prepared from the human hepatoma cell line, HuH-7. Total RNA
was made using Trizol reagent (Sigma) according to the manufacturer's
instructions. The hADRP cDNA was ligated to pGEM-T Easy (Promega)
and, from the resultant progeny after transformation, plasmids were
screened for the presence of hADRP sequences by restriction
endonuclease digestion. Plasmid DNA from one positive bacterial clone,
termed pLA1, was isolated and the nucleotide sequence of the hADRP
cDNA was determined. For the mouse ADRP (mADRP) cDNA, an
amplified product was generated using mRNA isolated from mouse L
cells and primers 3 and 4 (Table I) that were derived from published
murine sequences (GenBankTM accession number NM007408; Ref.
12). The PCR product was inserted into pCRII-TOPO (Invitrogen). A clone
containing a mADRP cDNA insert, pCRII/mADRP, was selected by
restriction enzyme analysis and the nucleotide sequence of the inserted
fragment was determined.
Construction of Plasmids Expressing DNase X, Human and Mouse ADRP
Fused to Fluorescent Proteins--
The hADRP ORF was excised from pLA1
using EcoRI and ligated to EcoRI linearized
pEGFP-C1 (Clontech) to generate a plasmid termed
pLA4 that encoded a GFP-hADRP fusion protein. The same strategy was
employed to fuse the hADRP ORF to YFP in plasmid pEYFP-C1
(Clontech). Plasmid pLA5, encoding the N-terminal
half of hADRP linked to the C terminus of GFP, was constructed by
digesting pLA4 with BamHI, which removed the 3' terminal
region of the hADRP ORF, followed by re-circularization of the digested
plasmid. To fuse the C-terminal region of hADRP to GFP, the smaller DNA
fragment liberated upon digestion of pLA4 with BamHI was
purified and ligated to BamHI linearized pEGFP-C1. This
construct was named pLA14. Constructs pLA10, pLA9, and pLA13 encoded
GFP-hADRP fusion proteins in which premature stop codons were inserted
in the coding sequence by site-directed mutagenesis using the Altered
Sites II mammalian in vitro mutagenesis system (Promega).
This system required the use of oligonucleotides containing single
nucleotide mismatches corresponding to the region of the hADRP ORF to
be mutated. Oligonucleotides Mut1, Mut2, and Mut3 (Table I) were used
to generate the GFP-hADRP fusion proteins encoded by pLA10, pLA9, and
pLA13, respectively, and premature stop codons were inserted at
nucleotide positions 671-673 (pLA10), 593-595 (pLA9), and 503-505
(pLA13) within the hADRP ORF (nucleotides numbered according to the
ADRP sequence in BC005127). N-terminal deletions of the hADRP ORF were
created by using Mut4 and Mut5 oligonucleotides (Table I) to amplify regions of the ADRP ORF from internal ATG codons situated at
nucleotides 230-232 (pLA11) and 170-172 (pLA12), respectively. In
both PCR reactions, the downstream primer was MutB (Table I), which
terminated amplification of sequences after the BamHI site
was located within the hADRP ORF. The resulting hADRP DNA fragments
were ligated into pGEM-T Easy. EcoRI digestion was used to
liberate the hADRP DNA fragments from pGEM-T Easy, which were then
inserted into EcoRI-linearized pEGFP-C1. To generate pLA17,
ligation was performed with hADRP DNA fragments that were liberated
upon digestion of pLA12 with EcoRI and BamHI and
pLA4 with BamHI and SalI together with pEGFP-C1
linearized with EcoRI and SalI. pLA22 was made by digesting pLA4 with MscI, which removed the region of the
hADRP ORF between nucleotides 267 and 476 (inclusive), followed by
purification and re-circularization of the digested plasmid using T4
DNA ligase. Construct pLA29 was created by digesting pLA4 with
MscI and BamHI, which removed the region of the
hADRP ORF between nucleotides 267 and 746 (inclusive). The digested
plasmid was purified, treated with Klenow enzyme, and re-circularized
using T4 DNA ligase.
For expression of mADRP, a BamHI/SpeI fragment
from pCRII/mADRP containing mADRP nucleotide sequences was inserted
first into pGEM-1 (Promega) cleaved with
HindIII/XbaI along with oligonucleotide mADRP1
(Table I). Because the BamHI/SpeI fragment
removed part of the mADRP coding region (the BamHI site lies
15 nucleotides downstream of the ATG initiation codon), oligonucleotide
mADRP1 restored these sequences. Inserting this oligonucleotide also abolished the BamHI site in the mADRP coding region without
altering the predicted amino acid sequence and placed a novel
BamHI site immediately upstream of the ATG codon (Table I).
The resulting clone was termed pGEM/mADRP. A BamHI fragment
from pGEM/mADRP that was introduced into pEGFP-C1 also cleaved with
BamHI to give plasmid pGFP-mADRP.
The vector pGFP-DNase X, which directs the synthesis of a GFP-DNase X
fusion product, was obtained by initially subcloning a PCR-generated
cDNA fragment containing the complete coding region of DNase X (25)
into the mammalian expression vector pcDNA3.1 (Invitrogen). The
DNase X coding region was fused N-terminal to GFP in pEGFP
(Clontech), to give pGFP-DNase X.
Maintenance of Tissue Culture Cells and Generation of Cells
Expressing GFP-mADRP--
HuH-7 and Vero cells were propagated in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 2 mM L-glutamine, non-essential amino
acids, and 100 IU/ml penicillin/streptomycin. To generate cells
constitutively expressing GFP-mADRP, cells were transfected with
plasmid pGFP-mADRP followed by selection with 800 µg/ml G418
(Clontech). Clones producing GFP-mADRP were
selected first by pooling GFP fluorescent cells isolated by
fluorescent-activated cell sorting followed by growth of individual
colonies. Cloned cell lines were maintained in media containing 500 µg/ml G418.
Microinjection and Transfection of Plasmid DNAs--
For live
cell studies, cells were plated onto either 13-mm sterile coverslips or
35-mm live cell dishes (MatTek Corp.) at ~10% confluency. The
following day, cells in each well were transfected with 1 µg of the
appropriate plasmid DNA using TransFast transfection reagent (Promega)
according to the manufacturer's instructions. Transfected cells were
incubated overnight at 37 °C and analyzed ~16 to 18 h after
transfection. For microinjection, plasmids were diluted to a final
concentration of 100 µg/ml in double distilled H2O and
injected with borosilicate capillaries (Harvard Apparatus) using an
Eppendorf injection system. Cells were maintained at 37 °C for 5-8
h before analysis.
Preparation of Cell Extracts, Polyacrylamide Gel Electrophoresis,
and Western Blot Analysis--
To prepare extracts, transfected HuH-7
cells were harvested by removing growth medium and washing the cell
monolayers with PBS. Cells were solubilized in sample buffer,
consisting of 160 mM Tris-HCl, pH 6.7, 2% SDS, 700 mM Indirect Immunofluorescence, Staining of Lipid Droplets, and GFP
Fluorescence--
Cells on 13-mm coverslips were fixed for 20 min in
methanol at
Lipid droplets were stained with oil red O (Sigma) in
paraformaldehyde-fixed cells as described previously (11, 12). For staining of lipid droplets with Bodipy 558/568 C12
(Molecular Probes Europe BV, The Netherlands), cells were incubated
with the dye at a final concentration of 20 µg/ml for 45 min at
37 °C in PBS, rinsed to remove excess stain followed by a further incubation of 60 min at 37 °C in cell culture media. Cells were either viewed live or fixed with 4% paraformaldehyde at 4 °C for 30 min. Fixed cells were washed with PBS prior to mounting as described above.
To examine GFP fluorescence, cells were either viewed live or after
fixation with 4% paraformaldehyde at 4 °C for 30 min. The
distribution of proteins made by the various GFP-hADRP plasmids was
determined by observation with a mercury lamp connected to a Zeiss
LSM510 confocal microscope to provide standard viewing conditions. The
intracellular distribution of GFP was determined for 100 cells
expressing each of the GFP-hADRP variants.
Acquisition of Live Cell Data--
To obtain still images of
live cells and for FRAP analysis, coverslips were placed inverted on a
microscope slide and analyzed using a Zeiss LSM510 confocal microscope.
For photobleaching studies, selected regions of cells were bleached at
100% laser power (488 nm laser line). Before and after photobleaching,
images were taken at 7-s intervals using 2% laser power. Time-lapse
imaging was performed with an Ultraview Realtime Confocal imaging
system (PerkinElmer Life Sciences) in which the microscope was
contained within a temperature-controlled chamber. Imaging was
performed at 37 °C in pre-warmed minimal essential medium lacking
phenol red supplemented with 30 mM HEPES.
Data Analysis--
Quantitative analysis of photobleaching
experiments was performed using Zeiss LSM510 software. Images
captured using the Ultraview system were converted to 8-bit TIF files
using a routine written in Interactive Data Language (created by T. Zimmerman, EMBL) and thereafter analyzed with ImageJ software
(National Institutes of Health, Bethesda, MD). Quicktime
videos were assembled using ImageJ and still images compiled using
Adobe Photoshop version 6 and Adobe Illustrator version 10.
On-line Supplemental Material--
Videos 1 and 2 were assembled
from images of HuH-7 cells expressing GFP-mADRP taken at a frame rate
of 1 per s and an exposure time of 250 ms. Video 3 was assembled from
images of Vero cells that had been microinjected with plasmids
expressing GFP-hADRP (pLA4) and GFP-DNase X. 8 h after injection,
images were taken at 6-s intervals with an exposure time of 600 ms.
Cells were maintained in a humidified atmosphere at 37 °C during the
imaging process. Imaging was performed using an Ultraview Realtime
Confocal imaging system (PerkinElmer Life Sciences).
ADRP Fused to EGFP Associates with Lipid Droplets--
To clone
both the human and mouse cDNA sequences for ADRP, primers were
derived from the nucleotide sequences flanking the coding region for
human and mouse ADRP (Table I). Template
mRNA for reverse transcription was prepared from human hepatoma
HuH-7 and mouse L cells. Following reverse transcription, amplification by PCR and cloning, full-length copies of the coding region for both
human and mouse ADRP were obtained. Sequence analysis of the entire
coding region for one clone of both human and mouse ADRP revealed only
a single nucleotide change in each that was different from sequences
deposited in the GenBankTM data base but these did not
alter the predicted amino acid sequences. For localization studies, DNA
fragments encoding human and mouse ADRP were ligated in-frame to the 3'
end of EGFP, thereby generating GFP-hADRP (expressed by plasmid pLA4)
and GFP-mADRP fusion products. Previous studies have shown that ADRP is
located at the surface of cytoplasmic lipid droplets (6, 15). To
examine whether the GFP-ADRP products for both species retained the
capacity to associate with these structures, constructs expressing the
fusion proteins were introduced into HuH-7 cells by transfection. In the case of GFP-mADRP, transfected cells were selected by G418 selection to isolate clonal cell lines that constitutively expressed the fusion protein. HuH-7 cells contain numerous lipid droplets that
can be identified by staining with lipophilic dyes and detection by
antisera that is specific for ADRP at the surface of the structures (Fig. 1A, panels i
and v). Compared with EGFP, which shows staining throughout
HuH-7 cells (Fig. 1A, panel ii), GFP-mADRP was
located at the surface of spherical intracellular structures in live
cells (Fig. 1A, panel iii). To demonstrate that
these structures represented lipid droplets, fixed cells were probed
with Living ColorsTM antiserum and an antiserum that could
recognize the endogenous human form of ADRP but not the corresponding
mouse protein that was a component of the GFP fusion product (Fig.
1A, panels iv-vi, and data not
shown). The localization for endogenous hADRP and the GFP-mADRP
products coincided (Fig. 1A, panel vi),
indicating that GFP-mADRP was located at the surface of lipid droplets.
This result also demonstrated that it was possible to detect tagged and
untagged ADRP on the same lipid droplet. Therefore, sufficient attachment sites are present on lipid droplets to accommodate both
forms of ADRP. To verify the localization for GFP-mADRP, cells
expressing the fusion protein were incubated with Bodipy 558/568
dodecanoic acid, a fluorescent fatty acid analogue that is sequestered
in lipid droplets (26, 27). This dye was selected because its spectral
properties allow discrimination from EGFP by confocal microscopy and
staining of droplets does not require treatment of cells with alcohols,
which is necessary for oil red O staining and induces fusion of lipid
droplets. Results in both live cells (data not shown) and cells fixed
with paraformaldehyde showed specific localization of GFP-mADRP at the
surface of lipid droplets stained with the Bodipy dye (Fig.
1A, panels vii-ix). The apparent
molecular weight for GFP-mADRP was estimated at about 80,000, which approximates to the predicted size for the fusion protein (Fig.
1B, lane 1). GFP-hADRP, produced by plasmid pLA4, also was detected at the surface of lipid droplets and had an estimated
size that agreed with its predicted molecular weight (Figs.
2A, panel i, and
3, lane 2). Localization of
human and mouse ADRP fusion proteins was neither influenced by the
methods used to introduce plasmids into cells (e.g.
microinjection and electroporation; data not shown) nor by cell type
(Fig. 5). Therefore, linking EGFP to the N terminus of ADRP does not
affect its intracellular localization. Attempts to fuse EGFP to the C
terminus of ADRP gave few cells that produced fluorescence of the
chimeric protein, and the fluorescent protein that was expressed was
not present at the surface of lipid droplets (data not shown).
Consequently, ADRP with EGFP fused to the N terminus was used in
further studies of the human and mouse forms of the protein.
Efficient Localization to Lipid Droplets Requires Discontinuous
Sequences within the ADRP Coding Region--
In the absence of any
previous analysis of the sequences required for lipid droplet
association, we made a series of constructs that removed portions of
the hADRP coding region and examined the intracellular distribution of
the resultant GFP-hADRP variants by fluorescence microscopy. From
observations over a series of experiments, three distinct patterns of
GFP-hADRP fluorescence were identified and these were used to
categorize the behavior of the fusion proteins (Table
II). Cells with Class A distribution represented association of GFP-hADRP with the surface of lipid droplets
with little or no fluorescence elsewhere within the cell (Fig.
2A, panel i). For Class C distribution,
fluorescence was observed throughout the cell with no specific staining
around lipid droplets (Fig. 2A, panel iii). Class
B distribution was a combination of fluorescence at the surface of
lipid droplets and diffuse intracellular fluorescence (Fig.
2A, panel ii). It was assumed that this
distribution was a partial phenotype and that targeting was less
efficient but not abolished with such GFP-hADRP species. The three
types of distribution were not time-dependent as identical
patterns of fluorescence for individual mutants were observed over a
range of times from 6 to 18 h after transfection (data not shown).
Disruption of targeting also was not because of loss of lipid droplets
in cells since they were readily detected in cells that expressed
GFP-tagged proteins with all three types of localization (Fig.
2A, panels iv-vi).
Using the above system to categorize localization, 96% of cells
expressing full-length GFP-hADRP had either exclusive localization on
lipid droplets (70%) or fluorescence around lipid droplets as well as
some diffuse intracellular fluorescence (26%; Table II). Only 4% of
cells showed no specific targeting to lipid droplets. This compares
favorably with results from clonal cell lines that constitutively
express GFP-hADRP where there is a similar ratio of Class A to Class B
intracellular distribution and no cells had Class C
fluorescence.2 Separating the
hADRP coding region into N- and C-terminal segments of approximately
equal length gave two plasmids, pLA5 and pLA14 (Fig. 2B).
The fusion protein made by pLA5 retained the capacity to target to
lipid droplets with roughly equal proportions of cells showing either
Class A or Class B distribution and only 8% of cells with a Class C
distribution (Table II). By contrast, pLA14 produced a protein that had
diffuse fluorescence alone in 94% of cells examined, and only a small
proportion of cells with some targeting to lipid droplets. Hence, the
N-terminal 222 residues of hADRP are sufficient for localization with
lipid droplets with an efficiency that is only slightly lower than that
for the full-length protein. To further define the regions of ADRP
responsible for this distribution, three other C-terminal truncated
GFP-hADRP constructs, pLA10, pLA9, and pLA13, were generated that had
stop codons inserted following nucleotides encoding amino acid residues 195, 169, and 139, respectively (Fig. 2B). The fusion
protein made by pLA10 behaved identically to that made by pLA5 (Table II). However, there was a change in the overall distribution with the
protein produced by pLA9 such that 57% of cells displayed only diffuse
fluorescence (Table II). Targeting to lipid droplets along with diffuse
fluorescence was found in 43% of cells but no cells displayed
exclusive localization of GFP-tagged protein on lipid droplets. Further
truncation of the coding region to amino acid residue 139 (pLA13)
effectively abolished targeting to lipid droplets (Fig. 2B,
Table II). We conclude from this data that sequences between amino
acids 139 and 195 have a role in the association of ADRP with lipid droplets.
The above results identified the C-terminal boundary for sequences that
are sufficient to direct ADRP to lipid droplets. To examine whether the
N-terminal region of ADRP had a role in targeting, two constructs,
pLA11 and pLA12, which lacked sequences for amino acids 1-48 and
1-28, respectively, were derived from plasmid pLA5 (Fig.
2B). Proteins made by these constructs failed to show any association with lipid droplets and were distributed throughout the
cell (Table II). The requirement for N-terminal sequences in targeting
was further tested by restoring the C-terminal coding region to plasmid
pLA12, generating pLA17 (Fig. 2B). The resultant protein was
found at the surface of lipid droplets in 59% of cells examined but
diffuse fluorescence was also present in these cells (Table II). The
remaining 41% of cells showed no targeting to lipid droplets. We
conclude that sequences within the N-terminal 28 amino acids have a
role in efficient association of ADRP with lipid droplets. However, the
data also suggest that there is redundancy in the sequences that are
necessary for lipid droplet association. In particular, it would appear
that there are targeting elements in the C-terminal domain but these
sequences alone are insufficient to direct ADRP to lipid droplets.
Our results above showed that the signals required for efficient
association of ADRP with lipid droplets were distributed along the
protein and it was possible that these were organized as a contiguous
targeting element. Therefore, pLA22 was constructed that lacked amino
acids 61-130 (Fig. 2B). The protein made by pLA22 behaved
almost identically to pLA4, which expressed full-length hADRP. 66% of
cells had GFP-hADRP made by pLA22 exclusively on lipid droplets and a
lower proportion (31%) in which the protein was both present on lipid
droplets and diffuse within the cell (Table II). This showed that the
sequences for targeting to lipid droplets are not continuous within
ADRP. As a further test of a role for amino acids between residues 131 and 220 in directing efficient association with lipid droplets, we made
plasmid pLA29, which lacked amino acids 61-220 (Fig. 2B).
The resultant protein was found with lower abundance at the surface of
lipid droplets but targeting was not abolished (Table II). This
highlights again the redundancy in the sequences within ADRP that are
needed for efficient association with lipid droplets because the
results with pLA13 would predict that removal of a region between amino acids 139 and 220 should abrogate targeting.
To verify that any reduced association of the variant forms of
GFP-hADRP with lipid droplets was not a consequence of proteolysis, the
fusion proteins produced by each of the constructs were examined by
Western blot analysis using Living ColorsTM antiserum to
estimate their apparent molecular weights (Fig. 3). Our analysis showed
that the apparent molecular weights of the proteins correlated with
their predicted molecular weights. We conclude that any disruption to
the ability of GFP-hADRP mutants to associate with lipid droplets is
not a consequence of cleavage within the fusion proteins that may
remove targeting sequences. The differences in abundance of GFP-hADRP
between individual mutants was because of either variable transfection
efficiency or different levels of transcription for individual plasmids
because the amount of RNA detected in transfected cells correlated with
the relative abundance of the proteins seen in Fig. 3 (data not shown).
Mobility of Lipid Droplets in Live Cells--
Previous studies in
live baby hamster kidney cells using an EGFP-tagged caveolin mutant
indicated that lipid droplets are relatively immobile structures (2).
Given that droplets can provide lipid precursors for specific purposes
in certain cell types, including hepatocytes, their behavior may differ
between cell types. Hence, we sought to re-examine their mobility using GFP-mADRP as a marker protein in stably transfected HuH-7 cells. The
majority of lipid droplets, particularly those that were larger in
size, tended to oscillate around a point of origin but did not migrate
by any substantial distance (Fig.
4B, panels
i-x, lipid droplets indicated by open
arrows; Supplemental Materials, Videos 1 and 2). However, a small
number of droplets did display rapid, unidirectional movement that was
transient (Fig. 4, A and B, indicated by
filled arrows; Supplemental Materials, Videos 1 and 2). For
example, the lipid droplet indicated in Fig. 4B moves
rapidly for a period of 5 s (panels i-vi),
remains relatively immobile for 2 s (panels
vi-viii) and then moves again for a further 2 s
(panels viii-x). We estimate that the speed of
such droplets is in the range 2-2.5 µm/s and indicates motion along
a cellular transport network. In cells treated with nocodazole, we
failed to detect any similar rapid movements of lipid droplets (data not shown). Hence, we conclude that such movement is most likely mediated by interaction with microtubules.
Association of Lipid Droplets with the ER--
It was considered
likely that most lipid droplets, which moved only within a limited
area, were attached to another subcellular network other than
microtubules. Recent evidence has indicated association between lipid
droplets and the ER (2). To examine this possibility directly under
live cell conditions, HuH-7 cells were microinjected with plasmids
expressing a commercially available YFP-tagged protein that targets the
ER and YFP-hADRP. Constructs expressing YFP-tagged proteins were
utilized because no plasmid expressing EGFP-ER marker protein was
available. Given that the ER and lipid droplets have distinct
morphologies, the targeting of the different YFP-tagged proteins was
readily discriminated. Imaging in HuH-7 cells indicated that, whereas
it was difficult to identify individual strands of the ER membrane in
this cell type (Fig. 5, panel
i), there was close association between ER and lipid droplets
(Fig. 5, panels iii and iv). By comparison, strands of the ER membrane were more readily identified in Vero cells
(Fig. 5, panel v) and the cells contained fewer lipid
droplets (Fig. 5, compare panels ii and vi).
Co-expression of YFP-hADRP and the YFP-ER targeting protein in Vero
cells again revealed co-localization of lipid droplets with the ER
(Fig. 5, panels vii and viii). This
co-localization was particularly evident at peripheral regions of the
cell where packing of the ER and lipid droplets was less dense (Fig. 5,
panel viii). We further analyzed the extent of this
association between lipid droplets and the ER by time-lapse microscopy.
Bleaching of EYFP is more rapid than that for EGFP and therefore we
used plasmids expressing GFP-hADRP (pLA4) and a GFP-DNase X fusion
protein that is directed to the ER.3 Analysis of individual
images along with animations from Vero cells expressing both proteins
indicated concurrent movement of both the ER and attached lipid
droplets (Fig. 6, Supplemental Materials,
Video 3; data not shown). This reveals that there is intimate
association between the ER and lipid droplets in live cells.
ADRP Does Not Diffuse Rapidly between Adjacent Lipid
Droplets--
Organelles such as the ER, Golgi, and plasma membrane
form continuous intracellular structures that permit rapid diffusion of
proteins along membranes. Frequently, lipid droplets are found in
tightly packed clusters (see Fig. 5, panel iii, top
right of cell) that could also form a network allowing transfer of
protein between individual droplets. To examine such a possibility,
FRAP analysis was performed on live HuH-7 cells expressing GFP-mADRP. In this cell type, we find that GFP-tagged hepatitis C virus (HCV) non-structural protein NS4B, which is associated with the ER membrane, recovers fluorescence within 10 s after
bleaching.4 Using identical
conditions, we photobleached regions at the periphery of HuH-7 cells
where lipid droplets were clustered and in areas distant from the
plasma membrane where droplets were apparently unconnected (Fig.
7A, boxed
regions 1 and 4, respectively). After photobleaching, there was little recovery of fluorescence (Fig. 7B); the small amount of recovered fluorescence was observed
also in fixed cells and therefore represents refolding of GFP-mADRP (data not shown). This lack of fluorescence recovery was consistent in
all other experiments, including conditions where fluorescence was
measured up to 10 min after bleaching (data not shown). Hence, we
conclude that ADRP does not readily diffuse between droplets.
ADRP is a component of lipid droplets found in a broad range of
cultured cells and tissues. Accordingly, it is a useful model protein
to examine the properties of proteinaceous components of lipid droplets
and the innate behavior of these organelles. In our study, we have
employed fusion of both human and mouse forms of ADRP with EGFP to
study both of these aspects.
Analysis of the sequences required to direct hADRP to lipid droplets
revealed that targeting signals are distributed throughout the
polypeptide coding region (Fig. 8). In
the context of our study, we define targeting as the process through
which ADRP is directed to lipid droplets and then forms a stable
association with the organelles. Therefore, reduced targeting
efficiency could result from the inability of the tagged proteins to
attach stably to lipid droplets, possibly through competition with
endogenous ADRP. We found that removal of the N-terminal 28 amino acids
impaired lipid droplet localization and a second targeting element was located between residues 139 and 220. Sequences that can contribute to
efficient association with lipid droplets also were present in the
C-terminal region from amino acids 221 to 437 but this segment of the
protein alone was not capable of directing the protein to droplets.
These regions of the protein do not form a contiguous targeting element
because sequences between amino acids 61 and 130 can be removed with no
significant impact on the efficiency of lipid droplet association. Our
results indicate that removing one of these targeting sequences can
impair but not abolish lipid droplet localization, suggesting that
there is redundancy in the sequence requirements for efficient
association. Thus, identification of indispensable sequences has not
been possible. A recent study also indicated redundancy within the
sequences that were necessary for directing perilipin to lipid droplets (28). Moreover, simple targeting signals for peroxisomal membrane proteins have not been readily identified and it appears that more than
one targeting element may reside within such proteins (29). Therefore,
sequence redundancy may be a general feature for directing proteins to
the surface of organelles such as peroxisomes and lipid droplets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 10% glycerol, and 0.004%
bromphenol blue, at a concentration of ~2 × 106
cells/ml sample buffer. The samples were heated at 100 °C for 5 min
to denature proteins and nucleic acids prior to electrophoresis through
a 10% polyacrylamide gel prepared using a 30% acrylamide, 0.8% bisacrylamide (37.5:1) stock solution (Bio-Rad). For
Western blot analysis, proteins, separated on polyacrylamide gels, were transferred to nitrocellulose membrane. After blocking with PBSA containing 5% milk powder (Marvel) and 0.05% Tween 20, membranes were
incubated with the Living ColorsTM full-length A.v.
polyclonal antibody (Clontech), diluted to 1:2000 in PBSA containing 5% milk powder (Marvel) and 0.05% Tween 20. After
washing, bound antibody was detected using protein A-peroxidase (Sigma)
followed by enhanced chemiluminescence.
20 °C. Following washing with PBS and blocking with
PBS/CS (PBS containing 1% newborn calf serum), cells were incubated
with primary antibody (diluted in PBS/CS at 1:200 and 1:500 for Living ColorsTM and ADRP antisera, respectively) for 2 h at
room temperature. Cells were washed extensively with PBS/CS and then
incubated with conjugated secondary antibody (either anti-rabbit or
anti-sheep IgG) for 2 h at room temperature. After washing with
PBS/CS and PBS, cells were rinsed finally with H2O before
mounting on slides using Citifluor (Citifluor Ltd.). Samples were
analyzed using a Zeiss LSM confocal microscope. ADRP antiserum was
raised in sheep against a synthetic peptide comprised of amino acid
residues 5-27 of hADRP conjugated to keyhole limpet hemocyanin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides used for cloning and mutation of human and mouse ADRP
cDNAs
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Fig. 1.
Localization and detection of GFP-mADRP in
HuH-7 cells. A, cells constitutively expressed
EGFP (panel ii) and GFP-mADRP (all panels
except for panel ii). Panels show cells viewed
under the following conditions: panel i, cells were fixed
with paraformaldehyde and stained with oil red O; panels ii
and iii, cells were viewed live and protein was detected
directly by GFP fluorescence; panels iv vi,
cells were fixed with methanol and probed with anti-Living
ColorsTM (panel iv) and anti-ADRP antisera
(panel v); panels vii and viii, cells
were incubated with Bodipy 558/568 C12 and then fixed with
4% paraformaldehyde prior to analysis of GFP (panel vii)
and Bodipy fluorescence (panel viii). Panels vi
and ix are merged images of panels iv and
v and panels vii and viii,
respectively. Cells in panels i-vi are at the
same scale and the size bar in panel i represents
10 µm. The size bar in panel vii represents 5 µm. B, Western blot analysis of GFP-mADRP and EGFP
expressed in HuH-7 cells. Extracts from cells constitutively producing
GFP-mADRP (lane 1) and EGFP (lane 2) were run on
a 12% polyacrylamide gel and the proteins were transferred to
nitrocellulose membrane following electrophoresis. The membrane was
probed with anti-Living ColorsTM antiserum. Protein size
markers are indicated to the left of the blot and bands
corresponding to GFP-mADRP and EGFP are indicated by filled
boxes.
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Fig. 2.
A, representative images of the
distributions of wild type and mutant GFP-hADRP. HuH-7 cells were
transfected with plasmid DNA for 18 h, stained with Bodipy 558/568
C12, and then fixed with 4% paraformaldehyde. Plasmids
used for transfection were: panels i and iv,
pLA4; panels ii and v, pLA17; panels
iii and vi, pLA12. Panels i-iii
show GFP fluorescence and Bodipy staining is illustrated in
panels iv vi. Arrows indicate
corresponding regions in each cell with lipid droplets. All images are
at the same scale and the size bar in panel i
represents 10 µm. B, schematic diagram of the regions of
hADRP expressed in each of the plasmids. The segments that correspond
to PAT-1 (dark boxed region), PAT-2 (light boxed
region), and that have sequence identity with tandem repeats in
the S3-12 membrane protein are shown. Numbers indicate
amino acid residues in the hADRP coding region.
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Fig. 3.
Apparent molecular weight and
relative abundance of wild-type and variant forms of GFP-hADRP.
HuH-7 cells were transfected with plasmid DNAs and cell extracts were
prepared at 18 h after transfection. Samples were run on a 10%
polyacrylamide gel and thereafter, proteins were transferred to
nitrocellulose membrane. The membrane was probed with anti-Living
ColorsTM antiserum. Extracts were prepared from cells
transfected with the following constructs: lane 1, pEGFP-C1;
lane 2, pLA4; lane 3, pLA5; lane 4,
pLA14; lane 5, pLA10; lane 6, pLA9; lane
7, pLA13; lane 8, pLA11; lane 9, pLA12;
lane 10, pLA17; lane 11, pLA22; lane
12, pLA29. Protein size markers are indicated to the
left of the blot and species corresponding to GFP-hADRP
proteins are indicated with arrowheads.
Distribution of GFP-hADRP in transfected cells
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Fig. 4.
Transient association of lipid droplets with
intracellular transport networks. For A and
B, images of HuH-7 cells constitutively expressing GFP-mADRP
were taken at 1-s intervals by confocal microscopy. Lipid droplets that
displayed rapid, unidirectional movement are indicated by filled
arrowheads and those that were relatively immobile are shown by
open arrowheads. Images in A and B are
at the same scale and the size bar in panel i of
A represents 5 µm. Images in A and B
are individual frames taken from Videos 1 and 2, respectively (in
Supplemental Materials).
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Fig. 5.
Association of lipid droplets with the ER
membrane. Live images were taken by confocal microscopy of HuH-7
(panels i-iv) and Vero cells (panels
v-viii) microinjected with plasmids that expressed YFP
fusion proteins. Cells were microinjected with the following constructs
for 6 h before imaging: panels i and v,
YFP-ER (Clontech); panels ii and
vi, YFP-hADRP; panels iii and vii,
YFP-ER and YFP-hADRP. Panels iv and viii show
magnified images of the boxed regions in panels
iii and vii, respectively. Images in panels
i-iii and v-vii are at the same
scale and the size bar in panel iii represents 10 µm. Images in panels iv and viii are at the
same scale and the size bar in panel iv
represents 5 µm.
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Fig. 6.
Coincident movement of lipid droplets and ER
membranes in Vero cells. Cells were microinjected with plasmids
expressing GFP-hADRP and GFP-DNase X and imaged 6 h after
injection. Images were taken at 6-s intervals by confocal microscopy.
Large and small arrowheads indicate lipid
droplets and the ER membrane, respectively. The size bar in
the first panel represents 5 µm. Images are individual frames taken
from Video 3 (in Supplemental Materials).
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Fig. 7.
FRAP analysis of GFP-mADRP attached to lipid
droplets. A, an HuH-7 cell that constitutively produced
GFP-mADRP was photobleached in boxed regions 1 and
4. Images were taken at the times indicated by confocal
microscopy and bleaching was performed after the image was taken at
7 s. The scale bar in the first panel represents 5 µm. B, fluorescence intensities in photobleached and
equivalent non-bleached areas (boxed regions 2 and
3) were measured and plotted against time. Intensity was
quantified relative to the fluorescence measured at t = 0 s for each boxed region. Percentage fluorescence
intensity was plotted for each region as follows: region 1, open
circles; region 2, open boxes; region 3, closed
boxes; region 4, closed circles.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Schematic diagram showing regions in hADRP
that contain targeting signals. The segments that correspond to
PAT-1, PAT-2, and that have sequence identity with tandem repeats in
the S3-12 membrane protein are shown. The hADRP coding region is
represented by four domains (I-IV) and
numbers indicate the amino acid residues that limit each
domain. The contributions of combinations of domains I-IV to
intracellular distributions that correspond to Class A (exclusive
localization on lipid droplets), Class B (a combination of association
with lipid droplets and diffuse localization throughout the cell), and
Class C (no specific targeting to lipid droplets) are shown. Regions
that were present (+) or absent ( ) in the fusion proteins tested are
indicated.
It has been proposed that ADRP can be separated into two domains, PAT-1 and PAT-2, based on sequence identity with other lipid droplet-associated proteins (14). PAT-1 is considered to consist of approximately the N-terminal 100 amino acids of the protein and the remaining C-terminal segment is PAT-2 (Fig. 8). Sequence identity between PAT-1 domains for lipid droplet proteins is greater than that for PAT-2. Indeed, using PAT-1 sequences, lipid droplet proteins have been identified putatively in Drosophila melanogaster and Bombyx mori (14). Our analysis shows that the N-terminal 28 amino acids of hADRP, which are necessary for efficient targeting, are contained within PAT-1. However, the other sequences that have been identified as important for efficient lipid droplet association are located in PAT-2. The region within perilipin that has been identified as important for localization to lipid droplets also lies in PAT-2 (28). This indicates that the higher sequence identity in PAT-1 between lipid droplet-associated proteins such as ADRP and perilipin does not necessarily represent conserved targeting sequences. Hence, the sequences that direct these proteins to lipid droplets may be distinct. It has also been observed that a region in the PAT-1 domain of ADRP has sequence identity with a tandem repeat in the plasma membrane protein, S3-12 (20, 30). Our results show that removing this segment from ADRP has no effect on lipid droplet association (Fig. 8), leading us to conclude that any sequence identity with this tandem repeat is not related to targeting of ADRP.
The discontinuous nature of the targeting sequences in ADRP differs from the characteristics of other lipid droplet-associated proteins apart from perilipin. Association of HCV core protein with lipid droplets requires a domain of about 55 amino acids that is present also in a related virus, GB virus-B but not in pesti- and flaviviruses that share the same genomic organization as HCV (11, 12). Apart from a short stretch of amino acids, removal or substitution of amino acids along the length of the domain abolishes lipid droplet localization. In addition, plant oleosins have a central region of 85 amino acids that has all of the sequences required for efficient attachment in mammalian and plant cells; this central region is flanked by domains that appear to be dispensable for lipid droplet association (12, 31). Moreover, the targeting sequences in the HCV core and plant oleosin proteins are hydrophobic, whereas no corresponding segments with similar characteristics are found in either human or mouse ADRP. This suggests that processes, which are different from those for the viral and plant proteins, may guide localization of ADRP to lipid droplets. In the case of HCV core, trafficking of the protein to lipid droplets involves initial targeting and cleavage of a precursor protein at the ER and core is found both at the ER membrane as well as at the surface of lipid droplets (32). It is likely that transfer of core between the two organelles is facilitated by the attachment of lipid droplets to the ER, which we describe from our live cell studies. However, for ADRP, no staining of the ER is found by either indirect immunofluorescence or live cell analysis. The mechanism that controls partitioning of ADRP is presumably a reflection of differences between either components or compositions of lipid droplets and the ER that favor interaction with lipid droplets. In addition, the literature indicates that ADRP is synthesized on free and not ER-bound polyribosomes (20). A similar situation has been demonstrated for the perilipins (33). It may be that association of ADRP and perilipin with the ER is not favored to prevent fusion of lipid droplets with the ER after their formation.
Lipid droplets often occur as clusters in a number of cell types. This may indicate that they form a connected network in which transfer of macromolecules between droplets could occur. FRAP is gaining wide use to determine the mobility of GFP-tagged proteins in organelles with continuous surfaces such as the ER, Golgi, and plasma membranes (Refs. 34-36, reviewed in Ref. 37). More recently, FRAP has revealed that clusters of mitochondria are functionally distinct and unconnected (38). From our data, ADRP does not rapidly diffuse between lipid droplets that are apparently in contact with each other. This suggests that droplets exist as discrete entities and that there is little or no transfer of surface proteins between them. As a consequence, there may be little turnover of ADRP at the surface of lipid droplets after their formation.
Analysis of live cells offers advantages for determining the characteristics of lipid droplets. Lipid droplets are heterogeneous in size both between different cell types and even in individual cells. The factors that govern their size are not known but could be determined during their biogenesis. It is also possible that additional neutral lipid is added after formation, either through transfer from the ER or from other lipid droplets. In HuH-7 cells, lipid droplets were no greater than about 1 µm in diameter in live cells using GFP-ADRP as a marker. However, upon fixation and subsequent treatment with alcohol to stain lipid droplets with oil red O, their maximum size increased to 3 µm (data not shown). We propose that this discrepancy results from fusion of closely associated droplets and the size distribution in cells, which are stained using alcoholic solutions, is not authentic. Such an observation may have a bearing on the interpretation of lipid droplet size in cells that store large quantities of neutral lipid and in pathological conditions where lipid storage is assessed. For example, steatosis of the liver can be described as either micro- or macrovesicular depending on the diameter of lipid droplets (39). It is possible that the presence of lipid droplets with larger diameters does not reflect their size in vivo and is an artifact of the fixation process.
Formation of lipid droplets is considered to occur by a budding process at the ER (1, 40). From live cell studies, our results reveal that, following their biogenesis, droplets continue to attach to the ER membrane. As lipid droplets are a reservoir, their close association with the ER may be important for maintenance and expansion of the organelle during the cell cycle. Such an association also allows ready access to a storage reservoir under conditions where excess lipid is either synthesized or absorbed by cells. Thus, lipid droplets may be important to maintain lipid homeostasis and permit normal function of the ER in abnormal situations where lipid is either limiting or in excess. In HuH-7 cells, we observed that a proportion of lipid droplets had movements that were consistent with transient attachment to the microtubule network. Such trafficking was clearly distinct from the motion of droplets that were ER-associated. We presume that brief association of lipid droplets with microtubules indicates movement from one site on the ER to a second site. However, the factors that control lipid droplet association with microtubules are not known. We did not observe similar rapid movement in Vero cells. This cell type contains considerably fewer lipid droplets compared with HuH-7 cells and, because the proportion of lipid droplets that move rapidly in HuH-7 cells is low, it is possible that observations over longer time periods are necessary to identify microtubule-associated movement of lipid droplets in Vero cells. Alternatively, microtubule-related trafficking could be cell type-dependent. As well as acting as a source of lipid for membranes, lipid droplets have specialized functions in certain cell types, which may affect their interactions with cellular processes. For example, milk fat globules released by mammary epithelial cells are derived from lipid droplets secreted from the apical surface and it is proposed that movement of droplets to sites of secretion involve trafficking along microtubules (41). In addition, hepatocytes are a primary source for the production of very low density lipoprotein, and triglycerides stored in lipid droplets provide much of the lipid content in these particles (5). Mechanistically, this process is poorly understood but it may require mobilization of lipid droplets to regions of the ER where lipoprotein assembly occurs. Hepatocytes are the progenitor cells for the human hepatoma from which HuH-7 cells are derived and thus may maintain characteristics for the very low density lipoprotein assembly pathway that involve lipid droplets. Therefore, the association of lipid droplets with microtubules in HuH-7 cells may not be found more generally in other cell types.
In summary, we have identified sequences within ADRP that are important
for association with lipid droplets and have demonstrated that the
protein can be utilized to examine the properties of these organelles.
This has allowed analysis of the interaction between lipid droplets and
other cellular processes. From our work and other studies where
association with mitochondria, intermediate filaments (6, 42), and
peroxisomes (43) have been observed, it appears that lipid droplets are
not inert sites of lipid storage but actively interconnect with other
organelles. Further studies on the consequences of these interactions
will help to elucidate the full extent of the contribution of lipid
droplets to cell metabolism.
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ACKNOWLEDGEMENTS |
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We thank Drs. Rainer Pepperkok, Timo Zimmerman, Jens Rietdorf, and Lorraine Anderson for valuable discussions and assistance with obtaining live cell imaging data. We are grateful to Dr. Reg Clayton for isolation of HuH-7 cells by fluorescent-activated cell sorter and Dr. Frazer Rixon for valuable comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a short-term fellowship from the Transnational Access to Major Research Infrastructures program (European Union) to enable use of the EurALMF (EMBL, Heidelberg) (to J. McL.) and a Realising Our Potential Award from the Medical Research Council (UK).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.
The on-line version of this article (available at
http://www.jbc.org) contains Videos 1-3.
** To whom correspondence should be addressed. Tel.: 44-141-330-4028; Fax: 44-141-337-2236; E-mail: j.mclauchlan@vir.gla.ac.uk.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211289200
2 P. Targett-Adams and J. McLauchlan, unpublished data.
3 J. F. Coy and A. Girod, unpublished results.
4 A. I. Taylor and J. McLauchlan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ADRP, adipocyte differentiation-related protein; hADRP, human ADRP; mADRP, mouse ADRP; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; ORF, open reading frame; PBS, phosphate-buffered saline; RT, reverse transcriptase; HCV, hepatitis C virus; FRAP, fluorescent recovery after photobleaching.
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