Download Cellular Internalization of Fluorescent Proteins via Arginine

Plant Cell Physiol. 46(3): 482–488 (2005)
doi:10.1093/pcp/pci046, available online at www.pcp.oupjournals.org
JSPP © 2005
Cellular Internalization of Fluorescent Proteins via Arginine-rich
Intracellular Delivery Peptide in Plant Cells
Microsugar Chang 1, 2, Jyh-Ching Chou 1 and Han-Jung Lee 1, 3
1
2
Institute of Biotechnology and Department of Life Science, National Dong Hwa University, Hualien 974, Taiwan
Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan
;
Recently, direct delivery of peptides or proteins into animal cells in vivo has been achieved by protein transduction
domains (PTDs) (Schwarze et al. 1999). The three most widely
studied PTDs are from the human immunodeficiency virus type
1 (HIV-1) transcriptional activator Tat protein (Frankel and
Pabo 1988, Green and Loewenstein 1988), the Drosophila
homeodomain transcription factor antennapedia (Joliot et al.
1991) and the herpes simplex virus structural protein VP22
(Elliott and O’Hare 1997). These domains are capable of delivering biologically active proteins across membrane barriers and
into animal cells, when synthesized as recombinant fusion proteins or covalently cross-linked to full-length proteins (Wadia
and Dowdy 2002). PTD–cargo conjugates can be found within
both the cytoplasm and the nucleus in a variety of animal cell
lines. Though the mechanism of protein transduction delivering molecules into animal cells remains unsubstantiated, it
appears to be independent of endocytosis, receptors and active
transporters (Lindsay 2002).
Derived from HIV Tat PTD peptide, many basic peptides
have been generated for protein transduction in animal cells
(Morris et al. 2001, Futaki 2002). The cellular uptake of pol-
The protein delivery across cellular membranes or
compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell
membranes. Usually the delivery processes utilize passive
protein channels or active transporters to overcome the
membrane impediment. In this report, we demonstrate that
arginine-rich intracellular delivery (AID) peptide is capable
of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot
plant) and onion (a monocot plant) in a fully bioactive
form. Thus, cellular internalization via AID peptide can be
a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins
in planta or in plant cells in vivo. This novel method may
alternatively provide broader applications of AID chimera
in plants without the time-consuming transgenic approaches.
Keywords: Cellular internalization — Green fluorescent protein — Protein transduction — Recombinant protein — Transgenic plant.
Abbreviations: AID, arginine-rich intracellular delivery; GFP,
green fluorescent protein; HIV-1, human immunodeficiency virus type
1; IPTG, isopropyl-β-D-thiogalactopyranoside; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NEM, N-ethylmaleimide; PTD, protein transduction domain; R9, nona-arginine; RFP, red
fluorescent protein.
Introduction
The major challenge of intracellular trafficking and even
nuclear delivery of biomolecules into target cells resides in the
design of efficient carriers that can overcome the low permeability of the cell membrane. Research in the field of gene therapy in animals has used numerous systems for the delivery of
sense and antisense nucleic acids into cells, including viral and
non-viral vectors (Wagner 1999, Cavazzana-Calvo et al. 2000).
In general, non-viral systems present several advantages over
viral systems in that they are simple to use, easy to produce,
less toxic and do not induce specific immune responses (Morris et al. 2000).
3
Fig. 1 Schematic structure of plasmids used in protein transduction.
pQE8-GFP plasmid contains an N-terminal His6-tagged (6His)
GFPmut2 under the control of the T5 promoter/lac operator. pTATGFP plasmid consists of 11 amino acids of HIV-Tat-PTD (Tat) inframe with six histidine residues, a hemagglutinin (HA) tag and GFP
under the control of the T7 promoter. pR9-GFP plasmid contains nonaarginine (R9) in-frame with six histidine residues, an HA tag and GFP
under the control of the T7 promoter. pR9-RFP plasmid was generated
by the replacement of the RFP coding region of pDsRed with GFP in
pR9-GFP. All constructions were verified by DNA sequencing.
Corresponding author: E-mail, [email protected]; Fax, +886-3-8630340.
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Protein transduction in plant cells
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Fig. 3 Protein internalization in tomato roots. Tomato roots were
treated with 2 µg of GFP for 5 min, washed with water as a control and
recorded by the TCS SL confocal microscope system (Leica, Wetzlar,
Germany) (A). Tomato roots incubated with 2 µg of Tat–GFP protein
for 5 min and washed with water are shown at a magnification of either
200× (B) or 400× (C). Tomato roots treated with 2 µg of R9–GFP protein are shown at a magnification of either 200× (D) or 400× (E).
Fig. 2 Morphologies of cellular boundaries and nuclei. Plant cells
(onion epidermal and root tip cells) were stained with crystal violet
(1%) and washed with water in order to observe their cell boundaries
and nuclei. Images were observed under an Eclipse E600 microscope
(Nikon, Melville, NY, U.S.A.) and recorded by a Penguin 150CL
cooled CCD camera (Pixera, Los Gatos, CA, U.S.A.). Onion epidermal cells are shown at a magnification of either 100× (A) or 400× (B).
Onion root tip is shown at a magnification of 400× (C).
yarginine tends to be more efficient than that of polylysine,
polyhistidine or polyornithine (Futaki 2002). Various chain
lengths of polyarginine were examined for cellular internalization. The highest translocation efficiency was reached by using
octa-arginine or nona-arginine (R9) peptides (Futaki 2002). In
addition, the arginine residues do not necessarily need to be linearly arranged. Peptides with branched chain structure suggested that considerable flexibility in the location of the
arginine residue resides in transduction molecules (Futaki et al.
2002, Tung et al. 2002). Furthermore, backbone spacing
between arginine residues is important for transport, since
increased flexibility among residues may allow the guanidine
headgroups to interact more effectively with negative charges
on the cell surface (Rothbard et al. 2002).
Successful overexpression of rotavirus NSP4 enterotoxin
fusion protein with PTD of HIV-1 Tat in transgenic potato has
been reported (Kim and Langridge 2004). However, there is no
report to investigate the PTD peptide transduction activity in
plant cells. In this study, we are interested in the application of
arginine-rich intracellular delivery (AID) peptide transduction
activity in plants, and found that the AID peptide is able to
deliver green fluorescent protein (GFP) and red fluorescent
protein (RFP) efficiently into plant cells in a fully biologically
active form. This result will provide a new approach for plant
biologists to investigate interesting proteins by direct transduction without time-consuming gene transformation procedures.
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Protein transduction in plant cells
Fig. 4 Protein internalization in onion epidermal cells. Green fluorescent images were recorded by the TCS SL confocal microscope system (Leica, Wetzlar, Germany). Onion skin epidermal cells were
treated with control GFP protein (A), with Tat–GFP protein shown at a
magnification of either 200× (B) or 400× (C), or with R9–GFP protein shown at a magnification of either 200× (D) or 400× (E).
Results
Plasmid constructions for protein Internalization
To examine protein transduction in plant cells, we have
prepared several GFP-containing plasmids (Fig. 1). Various
GFP proteins were subsequently overexpressed and purified
from pQE8-GFP, pTAT-GFP, pR9-GFP and pR9-RFP plasmids
in Escherichia coli. The expression of pTAT-GFP, pR9-GFP
and pR9-RFP plasmids generated Tat–GFP, R9–GFP and R9–
RFP fusion proteins containing HIV Tat PTD-tagged GFP,
AID-tagged GFP, and AID-tagged RFP proteins, respectively.
On the other hand, the expression of pQE8-GFP plasmid produced GFP protein alone, which can serve as a negative control.
Cellular internalization of Tat-GFP and R9-GFP
Onion (Allium cepa L.) epidermal cells and root tip cells
were stained with 1% crystal violet and washed with water in
order to observe their cellular boundaries and nuclei (Fig. 2).
Onion epidermal cells are shown in Fig. 2A and B, while onion
root tip is shown in Fig. 2C. Giant nuclei were found in the
Fig. 5 Protein internalization in onion root tip cells. Onion root tip
cells were treated with control GFP protein (A), with Tat–GFP protein
shown at a magnification of either 200× (B) or 400× (C), or with R9–
GFP protein shown at a magnification of either 200× (D) or 400× (E)
by confocal microscopy.
onion root tip. In order to investigate the potential protein
transduction, tomato (Lycopersicon esculentum M.) roots were
treated with different GFP-containing proteins. As shown in
Fig. 3A, no green fluorescence was detected in the tomato roots
incubated with GFP protein as a control. In contrast, tomato
roots incubated with either Tat–GFP (Fig. 3B, C) or R9–GFP
(Fig. 3D, E) protein clearly exhibited green fluorescence with a
high efficiency. The signal was distributed evenly in almost all
cells observed.
In order to test the validity of Tat–GFP and R9–GFP in
different plants, onion epidermal cells were treated with the
same protocol. As shown in Fig. 4A, no signal was observed in
onion epidermal cells treated with GFP protein only. However,
green fluorescence was consistent with onion epidermal cells
incubated with either Tat–GFP (Fig. 4B, C) or R9–GFP (Fig.
4D, E). The signal was also distributed evenly in almost all
cells. These results demonstrated that PTD or AID fused with
Protein transduction in plant cells
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Fig. 6 Time course analysis of protein internalization under different conditions. Onion epidermal cells were treated with either Tat–GFP (A) or
R9–GFP (B) in the presence or absence of endocytotic inhibitors (1 mM NEM and 1.5 µM okadaic acid) at different temperatures (room
temperature or 4°C). Relative fluorescent intensities (y-axis) at different times (x-axis; 1, 2, 3, 4,
5 min, 24 h and 48 h) after protein internalization were analyzed by UN-SCAN-IT software.
GFP was efficiently and rapidly transduced into all tested plant
cells in vivo. Additionally, protein transduction of Tat–GFP
and R9–GFP proteins was neither species specific nor tissue
specific in the plants tested.
Cellular localization of Protein internalization
To identify further their precise localization after protein
transduction, onion root tip was treated with a similar protocol.
As shown in Fig. 5A, no signal was detected in the onion root
tip treated with GFP protein. In sharp contrast, elevated green
fluorescence was easily visualized in onion root tip incubated
with either Tat–GFP (Fig. 5B, C) or R9–GFP (Fig. 5D, E) protein. The signal was also distributed evenly in almost all cells
in the cytoplasm, and was located predominantly at higher levels in the nuclei. These results revealed again that protein internalization could be mediated by fusion with AID peptides.
Mechanism of cellular internalization
The kinetics of protein internalization were studied by
varying the time of incubation of the cells with Tat–GFP or
R9–GFP protein. Onion epidermal cells were treated with
either Tat–GFP (Fig. 6A) or R9–GFP (Fig. 6B) for different
time courses at room temperature. The relative intensities were
analyzed by UN-SCAN-IT software from >30 fields of confocal microscopic images. As shown in Fig. 6, protein internalization occurred as fast as 1 min after the incubation of the cells
with Tat–GFP or R9–GFP protein. The protein was taken up
efficiently, and the maximal internalization was reached after
5 min of incubation. Internalized protein could stably remain in
plant cells up to 2 days after entry into the cells.
To reveal the mechanism through which these Tat–GFP
and R9–GFP proteins could be internalized, experiments were
performed at different time courses at 4°C. As shown in Fig. 6,
low temperature incubation did not alter cellular uptake, which
precludes the possibility of an endocytotic mechanism. These
data are in agreement with the protein transduction observed in
animal cells (Vives et al. 1997, Wadia and Dowdy 2002).
Although the translocating activity of PTD peptides was
not inhibited at 4°C, uptake via endocytosis involving binding
of plasma membrane can be greatly inhibited by N-ethylmaleimide (NEM) and okadaic acid (Vives et al. 1997). We treated
onion epidermal cells with either Tat–GFP (Fig. 6A) or R9–
GFP (Fig. 6B) for different time courses in the presence or
absence of endocytotic inhibitors (1 mM NEM and 1.5 µM
okadaic acid). Treatment with endocytosis inhibitors did not
interfere with protein internalization. These data suggested that
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Protein transduction in plant cells
Fig. 7 Cytotoxicity of protein fused with
AID peptide. Onion epidermal cells were
washed with water and either stained directly
with trypan blue (A) or treated with alcohol
and then stained with trypan blue (B). Onion
epidermal cells were treated with either Tat–
GFP (C) or R9–GFP (D) protein, and stained
with trypan blue. All the images were collected at a magnification of 100×.
internalization of TAT–GFP and R9–GFP was independent of
energy and endocytosis.
Cytotoxicity of Tat-GFP and R9-GFP
To investigate any possible cytotoxicity associated with
protein internalization, cell viability was tested with trypan
blue after the treatment with Tat–GFP or R9–GFP protein.
Onion epidermal cells were washed with water and stained
with 50% trypan blue as a negative control, since living cells
cannot be stained (Fig. 7A). Onion epidermal cells sterilized
with 70% alcohol and stained with trypan blue served as the
positive control (Fig. 7B). We found that onion epidermal cells
could not be stained after the treatment with either Tat–GFP
(Fig. 7C) or R9–GFP (Fig. 7D). This result clearly demonstrated that cells remain alive after protein internalization.
Cellular internalization of R9-RFP
To extend this study to other proteins, we constructed a
pR9-RFP plasmid which fused RFP with the AID peptide for
protein internalization experiments (Fig. 1). The expression of
pR9-RFP plasmid generates R9–RFP fusion protein containing
AID-tagged RFP protein. As shown in Fig. 8A, no signal was
observed in onion epidermal cells treated with RFP protein
only. In contrast, tomato roots (Fig. 8B) or onion epidermal
cells (Fig. 8C) incubated with R9–RFP exhibited red fluorescence with a high efficiency. Similar to the results of the Tat–
GFP and R9–GFP experiments, the signal was distributed
evenly in almost all cells observed. This result demonstrated
that RFP could also be internalized into plant cells as well as
GFP when fused with AID peptide.
Discussion
This is the first report to demonstrate that short AID peptides are able to deliver bioactive protein into plant cells efficiently when fused with the target proteins. Based on the lipid
bilayer model of biomembranes, there is an inner hydrophobic
Fig. 8 RFP protein internalization in plant cells. (A) Onion epidermal cells were treated with control RFP protein. Tomato roots (B) and
onion epidermal cells (C) were incubated with R9–RFP, shown at a
magnification of 200× by confocal microscopy.
Protein transduction in plant cells
layer between two outer hydrophilic surface in the membranes
of plant cells to serve as a screening barrier for transport of
materials across the biomembranes. Only specific biomolecules and ions may pass through via specific transporters or
protein channels. Therefore, highly basic and hydrophilic AID
peptides should not pass through the membranes. In fact, however, these AID peptides quite easily pass through the membranes within 5 min in the present study (Fig. 6). The HIV Tat
peptide has been shown to be internalized and reached the
nuclei within 5 min in animal cells (Wadia and Dowdy 2002).
Liposomes >200 nm diameter can be delivered into cells by
anchoring Tat PTDs to their surface (Wadia and Dowdy 2002).
The strong features of the AID peptides include their stability
in physiological buffer, lack of toxicity, and lack of sensitivity
to serum (data not shown). The AID–GFP peptides can stably
remain in plant cells as well as animal cells for up to 2 days
after treatment (Fig. 6). This long period for active AID fusion
protein to stay in plant cells may provide enough time to
observe many biological functions of important proteins in
plants. As determined by the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) assay, >85% of animal
cells survived for 24 h in the presence of 100 µM Tat PTD peptides (Futaki 2002). Our data showing that plant cells remain
alive after protein internalization are in agreement with the
MTT assay in animal cells (Fig. 7). Like Pep-1, the efficiency
of AID-mediated peptide delivery is not affected by the presence of 10% serum (Morris et al. 2001). Thus, the damage
caused by internalization of PTD peptides to cells and the cell
membranes is assumed to be relatively minor (Futaki 2002).
Plasma membranes of plant or animal cells are generally
impermeable to peptides or proteins. The precise mechanism of
the AID peptide-mediated cellular delivery system remains
largely unknown (Snyder and Dowdy 2004). It has been commonly accepted that the membrane translocation mechanism
involves a passive membrane diffusive or de-stabilization process that does not require binding to animal cell surface receptors (Lindsay 2002, Wadia and Dowdy 2002). Circular
dichroism spectra of basic PTD peptides in methanol do not
share any typical secondary structure (Futaki 2002). The HIV
Tat PTD peptide showed a random structure, while the Rev
peptide had a helical structure. In addition, AID peptides may
exert their effects at 4°C and internalization is unaffected by
endocytotic inhibitors in cells, indicating that cell entry is in an
endocytotic- and energy-independent pathway in plants (Fig. 6).
To identify protein localization after cellular internalization, elevated green fluorescence tends to stay in the cytoplasm (Fig. 3, 4) and at higher levels in the nuclei (Fig. 5).
Although the precise mechanism of nuclear trafficking remains
to be clarified, a sequence with 27 amino acids of HIV-1 Vpr
protein that enables nuclear trafficking of proteins has been
reported recently (Taguchi et al. 2004). Interestingly, Vpr does
not have a classical nuclear localization signal. For cellular
entrance, Vpr may form ion channels in membranes. It subsequently binds to karyopherin, members of nuclear pore com-
487
plex proteins, or nucleoporin hCG1 for nuclear shuttling
(Taguchi et al. 2004). Additionally, we observed that some
cells exhibited marked fluorescence, but some did not (Fig.
5B). That is probably due to different physiological conditions
of every single cell. Protein internalization occurs quickly, and
reaches maximal activity after 5 min. However, the efficiency
will not reach 100%.
In conclusion, we have described a novel strategy for protein internalization mediated by AID peptides in plant cells.
Cellular delivery via short AID peptides is characterized by its
simplicity, non-invasion and powerful efficiency. These significant advantages of protein transduction may be applied to those
bioactive proteins needed to be expressed and to function in
plant cells in vivo for basic research. This novel method alternatively could be considered to replace time- and labor-consuming transgenic plant technology. Furthermore, AID
peptides may serve as a vector for broader applications in
plants, such as to shuttle some important peptides or proteins
into plant tissues in hydroponic culture. We believe this concept new to plant biology will help to simplify many experimental procedures and open up new thinking in designing
experiments related to protein action in plants in vivo.
Materials and Methods
Plasmid constructions
pTAT-HA vector (kindly provided by Dr. Steven F. Dowdy,
Washington University at St Louis, MO, U.S.A.) contains 11 amino
acids of HIV Tat PTD in-frame with six histidine residues and a
hemagglutinin tag under the control of the T7 promoter. pTAT-GFP
plasmid (also provided by Dr. Dowdy) consists of the GFP coding
region in pTAT-HA. pQE8-GFP plasmid contains an N-terminal polyhistidine (His6)-tagged GFPmut2 under the control of the T5 promoter/
lac operator (kindly provided by Dr. Michael B. Elowitz, Rockefeller
University, NY, U.S.A.). pR9 vector was generated by cutting pTATHA with BamHI restriction enzyme to remove the DNA sequence of
Tat PTD followed by insertion of the DNA sequence encoding nine
arginines that were generated by annealing of primers R9-up (5′GATCTAGATCTCGACGACGACGACGACGACGACGACGAGAATTCA-3′) and R9-down (5′-TGAATTCTCGTCGTCGTCGTCGTCGTCGTCGTCGAGATCTAGATC-3′). pR9-GFP and pR9-RFP plasmids
were generated by the insertion of the GFP and RFP coding regions
into pR9 vector, respectively. All constructions were verified by DNA
sequencing.
Protein expression and purification
Different GFP- and RFP-containing clones were transformed into
E. coli BL21(DE3)pLysS strain (Invitrogen, Carlsbad, CA, U.S.A.),
and then cultured aerobically at 37°C in 500 ml of LB broth (1% tryptone, 0.5% yeast extract and 200 mM NaCl, pH 7.5) containing ampicillin at a final concentration of 200 µg/ml. Bacteria were grown until
the OD600 reached 0.5, and isopropyl-β-D-thiogalactopyranoside
(IPTG) was added to a final concentration of 0.1 mM for protein
induction. After 12 h of induction with IPTG, cells were harvested by
centrifugation and the pellet could be resuspended in 10 ml of binding
buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9)
according to the manufacturer’s protocol (Novagen, Madison, WI,
U.S.A.). Cells were disrupted by sonication (Microson XL, Misonix,
NY, U.S.A.), and the insoluble debris was removed by centrifugation
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Protein transduction in plant cells
(4,000×g for 1 h at 4°C). The clarified supernantant was applied
directly onto a His-Bind column (Bio-Rad, Hercules, CA, U.S.A.) containing chelating Sepharose Fast Flow resins (Amersham Biosciences,
Piscataway, NJ, U.S.A.). A 3 ml aliquot of resins was packed into the
column, washed with 4 ml of water, charged by 4 ml of charge buffer
(50 mM NiSO4) and equilibrated with 10 ml of binding buffer. After
loading the sample, the column was washed again with 10 ml of binding buffer followed by 10 ml of wash buffer (40 mM imidazole, 0.5 M
NaCl, 20 mM Tris–HCl, pH 7.9). His6-tagged products were eluted
with 5 ml of elute buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris–
HCl, pH 7.9). Images of SDS–PAGE were analyzed with Gel Catcher
(MedClub, Taiwan) for protein purity. Protein was then quantified by a
Protein Assay kit (Bio-Rad, Hercules, CA, U.S.A.).
Protein internalization
Various fluorescent proteins were expressed and purified from
pQE8-GFP, pTAT-GFP, pR9-GFP, pR9-RFP and pDsRed plasmids in
the E. coli system as described above. For GFP or RFP analysis, plant
cells were administrated 2 µg of GFP for 5 min and washed with water
as a control at room temperature. To test protein transduction, plant
cells were mixed with 2 µg of Tat–GFP, R9–GFP or R9–RFP protein
for 5 min and washed with water in order to remove free protein. For
experiments at 4°C, the protocol was the same except that all incubations were performed at 4°C until the end of the procedure. Cells were
pre-incubated at 4°C for 30 min before being incubated with the protein solution. For the NEM and okadaic acid assay, onion epidermal
cells were treated with either Tat–GFP or R9–GFP for different times
in the presence or absence of 1 mM NEM for 5 min and 1.5 µM okadaic acid for 1 h, respectively. For the cytotoxicity assay, onion epidermal cells were washed with water and stained with 50% trypan blue.
Plant cell morphology and confocal microscopy
Plant cells were stained with crystal violet (1%) and washed with
water in order to observe their cell boundaries and nuclei. Images were
observed under an Eclipse E600 microscope (Nikon, Melville, NY,
U.S.A.) and recorded by a Penguin 150CL cooled CCD camera (Pixera, Los Gatos, CA, U.S.A.). Green fluorescent images were observed
by the TCS SL confocal microscope system (Leica, Wetzlar, Germany). This Leica confocal microscope was set up as follows: excitation was set at 488 nm and emission was collected using a 500 nm
dichroic mirror and a 505–530 nm bandpass barrier filter. To quantify
fluorescent images, relative intensities were converted by UN-SCANIT software (Silk Scientific, Orem, UT, U.S.A.) from >30 fields of
confocal microscopic images.
The authors wish to thank Dr. S.F. Dowdy for provision of the
pTAT-HA and pTAT-GFP plasmids, and Dr. M.B. Elowitz for the
pQE8-GFP plasmid. We are grateful to Drs. Yue-wern Huang, Ekkehard Sinn and Jason Elrod for critical reading and editing of this manuscript. This work was supported by the National Science Council,
Taiwan.
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(Received October 26, 2004; Accepted December 27, 2004)