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# 2008 The Authors
Journal compilation # 2008 Blackwell Publishing Ltd
doi: 10.1111/j.1600-0854.2007.00696.xTraffic 2008; 9: 528–539Blackwell Munksgaard
Tuning the Transport Properties of HIV-1 TatArginine-Rich Motif in Living Cells
Francesco Cardarelli1,2,*, Michela Serresi1,
Ranieri Bizzarri1,2 and Fabio Beltram1,2
1Scuola Normale Superiore and Italian Institute ofTechnology, Piazza dei Cavalieri, 7, I-56126, Pisa, Italy2Scuola Normale Superiore and NEST INFM-CNR,via della Faggiola, 19, I-56126, Pisa, Italy*Corresponding author: Francesco Cardarelli,f.cardarelli@sns.it
A large body of work is currently devoted to the rational
design of new molecular carriers for the controlled
delivery of cargoes (e.g. proteins or nucleic acids) to
relevant subcellular domains, particularly the nucleus. In
this article, we show that rational mutagenesis of the
human immunodeficiency virus type 1 Tat-derived pep-
tide (YGRKKRRQRRR) affords variants with finely tuned
intercompartmental dynamics and controllable transport
mechanisms. Our findings are made possible by the
demonstration that the Tat peptide possesses two com-
peting functionalities capable of active nuclear targeting
and additional binding to intracellular moieties. By alter-
ing the competition between these two functions, we
show how to control cargo localization of Tat peptide
chimeras. Our investigation provides a unified, coherent
description of previous conflicting in vivo and in vitro
results and lets the true nature of the Tat peptide emerge.
Key words: active import, FRAP, NLS, passive diffusion,
Tat peptide
Received 6 July 2007, revised and accepted for publica-
tion 19 December 2007, uncorrected manuscript pub-
lished online 21 December 2007, published online 13
January 2008
Human immunodeficiency virus type 1 encodes Tat, an
essential regulatory protein active in the cell nucleus (1–3).
This protein is an unusual transcriptional transactivator
that dramatically enhances the processivity of transcrip-
tion directed by the viral long terminal repeat promoter
element (4–6). Tat function involves its direct interaction
with an RNA target site, termed the trans-acting respon-
sive (TAR) element, that is mediated by an arginine-rich
RNA-binding motif (ARM; sequence: YGRKKRRQRRR,
basic residues in bold) (7–10). The same domain was also
indicated as a nuclear localization signal (NLS) (11,12). The
Tat ARM exhibits an additional important property: it is
readily taken up by cells through interactions with heparan
sulfate proteoglycans displayed on the cell membrane
(13). By means of this mechanism, this Tat-derived pep-
tide can determine cellular uptake of nonpermeant mole-
cules and lead to the expected biological response,
consistently with the internalization of intact peptide–
cargo fusions (14,15).
In the search for new therapeutic strategies, a large body
of work is currently devoted to determine whether cell-
penetrating peptides (CPPs, such as Tat-derived peptides)
can act as molecular carriers for delivery of cargoes (e.g.
proteins or nucleic acids) to relevant subcellular domains,
particularly the nucleus. Indeed, nuclear localization is very
significant because it is one of the fundamental steps for
gene therapy approaches and can be exploited to probe
and modify cellular processes of utmost importance. The
majority of nuclear proteins are targeted to the nucleus by
basic, generally lysine-rich NLSs. Nuclear localization signals
appear to fall into several classes, including those homol-
ogous to the NLS of the simian virus SV40 large tumor
antigen consisting of a single stretch of basic residues and
those termed bipartite NLSs comprising two clusters of
basic amino acids separated by a spacer of 10–12 amino
acids as typified by Xenopus laevis nucleoplasmin (16). All
these NLSs share analogous transport processes and
cytosolic factors mediating this transport (17). In detail,
they are usually recognized by the cytoplasmic receptor
proteins Imp-a and Imp-b and actively translocated through
the nuclear pore channel (NPC) (18). The directionality of
nuclear import is thought to be conferred by the asym-
metric distribution of the GTP- and GDP-bound forms of
protein Ran between cytoplasm and nucleus (19).
Tat ARM was ascribed to a novel class of basic NLSs, and
different detailed molecular mechanisms for its nuclear
active import were described based on in vitro assays.
Truant and Cullen proposed an active nuclear import
mechanism involving the cytosolic factor Imp-b but not
Imp-a (11). In contrast, Efthymiadis et al. reported that
the Tat peptide can target a large heterologous protein
(b-galactosidase from Escherichia coli, 120 kDa) to the
nucleus in the absence of both Imp-b and Imp-a by a
novel carrier-independent, energy-consuming process
(12). More recently, contrary to these in vitro experiments,
we showed that in vivo, the dominant mechanism of Tat
peptide-mediated nuclear transport is passive diffusion
(20). In this latter work, we discussed the role played by
intracellular interactions in determining this behavior. In
particular, we emphasized the high affinity for RNA of the
Tat peptide (7–10). Also, Friedler et al. reported evidence of
this affinity and developed a functional backbone mimetic
of the Tat arginine-rich motif whose nuclear import/RNA-
binding properties were tested by in vitro assays (21).
Interestingly, for the homologous arginine-rich sequence
of the protein Rev, it was shown that the latter has a much
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higher affinity for RNA molecules than for the importins
(22) and that cellular RNAs are able to efficiently block its
Imp-b nuclear import (23). Additional Tat interactions must
also be considered, however. For example, de Mareuil
et al. (24) reported that Tat, through its residues 38–72,
interacts with the microtubule network (20).
In this article, we show that the Tat peptide sequence
possesses two competing functionalities capable of active
nuclear targeting and additional binding to intracellular
moieties. The role of the C-terminal basic ‘RRR’ stretch is
highlighted: it determines the passive diffusion behavior of
Tat peptide–cargo fusion proteins by inducing high-affinity
interactionswith intracellularmoieties such as RNAs. Based
on this knowledge, we show that the NLS function that is
suppressed in wild-type Tat peptides in vivo can be fully
recovered in engineered mutants. Finally, by scanning
mutagenesis, we show how to tune the Tat peptide-
sequence properties in terms of both its localization and
its dominant transport mechanism. Subcellular localization
in living cells was analyzed by confocal imaging, while
intracellular dynamics was investigated by fluorescence
recovery after photobleaching (FRAP) real-time imaging.
The impact of this tunability on Tat exploitation as a CPP
is also addressed and its implications discussed.
Results
Tat peptide sequence can be turned into a functional
NLS by scanning mutagenesis
In our previous work, we developed a method to screen
short peptide sequences for their ability to mediate nuclear
import based on in vivo studies and real-time imaging with
an array of cargoes and benchmark systems. Our approach
allowed us to conclude that passive diffusion drives
nucleus–cytoplasm trafficking of cargoes mediated by Tat
peptide (YGRKKRRQRRR, Table 1) (20). However, the
homologous basic domain of SV40 (YPKKKRKVEDP, Table
1) unambiguously showed different intracellular localization
and trafficking properties. Analysis of the two sequences
drew our attention to the last three C-terminal amino acids.
These are positively charged Arg-Arg-Arg (RRR) residues in
the Tat sequence (hereafter, TatRRR) and acidic Glu-Asp-Pro
(EDP) residues in the NLS of SV40 (hereafter, NLSEDP).
We investigated the properties of the ‘RRR’ stretch and
its role in determining TatRRR molecular interactions and
nuclear transport properties. First, scanning mutagenesis
of the Tat peptide sequence was performed by mutating
RRR into three (not charged) glycine residues: the resulting
mutant (TatGGG sequence in Table 1) was analyzed by
in vivo imaging techniques.
Remarkably, TatGGG fused to a 110-kDa cargo [hereafter,
TatGGG–green fluorescent protein (GFP)4], well above the
threshold for passive diffusion across the nuclear pore
(25), was almost exclusively detected into the nucleus
(Figure 1A). This indicates that the TatGGG sequence does
determine active nuclear import, analogously to its
NLSEDP-tagged counterpart. Conversely, as previously
reported and shown in Figure 1B, the wild-type Tat peptide
is not able to drive the active nuclear import of the same
cargo and is excluded from the nucleus. The integrity of
GFP4 constructs was checked by means of fluorescence
resonance energy transfer (FRET) imaging (Figure 1D; see
Materials and Methods for further details).
The role of active processes in determining the nuclear
permeation driven by TatGGG sequence was further verified
by analyzing the subcellular localization of TatGGG–GFP
(single GFP cargo, capable of crossing the nuclear envelope
by passive diffusion) in response to energy depletion (see
Materials andMethods). Figure 2A shows that TatGGG–GFP
is predominantly localized in the nucleus, with a nuclear-to-
cytoplasmic ratio Keq ¼ 2.1 � 0.1 when expressed in cells
under normal growing conditions (þATP panel). A sizable
GFP fluorescence signal is also observed within the
cytoplasm owing to the nucleus-to-cytoplasm TatGGG–GFP
Table 1: Transport parameters derived from FRAP measurements
Sequence Protein Keq t(C/N)
(seconds)
kDN(mm3/second)
kAT(mm3/second)
DC
(mm2/second)
DN
(mm2/second)
pbN
— GFP �1 77 � 19 13.6 � 3.7 0 20 � 5 19.8 —
YPKKKRKVEDP- NLSEDP-GFP 3.3 60 � 3 5.2 � 0.6 10–17.1 11.9 � 2.6 17.8 —
YGRKKRRQRRR- TatRRR-GFP �1 410 � 125 2.7 � 1.5 0 5.9 � 1.5 6 1
YGRKKRRQRRG- TatRRG-GFP 1.1 172 � 25 3.2 � 1.2 0.3–3.5 6.1 � 0.7 ND 0.95
YGRKKRRQRGG- TatRGG-GFP 1.6 95 � 15 5.2 � 1.6 3.0–8.3 6.9 � 1 ND 0.77
YGRKKRRQGGG- TatGGG-GFP 2.1 66 � 9 7.5 � 1.2 7.1–15.7 9.8 � 1.3 14.4 0.56
YPKKKRKVGGG- NLSGGG-GFP 2.4 66 � 11 5 � 2 7.2–12 11.2 � 1.6 19.6 —
YPKKKRKVRRR- NLSRRR-GFP 1.2 262 � 36 1.2 � 0.3 0.2–1.4 4.5 � 0.8 4.3 —
This table summarizes the main transport parameters derived from FRAP analysis: Keq, ratio between nucleoplasmic and cytoplasmic
fluorescence (mean value); t(C/N), time constant of nuclear fluorescence recovery (mean � SD); kDN, passive diffusion parameter
(mean � SD); kAT, active transport parameter (range of reported values); DC, cytoplasmic diffusion coefficient (mean � SD); DN,
nucleoplasmic diffusion coefficient (values derived from the equation: RM ¼ DN/DC, as described in Materials and Methods); pbN, molar
fraction of bound molecules within nucleoplasm; ND, no data. Italicized values represent previously reported data (see (20)). Tag-
sequences are reported in one-letter code with conserved residues outlined and introduced mutations in bold, respectively.
Traffic 2008; 9: 528–539 529
In Vivo Study of Tat Peptide-Targeting Properties
passive diffusion. When active processes were blocked
by ATP depletion, TatGGG–GFP passive diffusion from the
nucleus to the cytoplasm successfully restored a homoge-
neous intracellular distribution (Figure 2B, �ATP). Notably,
this behavior is reversible: within a fewminutes after bringing
cells back to physiological conditions, fluorescence was
again predominantly localized in the nucleus (data not
shown). This assay reveals that TatGGG–GFP nuclear
accumulation is driven by an energy-consuming process,
analogous to what we observed for NLSEDP–GFP (20). We
obtained analogous results for TatEDP–GFP construct in
which the ‘RRR’ stretch was replaced with the SV40-
derived ‘EDP’ one (data not shown).
We compared these results with similar measurements
performed on an NLSEDP-derived mutant in which the
C-terminal ‘Glu-Asp-Pro’ residues had been replaced by
‘Gly-Gly-Gly’ ones (hereafter, NLSGGG). As expected, this
protein maintained the wild-type nuclear active import prop-
erties (Figure 2B, þATP), with only a slight decrease in
efficiency (compare Keq values in Table 1). Accordingly, we
observed relocalization upon energy depletion (Figure 2B,
�ATP). This behavior is not unexpected because the
functional core domain of the NLS of SV40 (YPKKKRKV)
is conserved in this mutant (26). Notably, when we
replaced the C-terminal ‘EDP’ residues of the wild-type
NLS of SV40 sequence with the three arginines derived
from the Tat sequence, the characteristic nuclear accumu-
lation was almost completely abolished and a marked
nucleolar staining emerged (NLSRRR–GFP in Figure 2A). On
the contrary, untagged GFPs did not change their homoge-
neous intracellular distribution upon energy depletion treat-
ment (data not shown). Taken together, these results
indicate that the ‘RRR’ stretch can impair the potential active
nuclear import mechanism. In order to establish the trans-
port parameters of these mutants, we performed a FRAP
analysis of their nucleus/cytoplasm shuttling. Data were
analyzed under the framework of a general model of
nucleus–cytoplasm exchange that considers not only the
fluorescent construct but also its complexes with import
carrier(s) and/or other biomolecules (Figure 7). This approach
is described in the supplementary information and yields the
quantitative determination of the passive diffusion out of the
nucleus (expressed by the parameter kDN) and the concom-
itant estimate of the active transport into the nucleus
(expressed by the variability range of the parameter kAT).
The construct encoding for TatGGG–GFP was investigated
first and compared with the corresponding passive diffu-
sion benchmark TatRRR–GFP. The fluorescence of the
nuclear compartment was photobleached by irradiating
a single point with high laser power for 10 seconds, the
Figure 1: TatGGG-mediated active nuclear accumulation of GFP4 cargo. A) As expected, for a 110-kDa protein, GFP4 is excluded from
the nucleus (cargo molecular weight above the limit for passive diffusion through the NPC). B) TatRRR sequence is not able to drive GFP4
nuclear localization, as shown previously (18). C) Notably, TatGGG-tagged GFP4 is almost exclusively localized within the nucleus,
demonstrating that this Tat-derived mutant is able to perform active nuclear import. D) Integrity of GFP4 construct is shown here by FRET
imaging (see Materials and Methods for further details). Scale bar: 10 mm. ex, excitation.
530 Traffic 2008; 9: 528–539
Cardarelli et al.
subsequent recovery was recorded by time-lapse acquisi-
tion at 30-second intervals. Figure 2C shows the meas-
ured recovery curves: the characteristic TatGGG–GFP
recovery kinetics is considerably faster than that of
TatRRR–GFP. Table 1 summarizes our results for the whole
population of analyzed cells. We obtained t ¼ 66 � 9 sec-
onds for TatGGG–GFP and t ¼ 410 � 125 seconds for
TatRRR–GFP (20). Furthermore, by means of our kinetic
model, we can derive the TatGGG characteristic transport
parameter kAT ¼ 7.1 � 15.7 mm3/second for the active
transport component and kDN ¼ 7.5 � 1.2 mm3/second
for the nucleus-to-cytoplasm diffusion. Fluorescence
recovery after photobleaching analysis of NLSGGG–GFP
shows that substitution of the ‘EDP’ stretch does not alter
the kinetics of NLS-driven nuclear import (see recovery
curve in Figure 2D and transport parameters in Table 1).
Overall, the t values obtained for NLS-tagged proteins are
in good agreement with recently reported FRAP data in
COS-7 cells (27). At the same time, the mutant NLSRRR–
GFP showed a marked decrease in the nucleus/cytoplasm
shuttling rate (Figure 2D), confirming the hypothesis that
the C-terminal arginine-rich ‘RRR’ stretch has a dramatic
impact on active transport processes.
The role of the ‘RRR’ stretch in determining Tat
peptide interactions
Arginine-rich sequences are found in many RNA-binding
proteins and were proposed as the mediators of specific
RNA recognition processes: in particular, the number/
position of positively charged amino acids in the basic
domain of Tat is critical for its interaction with the specific
TAR RNA sequence (7–10). This prompted us to assess
the impact of the RRR/GGG replacement examined
above on Tat peptide-binding affinity toward RNAs
Figure 2: Influence of the ‘RRR’ determinant on nuclear localization and intercompartment dynamics of constructs. A) The
presence of the ‘RRR’ stretch is able to impair the active nuclear import driven by the NLS of SV40, leading to an intracellular localization
similar to that of wild-type Tat-tagged GFP (TatRRR–GFP). B) The corresponding mutants in which the ‘RRR’ residues were replaced by the
‘GGG’ ones (TatGGG–GFP and NLSGGG–GFP) showed a marked nuclear accumulation in normal growing conditions (þATP panels). Within
15 min after treatment with 2-deoxy-D-glucose and sodium azide, both constructs equilibrated between nucleus and cytoplasm (�ATP
panels). Scale bar: 10 mm. C) FRAP analysis of nucleus/cytoplasm exchange of expressed proteins. A typical TatGGG–GFP recovery curve
(filled red circles, t value of 53 � 1 seconds) is here compared with the slower passive diffusion kinetics of TatRRR–GFP (open red circles,
t value of 307 � 4 seconds). D) NLSGGG–GFP characteristic active import kinetics (filled dark-yellow circles, t value of 53 � 2 seconds)
compared with the slower recovery kinetics of NLSRRR–GFP (open dark-yellow circles, t value of 242 � 8 seconds). All recovery curves
were normalized by prebleaching values: the asymptotic value around 1 indicates that no immobile fraction of molecules can be detected
in the bleached compartment. Overall t values are reported in Table 1.
Traffic 2008; 9: 528–539 531
In Vivo Study of Tat Peptide-Targeting Properties
because a significant variation of this affinity could be at
the basis of the observed emergence of Tat–NLS proper-
ties in vivo.
We experimentally confirmed a large variation of the
binding affinity between Tat and RNAs when RRR is
replaced by GGG by an in vitro binding assay. In detail,
we used tetramethylrhodamine (TAMRA)-labeled peptides
and AlexaFluor647-labeled random oligo-RNAs. These
fluorophores were selected owing to their high FRET
efficiency. Increasing amounts of labeled peptide were
added to an RNA-containing solution, and the fluorescence
emission spectrum was recorded following excitation of
the ‘donor’ species (TAMRA-labeled peptide). In this way,
peptide–RNA interactions could be directly assessed
in vitro by FRET signal analysis (see eqn 1 in Materials
and Methods): this approach allowed us to quantitatively
compare TatRRR and TatGGG sequences for their ‘overall’
RNA-binding affinity. The substitution of the three
C-terminal arginines with glycines leads to a dramatic
decrease in FRET efficiency (Figure 3), i.e. in the Tat
peptide-binding affinity for RNA molecules. Ribonuclease
treatment (black box in Figure 3) brought the FRET signal
back to zero, further demonstrating the direct involvement
of RNAs in the observed interaction. As expected, addition
of a TAMRA-labeled glycine residue (black dots) did not
lead to a FRET signal. Our results are consistent with
analogous measurements reported so far in the literature
(7,8) and confirm the impact of the RRR/GGG substitu-
tion on RNA affinity of the peptide under study.
Single-point mutagenesis on TatRRR sequence:
modulation of nuclear import properties
The above set of experiments showed that the ‘RRR’
residues impair the potential nuclear-targeting properties
of the Tat sequence. Because the replacement of ‘RRR’
residues with ‘GGG’ allowed us to switch on the Tat
peptide nuclear import properties, we tried to tune them
by varying the number of positively charged C-terminal
arginine residues. To this aim, we engineered two novel
mutants in which the ‘RRR’ residues were replaced by
‘RRG’ and ‘RGG’, respectively (the corresponding se-
quences are given in Table 1). Hence, we studied their
intracellular localization and transport properties by confo-
cal imaging and FRAP analysis and compared them with
actively imported TatGGG–GFP (and NLSGGG–GFP) and
passively diffusing TatRRR–GFP (and NLSRRR–GFP). Nota-
bly, the one-by-one addition of glycine residues to the
wild-type Tat sequence gradually increases the effective
nuclear accumulation of constructs in comparison with
TatRRR, while it leads to a decrease in their nucleolar
localization (Figure 4A,B). We addressed the quantitative
aspects of the nucleus–cytoplasm exchange by means of
FRAP measurements: both bleaching time and sampling
Figure 3: Monitoring peptide–RNA interaction by FRET.
Increasing amounts of TAMRA-labeled peptide were added to
a solution containing AlexaFluor647-labeled random oligodeoxy-
nucleotides, and fluorescence emission spectrum was recorded,
as described in Materials and Methods. Complex formation
between Tat peptides and RNAs was monitored through FRET
calculation (eqn 1). Here, N_FRET is plotted against peptide
concentration. Wild-type Tat peptide (red dots) shows a much
higher affinity for RNA compared with TatGGG mutant (dark-yellow
dots), as highlighted by the hyperbolic fitting (solid lines). Ribonu-
clease addition abolishes the FRET signal (dots highlighted in the
black box), demonstrating the specificity of peptide–RNA interac-
tion. We performed similar measurements with TAMRA–glycine
(black dots): as expected, we observed no FRET signal.
Figure 4: Analysis of intracellular localization of Tat-derived
mutants. A) Ratio between nucleoplasmic and cytoplasmic
fluorescence (red symbols, corresponding to the Keq values
reported in Table 1) is here plotted together with the ratio between
nucleolar and nucleoplasmic fluorescence (black symbols). The
latter parameter indicates the extent of nucleolar accumulation of
the expressed protein. B) Intracellular distribution of Tat-derived
mutants imaged using the confocal microscope. Scale bar: 10 mm.
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Cardarelli et al.
rate were tailored to the speed of fluorescence recovery of
the tested protein. Collected recovery curves (shown in
Figure 5A) and the derived shuttling time constants (Table
1) clearly show that the addition of glycine residues can
progressively accelerate the intercompartment mobility of
TatRRR-derived mutants. We shall argue that this behavior
stems from the gradual change from passive diffusion to
active import. kAT and kDN values provide a quantitative
description of this trend (Figure 5B,C; Table 1) and are
higher for actively imported species (TatGGG–GFP and
NLSGGG–GFP) and lower for the passively diffusing ones
(TatRRR–GFP and NLSRRR–GFP). Under the assumption
that kDN values of GFP and TatRRR–GFP reflect the passive
diffusion dynamics of the totally free and totally bound
proteins, respectively, the kDN values found for the other
Tat constructs were used to obtain the molar fractions of
bound proteins in the nucleus (Table 1).
Disk FRAP measurements: protein mobility
within the cytoplasm
These results provide strong indications of the relevance of
the TatRRR–peptide interactions in determining the overall
construct properties: in particular, we showed how the
mechanism of TatRRR peptide nuclear entry strongly de-
pends on the presence and the number of positively
charged C-terminal arginine residues.
Figure 5: Kinetic parameters for all Tat-derived mutants. A) Kinetics of nucleoplasmic FRAP for all Tat-derived mutants. t values
(mean � SD) are 53 � 1 seconds (TatGGG–GFP; the same of Figure 2C), 108 � 3 seconds (TatRGG–GFP), 190 � 5 seconds (TatRRG–GFP)
and 307 � 4 seconds (TatRRR–GFP, Figure 2C). All FRAP curves are normalized by prebleaching values; the asymptotic value of 1 indicates
that no immobile fraction of proteins is present in the nuclear compartment. t values for the whole population of analyzed cells are reported
in Table 1. B–D) Averaged kAT, kDN and DC values for Tat-derived mutants (red symbols) and SV40-derived mutants (yellow symbols). kATand kDN transport parameters have been derived from nuclear FRAP analysis, as described in Materials and Methods. The effective
cytoplasmic diffusion coefficients (DC) have been derived from disk bleaching measurements, as described in Materials and Methods.
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In Vivo Study of Tat Peptide-Targeting Properties
To isolate the impact of these interactions from nuclear
envelope permeation, we investigated the intracytoplasmic
diffusion properties of constructs. Following the method
described in Cardarelli et al. (20), wemeasured the effective
cytoplasmic diffusion coefficient (DC) and the value of the
bleaching parameter K0 (related to the bleaching depth) for
all the tested proteins. As could be expected, the sub-
stitution of the C-terminal ‘RRR’ stretch with ‘GGG’ leads to
a considerable increase in Tat peptide intracytoplasmic
diffusivity: DC values are 5.9 � 1.5 mm2/second for
TatRRR–GFP and 9.8 � 1.3 mm2/second for TatGGG–GFP
(Figure 5D; Table 1). This large change confirms that the
presence of the ‘RRR’ stretch leads to increased molecular
interactions with RNAs and other intracellular moieties that
decrease protein diffusivity. At the same time, the almost
doubling of the diffusion coefficient of TatGGG–GFP with
respect to isolated GFP is consistent with the presence of
interactions with molecular components that are involved in
determining its nuclear active translocation. In fact, active
import benchmarks showed similar DC values (11.9 �2.6 mm2/second for NLSEDP–GFP and 10.8 � 1.6 mm2/sec-
ond for NLSGGG–GFP, respectively).
The one-by-one mutation of arginine residues to glycine
progressively increases protein intracytoplasmic diffusion:
we obtained DC ¼ 6.1 � 0.6 mm2/second for TatRRG–GFP
and 6.9 � 1 mm2/second for TatRGG–GFP (the overall data
plot is shown in Figure 5D).
Line FRAP method: seeking the mechanism
of nuclear entry
The uniform disk bleach method is a very effective tool for
measuring diffusion coefficients within the cytoplasm but
is not suitable for measurements in the nucleus. Indeed,
the lower volume of this compartment leads to consider-
able loss of overall fluorescence upon photobleaching and
prevents its use to evaluate diffusion parameters. In order
to do so, we took another approach based on the line scan
of the cell. In this experiment, we bleached a thin stripe
across both the nucleus and cytoplasm for 90 milliseconds
with laser light at full power and imaged the recovery
process at high speed along the same line (Figure 6A). The
characteristic recovery time was calculated by fitting
fluorescence recovery to double exponential equations
(Figure 6B) and taking the slower time constant as the
representative one, as described inMaterials and Methods.
This approach allowed us to compare the protein diffusion
properties in the nucleus and the cytoplasm. By taking the
ratio (RM parameter in Table 1) between nuclear and
Figure 6: Line FRAP measurement. A) The cell is repeatedly imaged along the dotted line at high frequency (400 Hz). Bleaching is
performed along a strip covering both the nucleus and cytoplasm for approximately 90 milliseconds using 488-nm laser at full power.
Fluorescence recovery is thenmonitored by imaging at 400 Hz for about 2.5 seconds. Collected data are analyzed as described inMaterials
and Methods. Scale bar: 10 mm. B) Nuclear (green dots) and cytoplasmic (red dots) fluorescence recovery from the cell shown in (A).
At this temporal resolution, the difference in mobility between the two compartments is clearly discernible: photobleaching within the
cytoplasm is larger and the corresponding recovery is slower, indicating lower mobility. C) Cumulative results. The ratio RM (mean � SD)
between nuclear and cytoplasmic recovery rates (slower time constant from double exponential fit, seeMaterials and Methods) is plotted
for all the analyzed constructs. Passively diffusing proteins are characterized by a RM value around 1, while actively imported proteins
yielded a RM value below 1.
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Cardarelli et al.
cytoplasmic characteristic recovery times, we can esti-
mate the diffusion coefficient ratio of the two compart-
ments (DN/DC) and highlight the differences in protein
mobility between these two compartments. Figure 6C
shows that the proteins that are supposed to cross the
nuclear membrane by passive diffusion (i.e. GFP, TatRRR–
GFP and NLSRRR–GFP) showed no significant difference in
mobility between the nucleus and the cytoplasm, corres-
ponding to RM values around unity (Table 1). On the
contrary, the proteins that enter the nucleus by active
import (NLSEDP–GFP, NLSGGG–GFP and TatGGG–GFP)
unambiguously showed a larger diffusivity in the nucleus
than in the cytoplasm (RM < 1). These results will be
correlated to the molecular models proposed for the
nuclear import of the tested proteins (see Discussion).
Discussion
Recently, we showed that the mechanism driving in vivo
TatRRR nuclear permeation is passive diffusion, while
interaction with import carriers plays no detectable role
(20). These findings appear in contrast with previous
in vitro studies that had highlighted the relevance of active
processes in TatRRR-driven nuclear import (11,12). In
particular, Truant and Cullen showed that the TatRRRpeptide directly interacts with Imp-b in vitro and that
Imp-b is necessary and sufficient for the nuclear import
of TatRRR into isolated nuclei (11). We discussed these
findings and emphasized the impact on in vitro studies of
the absence of a diverse range of biomolecules that are
physiologically present in live cells. In fact, among these
molecules, some may bind to the TatRRR peptide and
compete with and even prevent TatRRR interaction with
Imp-b (21).
The SV40 peptide is an accepted functional NLS, as seen
also in our in vivo studies where it was actually used as an
active transport benchmark (20). A careful comparison
between Tat and SV40 peptides does show significant
sequence similarities (Table 1) with a notable difference in
the last three C-terminal amino acids. In fact, the latter are
positively charged in the TatRRR peptide (RRR) but are
mainly hydrophobic in SV40EDP (EDP). Moreover, these
three residues are not an integral part of the NLS of SV40,
and the remaining eight amino acid sequence (YPKKKRKV)
fully retains active import properties (26). These consid-
erations prompted us to investigate the impact of these
three amino acids on the transport properties of the Tat
peptide. To this end, we engineered fluorescent mutants
in which the terminal ‘RRR’ of the Tat sequence was
replaced by the neutral triplet ‘GGG’, while the remaining
sequence (YGRKKRRQ) was left unaltered. As previously,
we investigated the intracellular localization of these
proteins by in vivo confocal imaging. Remarkably,
TatGGG–GFP4 was virtually exclusively localized within the
nucleus (Figure 1C), even if its molecular weight exceeds
the diffusion limit through the NPC. This behavior is in full
contrast with TatRRR–GFP4 (completely excluded from the
nucleus) and consistent with active transport being the
dominant nucleus-to-cytoplasm transfer mechanism for
the TatGGG peptide. Furthermore, TatGGG–GFP (similar to
the active import benchmark SV40EDP–GFP) was not im-
ported into the nucleus upon energy depletion (Figure 2B).
This is once again in contrast with the behavior of TatRRR–
GFP (and isolated GFP). In fact, our published data show
that subcellular localization of TatRRR–GFP is not affected
by the same treatment (i.e. relocalization was not ob-
served) (20). Altogether, these findings indicate that the
NLS function is suppressed in wild-type Tat peptide, but
can be made available in engineered mutants. The Tat
peptide NLS can be associated to the first eight amino
acids of the sequence. Remarkably, the ratio between
nuclear and cytoplasmic fluorescence (Keq) of TatGGG–GFP
is very close to that of SV40GGG–GFP that can be consid-
ered its active import benchmark. Consistently and sym-
metrically with these results, we observed that when the
C-terminal ‘EDP’ residues of the functional NLS of SV40
are replaced by the ‘RRR’ ones, active nuclear import is
almost completely suppressed (with a concomitant
increase in nucleolar stain) and nucleus/cytoplasm shut-
tling kinetics is considerably slowed down (Figure 2B,D).
These findings lead to the conclusion that the first eight
residues of Tat peptide can operate as a NLS, but the
remaining three arginine residues hinder active transport
by promoting binding to intracellular moieties. Among
these, RNAs play an important role. In fact, Tat is known
to interact with negatively charged biomolecules such as
RNAs (7–10). Here, by an in vitro binding assay (Figure 3),
we showed that the substitution of the three C-terminal
arginines with glycines leads to a dramatic decrease in
Tat peptide-binding affinity for a population of random
RNA molecules. Our data show the role of unspecific
interactions between Tat and RNAs that can take place
within a living cell where TAR RNA is not present. The
marked decrease found in affinity upon ‘RRR’ replace-
ment by ‘GGG’ shows that interaction with RNAs can
indeed play a significant role in the observed modulation
of intracellular localization and trafficking properties as
described above.
In agreement with our findings, Friedler et al. reported that
a functional backbone mimetic of the Tat arginine-rich motif
was capable to interact with both import carriers and RNA
by in vitro assays (21). RNAs, however, are not the only
intracellular moieties that can influence Tat peptide proper-
ties. For example, it has been reported that Tat peptide-
comprising truncations of full-length Tat are able to target
the microtubule network (24). Thus, tubulin may represent
another moiety that modulates Tat trafficking upon binding.
Preliminary results using the microtubule-damaging drug
nocodazole, however, showed no effect on TatRRR–GFP
subcellular localization and dynamics (data not shown).
Even if we cannot exclude that microtubule assembly and
Traffic 2008; 9: 528–539 535
In Vivo Study of Tat Peptide-Targeting Properties
Tat are related, our data suggest that Tat–tubulin interaction
is not relevant in determining Tat transport biology, in
agreement with other results on Tat–tubulin interaction (28).
The picture of two competing equilibria was further probed
by varying the number of arginine residues. As expected,
from our analysis, we were able to fully tune the nuclear
import mechanism of Tat peptide by replacing one by one
the positively charged arginine residues with uncharged
glycine ones. This induced an increase in nuclear accumu-
lation of TatRRR-derived mutants (Keq in Table 1) and pro-
gressively accelerated the rate of their nucleus–cytoplasm
exchange.
These findings confirm a model of nucleus–cytoplasm
exchange of fluorescent constructs that takes into account
the competing binding with active import carriers (e.g.
importins) and other biomolecules (e.g. RNA). The interac-
tion pattern is schematized in Figure 7: ‘P’ labels the free
construct, ‘PA’ the complex with active import carriers and
‘PB’ the complex with other intracellular moieties. Our
findings show that P and PB diffuse passively across the
nuclear envelope, each species with its own diffusion
characteristics. In contrast, PA is actively imported into
the nucleus where it dissociates into its two components,
in line with a characteristic NLS behavior (17,29). The
overall observed permeation phenomenology is the result
of these processes weighted according to the mole
fraction of the different species (for further details, see
Supplementary Information).
The analysis of FRAP data according to this model allows
us to calculate the transport parameters associated to
nucleus-to-cytoplasm passive diffusion (kDN) and active
nuclear import (kAT). Not surprisingly, we observed that
passing from TatGGG–GFP to TatRRR–GFP one residue at
a time, both kAT and kDN values decrease (Figure 5B,C).
This shows the loss of nuclear import capability and
a concomitant increased affinity for other molecular com-
ponents. Assuming GFP and TatRRR–GFP as references of
totally free and totally bound molecules, respectively, we
were also able to estimate the mole fraction of bound
protein in the nucleus (Table 1). Note that the mole fraction
is not linearly related to the number of arginine residues,
suggesting co-operative behavior of these charged amino
acids in determining molecular interactions.
Binding can be experimentally monitored by measuring
intracytoplasmic diffusion properties. This was performed
by disk photobleaching FRAP measurements, a powerful
tool to investigate even small differences in diffusivity. We
found that sequential arginine replacement does modulate
the intracytoplasmic absolute diffusion coefficient (DC).
Consistent with our hypothesis of arginine-promoted bind-
ing properties, TatRGG, TatRRG and TatRRR sequences show
progressively and considerably reduced diffusion coeffi-
cient values (Figure 5D and Table 1).
A protein with NLS capability should be characterized by
a lower mobility in the cytoplasm than in the nucleoplasm
owing to the larger number of binding interactions [e.g.
with import carrier(s) and with other related biomolecules].
We investigated this property by means of the line FRAP
method that allowed us to estimate the ratio between
cytoplasmic and nuclear mobility RM (Figure 6; Table 1).
Interestingly, TatGGG–GFP, NLSEDP–GFP and NLSGGG–GFP
are characterized by RM values smaller than 1, as expected
for proteins undergoing receptor-mediated import to the
nucleus (17,29). Conversely, GFP, TatRRR–GFP and
NLSRRR–GFP show RM close to unity, in agreement with
their full passive diffusion behavior.
In conclusion, we have shown that the Tat peptide
sequence possesses a dual functionality. It includes
a NLS together with an additional competing functionality
leading to binding to intracellular moieties, including RNAs
(20,21). Our findings highlight the impact of the competi-
tion between two functions and explain contrasting results
reported in the literature. In this way, they provide a unified
and coherent interpretation of Tat peptide intracellular
transport properties. We believe that these results can
be useful for the rational design of new molecular carriers
Figure 7: Model of nucleocytoplasmic exchange. The free
NLS-tagged construct (P) in the cytoplasm can bind to both import
carriers (PA) and other biomolecules (PB). We assume that P and
PB shuttle across the nuclear envelope (NE) by reversible passive
diffusion (bidirectional black arrows), each species with its proper
diffusion characteristics. Conversely, the PA complex is actively
imported into the nucleus (red arrow) where it dissociates out into
the two components: this energy-consuming and irreversible
process leads to the accumulation of the NLS-tagged construct
in the nuclear compartment. The directionality of the active import
process is determined by asymmetric distribution of RanGTP
between compartments (19).
536 Traffic 2008; 9: 528–539
Cardarelli et al.
for the controlled delivery of cargoes to specific subcellular
domains, particularly the nucleus.
Materials and Methods
Synthetic compoundsThe C-terminus-labeled peptides used in this study were purchased from
Sigma-Genosys. Their amino acidic sequence is reported in Figure 3.
Tetramethylrhodamine was chosen for peptide labeling.
AlexaFluor647-labeled oligo-RNAs were purchased from Invitrogen–
Molecular Probes. These are random RNA sequences of nine oligodeoxy-
nucleotides labeled at the 50 end.
For all spectroscopic measurements (see below), peptides and RNAs were
resuspended in a suitable reaction buffer (70 mM NaCl, 10 mM Tris–HCl
pH ¼ 7.5, 0.2 mM ethylenediaminetetraacetic acid and 5% glycerol).
The reaction between the succinimidyl ester derivative of TAMRA (Invitrogen–
Molecular Probes) and glycine (Sigma) has been performed in citrate buffer
at pH ¼ 9.
In vitro spectroscopic FRET measurementsFluorescence spectra were recorded at 238C on a Cary Eclipse spectroflu-
orometer (Varian) by setting the excitation and emissionmonochromator slits
to 5 and 10 nm, respectively; the scanning speed was set to 120 nm/min,
the data resolution to 1 nm and the time collection average to each
wavelength interval to 0.5 seconds. Emission spectra were recorded by
exciting at 520 nm and collecting the fluorescence between 550 and
750 nm. First, a fluorescence emission measurement was performed on
a solution of labeled RNA alone. Thus, increasing amounts of TAMRA-labeled
peptide were added, and the emission spectrum was recorded. Complex
formation between Tat peptides and RNAs was monitored through FRET
calculation by using the emission counts collected at 666 nm after excitation
at 520 nm. Normalized FRET counts (N_FRET) were calculated by
N FRET ¼ ðF666 � FACC � FDONÞFACC
½1�
where F666 are the overall counts recorded at 666 nm, FACC are the counts
recorded when only the acceptor is present in solution at 666 nm and
FDON are the predicted counts because of donor emission at 666 nm,
respectively. FDON is calculated each step from the ratio between the
fluorescence emission at 578 and 666 nm detected for the donor alone.
Finally, N_FRET is plotted against the donor concentration in fluorescence
units (counts/mL), and fitted to an hyperbolic function, as shown in Figure 3.
The dilution factor was considered for the calculation of both N_FRET and
donor concentrations.
DNA manipulations: cloning and mutagenesisPlasmids encoding for TatRRR–EGFP and NLSEDP–EGFP were described in
our previous work (20). These plasmids were used as templates to create
all mutants mentioned in this work, and mutations were introduced by site-
directed mutagenesis using a QuickChange kit (Stratagene).
The following primers (Sigma-Genosys) were used for mutagenesis reac-
tions: 50 GAA GCG GAG ACA GGA AGA CCC AAA GCT TAT AGT GAG C 30
for TatEDP–GFP; 50 GAA GCG GAG ACA GGG AGG AGG AAA GCT TAT AGT
GAG C 30 for TatGGG–GFP; 50 GAA GCG GAG ACA GCG AGG AGG AAA GCT
TAT AGT GAG C 30 for TatRGG–GFP and 50 GAA GCG GAG ACA GCG ACG
AGG AAA GCT TAT AGT GAG C 30 for TatRRG–GFP. Antisense primers had
respective reverse complementary sequences.
The TatGGG–E1GFP–EGFP–tdTomato construct was obtained by subcloning
E1GFP–EGFP–tdTomato in TatGGG–GFP plasmid cut byHindIII–XbaI in order
to remove the EGFP sequence. The cloning strategies used for the
plasmids encoding for E1GFP (an EGFP containing the mutations T65S
T203Y), EGFP and the dimeric red fluorescent protein tdTomato were
described in detail in Cardarelli et al. (20).
NLSRRR–EGFP plasmid was constructed by multiple point mutation reac-
tions starting from Tat11–EGFP sequence. The primer used for multiple
mutagenesis was 50 GGA TCC ATG TAT CCC AAG AAG AAG CGG AAA
GTG CGA CGA AGA 30. Subsequently, to obtain NLSGGG–GFP construct,
we used NLSRRR–EGFP as template and the following sense primer: 50 GAA
GCG GAA AGT GGG AGG AGG AAA GCT TAT AGT GAG C 30. Antisense
primer had respective reverse complementary sequence.
The expression vector encoding for tdTomato protein was generated by
polymerase chain reaction (PCR) amplification of tdTomato template
(obtained from Roger Y. Tsien’s laboratory) and was inserted into EcoRI–
XbaI sites of pcDNA3.1(þ) vector (Invitrogen). To obtain the construct
pE1GFP, the EGFP template (pEGFP-N1; obtained from Clontech) was
amplified by PCR introducingHindIII site in 50 and EcoRI sites in 30 extremity
and it was inserted into pcDNA3.1(þ) vector. By using two distinct steps
of site-directed mutagenesis, we introduced the mutations T65S T203Y
using respectively the appropriate primers 50 GTG ACC ACC CTG TCC TAC
GGC GTG CAG 30 and 50 CAA CCA CTA CCT GAG CTA CCA GTC CGCCCT
GAG 30.
Cell culture and transfectionsChinese Hamster Ovary (CHO-K1) cells were provided from the American
Type Culture Collection (CCL-61; ATCC) and were grown in Ham’s F12K
medium supplemented with 10% of fetal bovine serum (FBS) at 378C and
in 5% CO2.
Transfections were carried out using Lipofectamine reagent (Invitrogen)
according to the manufacturer’s instructions. For live imaging, 12 � 104
cells were plated 24 h before transfection onto 35-mm glass bottom dish
(WillCo-dish GWSt-3522).
For energy depletion studies, cells were incubated for 30 min at 378C in
glucose-free DMEM (Invitrogen) containing 10% FBS and supplemented
with 10 mM sodium azide and 6 mM 2-deoxy-D-glucose (Sigma). After this
treatment, energy depletion medium has been replaced by DMEM used in
normally growing conditions.
Fluorescence microscopy and image analysisCell fluorescence was measured using a Leica TCS SP2 inverted confocal
microscope (Leica Microsystems AG) interfaced with a diode laser (Hama-
matsu Photonics) for excitation at 403 nm, with an Ar laser for excitation at
458, 476, 488 and 514 nm and with a HeNe laser for excitation at 543 and
633 nm. Glass bottom Petri dishes containing transfected cells were
mounted in a thermostated chamber at 378C (Leica Microsystems) and
viewed with a � 40 1.25 NA oil immersion objective (Leica Microsystems).
The images were collected using low excitation power at the sample (10–
20 mW) andmonitoring the emission by means of an Acousto Optical Beam
Splitter (AOBS)-based built-in detectors of the confocal microscope. The
following collection ranges were adopted: 400–450 nm (Hoechst), 500–
550 nm (EGFP and E1GFP) and 580–680 nm (tdTomato). All images have
been subtracted by background signal. Data were analyzed by LEICA IMAGING
package version 2.61, by ORIGIN-PRO7 package and by an implementation of
IGOR-PRO software package.
In vivo FRET measurementsIntegrity of the 110-kDa GFP4 cargo construct was verified in living cells by
FRET technique. We took E1GFP as the donor fluorophore and tdTomato
as the acceptor fluorophore (the corresponding imaging settings were
described above). FRET signal was measured by collecting the tdTomato
fluorescence after excitation at 403 nm. We generated an image of
sensitized emission FRET (corrected pixel-by-pixel for donor and acceptor
bleed-through) with the IMAGEJ PixFRET plug-in (30).
Traffic 2008; 9: 528–539 537
In Vivo Study of Tat Peptide-Targeting Properties
FRAP experiments
Single-point bleach to study nucleus/cytoplasm
shuttlingEach FRAP experiment started with a four-time line-averaged image
(prebleach) of the cell followed by a single-point bleach (nonscanning)
near the center of the nucleus with laser pulse at full power (150 mW) for
the minimum time required to photobleach most of the nuclear fluores-
cence. Fluorescence recovery was measured by starting a time-lapse
acquisition within few milliseconds from the end of bleaching. Each
image of the recovery was four times line averaged, image size was
512 � 512 pixels and scan speed was usually set to 400 Hz. The pinhole size
was set to the optimal value of 1.0 Airy (corresponding to a 81.44-mm confocal
aperture).
According to the mathematical description of our nucleocytoplasmic
exchange model (Supplementary Information), collected FRAP curves for
both compartments were fitted to the monoexponential equations (eqns 15
and 16 in Supplementary Information):
FC ¼ FNC þ ðF0
C � FNC Þ � exp ð�t=tÞ
FN ¼ FNN þ ðF0
N � FNN Þ � exp ð�t=tÞ
where FC and FN are the fluorescence values at time t of cytoplasm and
nucleoplasm, respectively. The three fitting parameters in each equation
refer to the asymptotic fluorescence (FNC , FN
N ), the fluorescence immedi-
ately after bleaching (F0C , F
0N) and the time constant of exponential recovery
(t). Before fitting, the experimental values of fluorescence were normalized
by the fluorescence of the entire cell at the same time in order to minimize
the effect of cell motility and defocusing on the recovery curves and to
correct for bleaching caused by imaging (31). Moreover, data were
normalized by prebleach fluorescence values in order to verify the presence
of an immobile fraction of fluorescent molecules within the bleached
compartment.
The ratio between the protein concentration in the nucleoplasm and
cytoplasm at equilibrium (Keq) was determined by taking the ratio between
the nuclear (FpbN ) and cytoplasmic fluorescence (Fpb
C ) before bleaching, i.e.
Keq ¼ FpbN =Fpb
C . Nuclear volume (VN) was calculated by assuming an
ellipsoid shape for the nucleus with semiaxes dx, dy and dz by means of
the formula VN ¼ (4/3) � p � dx � dy � dz. The three axes were estimated
from confocal images of nucleus, and in most cases, we set dz equal to dy,
the smallest semiaxis in the horizontal plane.
Readers are referred to the Supplementary Information for equations 14,
17, 18 and 19 that were used to obtain the cytoplasmic volume VC, the
passive permeation parameters kDN and the variability range of the active
permeation parameter kAT from t, Keq and VN.
Uniform disk bleach to study protein mobility
within cytoplasmFRAP measurements were carried out as described in Cardarelli et al. (20).
Briefly, a circle area of 3.5-mm radius within the cytoplasm was photo-
bleached using 476-, 488- and 514-nm light at maximum power (87, 350,
350 mW, respectively), and fluorescence recovery was then monitored at
220-millisecond intervals for about 8 seconds using 488-nm excitation at
10 mW. Recovery curves have been normalized by a prior time-lapse
imaging of the same region without the bleaching step to verify the
presence of an immobile fraction of fluorescent molecules within cyto-
plasm. Collected data were analyzed as described in Cardarelli et al. (20)
from which we derived the effective diffusion coefficient in the cytoplasm
(DC) and the bleaching parameter [K0, related to the specific bleach rate of
the fluorophore and zoom setting of the Confocal Scanning Laser Micro-
scope (CSLM) (32)].
Line bleach to study differences in protein mobility
between compartmentsIn these measures, we performed the bleach along a line (covering both
nucleus and cytoplasm) for 90 milliseconds with 488-nm laser pulse at full
power. Fluorescence recovery was imaged along the same line at high
frequency (400 Hz). Collected data were corrected for background and
normalized by a prior time-lapse imaging of the same region without the
bleaching step. The recovery of all imaged cells was fitted to a double
exponential equation to provide an empirical quantification of the time
dependence of the process. We took the slower time constant value as the
representative dynamic parameter of intracompartment mobility because
it was calculated on a larger number of reliable recovery points. The dif-
ference in mobility between compartments was calculated by taking the
ratio between nuclear and cytoplasmic characteristic recovery rates (RM
parameter). Under the assumption that the mobility and diffusivity are
inversely related, we stated RM ¼ DC /DN where DN is the diffusion co-
efficient in the nucleus.
Acknowledgments
We thank Roger Y. Tsien (University of California, CA, USA) for kindly making
available the tdTomato template. Authors also gratefully acknowledge partial
financial support by the Italian Ministry for University and Research (FIRB no.
RBLA03ER38) and by Fondazione Monte dei Paschi di Siena.
Supplementary Material
Supplementary Information: Mathematical model of nuclear/cytoplasmic
exchange in a FRAP experiment.
Supplemental materials are available as part of the online article at http://
www.blackwell-synergy.com
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