TRPV1 acts as a synaptic protein and regulates vesicle recycling.

Research Article 2045
Introduction
Transient receptor potential vanilloid subtype 1 (TRPV1),
alternatively known as capsaicin receptor, is a member of the TRP
superfamily (Caterina et al., 1997). It can be activated by different
exogenous and endogenous ligands, and also by temperature
(Caterina et al., 1997). Although previously known to be expressed
exclusively in the dorsal root ganglion (DRG) neurons, later studies
revealed a widespread expression of TRPV1 in brain, albeit at a
much lower levels (Tóth et al., 2005; Roberts et al., 2004; Starowicz
et al., 2008). Comparative immunohistochemical analyses of
knockout and wild-type animals also confirmed that TRPV1 is
present in the brain (Cristino et al., 2006). Endogenous expression
of TRPV1 in different parts of the brain, in the spinal cord and in
peripheral nociceptive neurons (Steenland et al., 2006; Kauer and
Gibson, 2009) have been interpreted as showing involvement of
TRPV1 in cognition, pain perception and in neuropsychiatric
disorders (Di Marzo et al., 2008). Indeed, TRPV1 receptor in the
central nervous system is a target for TRPV1-antagonist-mediated
analgesia (Cui et al., 2006). In addition, TRPV1 knockout mice
have reduced anxiety and conditioned fear responses (Marsch et
al., 2007), reduced hippocampal long-term potentiation (LTP), and
reduced long-term depression (LTD) (Gibson et al., 2008).
Moreover, pharmacological inhibition of TRPV1 is known to
increase anxiety-like behaviors in rodents (Micale et al., 2009;
Aguiar et al., 2009). On the basis of the finding that neurogenesis
is significantly reduced in TRPV1 and cannabinoid receptor 1
(CB1) double-knockout animals (Cb1–/–, Trpv1–/–), it has been
postulated that TRPV1 is involved in neurogenesis (Jin et al.,
2004).
Several studies have suggested that TRPV1 is involved in
synaptic transmission, neurotransmitter release and plasticity (Kauer
and Gibson, 2009; Maione et al., 2009; Li, H. B. et al., 2008;
Marinelli et al., 2007; Starowicz et al., 2007b; Starowicz et al.,
2008). Involvement of TRPV1 in glutamatergic synaptic
transmission has been documented for rat sensory neurons and
also for the input into dorsolateral periaqueductal gray neurons
(Sikand and Premkumar, 2007; Medvedeva et al., 2008; Xing and
Li, 2007). Similarly, activation of TRPV1 is necessary and sufficient
to trigger LTD at glutamatergic synapses in the hippocampus and
the developing superior colliculus (Gibson et al., 2008; Alter and
Gereau, 2008; Maione et al., 2009). Capsaicin has been shown to
alter synaptic transmission in the rat medial preoptic nucleus, a
hypothalamic area that regulates body temperature (Kauer and
Gibson, 2009; Karlsson et al., 2005). N-arachidonoyl-dopamine
(NADA), an agonist for TRPV1, is known to modulate the
presynaptic Ca2+ levels and transmitter release in the hippocampus
(Huang et al., 2002; Starowicz et al., 2007b; Tóth et al., 2009;
Köfalvi et al., 2007). Although all these reports support a function
of TRPV1 at synapses, direct evidence for the presence of TRPV1
in synapses is lacking. It is also not clear whether TRPV1 is
associated with presynaptic and/or postsynaptic structures.
Moreover, the issues of TRPV1-mediated modulation of synaptic
functions and transmission are poorly understood.
Previously, we reported that TRPV1 interacts with microtubules
and regulates the function of growth cones by cytoskeletal
reorganization (Goswami et al., 2004; Goswami et al., 2006;
Goswami et al., 2007a; Goswami et al., 2007b). Expression of
TRPV1 in DRG-derived F11 cells, a fusion cell line developed
TRPV1 acts as a synaptic protein and regulates
vesicle recycling
Chandan Goswami1,2,*,‡, Nils Rademacher1, Karl-Heinz Smalla3, Vera Kalscheuer1, Hans-Hilger Ropers1,
Eckart D. Gundelfinger3 and Tim Hucho1
1Signal Transduction in Pain and Mental Retardation, Department for Molecular Human Genetics, Max-Planck Institute for Molecular Genetics
Ihnestrasse 73, 14195 Berlin, Germany
2School of Biological Sciences, National Institute of Science Education and Research (NISER), Institute of Physics Campus, Bhubaneswar,
Orissa 751005, India
3Leibniz Institute for Neurobiology, Department of Neurochemistry/Molecular Biology, Brenneckestrasse 6, 39118 Magdeburg, Germany
*Author for correspondence (chandan@niser.ac.in)
Accepted 1 March 2010
Journal of Cell Science 123, 2045-2057
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.065144
Summary
Electrophysiological studies demonstrate that transient receptor potential vanilloid subtype 1 (TRPV1) is involved in neuronal
transmission. Although it is expressed in the peripheral as well as the central nervous system, the questions remain whether TRPV1
is present in synaptic structures and whether it is involved in synaptic processes. In the present study we gathered evidence that TRPV1
can be detected in spines of cortical neurons, that it colocalizes with both pre- and postsynaptic proteins, and that it regulates spine
morphology. Moreover, TRPV1 is also present in biochemically prepared synaptosomes endogenously. In F11 cells, a cell line derived
from dorsal-root-ganglion neurons, TRPV1 is enriched in the tips of elongated filopodia and also at sites of cell-cell contact. In
addition, we also detected TRPV1 in synaptic transport vesicles, and in transport packets within filopodia and neurites. Using FM4-
64 dye, we demonstrate that recycling and/or fusion of these vesicles can be rapidly modulated by TRPV1 activation, leading to rapid
reorganization of filopodial structure. These data suggest that TRPV1 is involved in processes such as neuronal network formation,
synapse modulation and release of synaptic transmitters.
Key words: Capsaicin receptor, Synapse, Active zone, FM4-64 dye, Synaptic-vesicle recycling
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JCS ePress online publication date 18 May 2010

from rat DRG neurons and mouse neuroblastoma cells, results in
increased neuritogenesis and the formation of extensive filopodial
structures (Goswami and Hucho, 2007). Surprisingly, most of these
filopodia contain elevated levels of TRPV1 at their tips and
morphologically resemble elongated club-shaped spines. In the
present study we demonstrate that TRPV1 is present in pre- and
postsynaptic structures, and that it is transported to synaptic sites
by synaptic transport packets. We also provide evidence that TRPV1
regulates filopodial dynamics and vesicle recycling, processes that
are important for synaptogenesis.
Results
TRPV1 localizes to the spines of primary cortical neurons
and is present in synaptic protein preparations
On the basis of our previous work (Goswami and Hucho, 2007),
we hypothesized the presence of TRPV1 in synapses. Primary
cortical neurons were employed to test this hypothesis. Because
the expression level of endogenous TRPV1 in these neurons was
too low for immunocytochemical analysis, we expressed TRPV1
for 6 hours in cortical neurons and analyzed its localization. In
spite of the short expression period, TRPV1 became enriched in
distinct spots resembling synapses (Fig. 1A). To test whether these
puncta represent dendritic spines carrying synapses, we co-stained
TRPV1 with endogenous pre- and postsynaptic markers. TRPV1
colocalizes with endogenous pre- and postsynaptic marker proteins,
including the active-zone protein Bassoon and postsynaptic
elements such as GluR2-containing AMPA receptors, NMDA
receptors and PSD95 (Fig. 1A,B). We also observed that TRPV1
colocalizes with ProSAP1 (also known as Shank2; a protein that is
mainly present in the postsynaptic density) in distinct punctate
spots (Fig. 1C). We also noted that TRPV1 localizes in both
MAP2a/b-positive as well as MAP2a/b-negative neurites (Fig. 1D),
indicating that TRPV1 is present in both the axon and dendritic
structures. Altogether, these data indicate that TRPV1 colocalizes
with both pre- and postsynaptic marker proteins in these structures,
as visualized by confocal microscopy.
To verify that TRPV1 is associated with synaptic structures, we
purified synaptic proteins from rat spinal cord, where expression
of TRPV1 has been reported (Lewinter et al., 2004). We tested
individual protein fractions for the presence of TRPV1 and by
immunoblot analysis we detected TRPV1 in rat spinal-cord
homogenate, the crude membrane fraction, synaptosomal fraction
and also in the synaptic junctional protein fraction (Fig. 1E).
TRPV1 immunoreactivity is enriched in the synaptic protein
fractions and matched well with the molecular weight attributed to
the monomeric TRPV1. Interestingly, we also observed
immunoreactivities that matched with glycosylated (Fig. 1E, smeary
appearance in lane 3) and dimeric TRPV1 in synaptosomes and in
synaptic junctions, respectively (Fig. 1E). The purity of the fractions
was further confirmed by probing the same fractions for PSD95
and synaptophysin (Fig. 1E).
Activation of TRPV1 alters spine morphology in cortical
neurons
Previously, we reported that activation of TRPV1 elongated
filopodial structures in F11 cells (Goswami and Hucho, 2007).
Therefore, we tested whether brief activation of TRPV1 can also
alter the spine morphology in cortical neurons. To achieve this
objective, neurons were treated with NADA (0.2 M, 3 minutes),
an endogenous ligand of TRPV1 (Huang et al., 2002). We
observed that a large number of spines developed from TRPV1-
expressing neurons were elongated (Fig. 2A). Spines from neurons
that did not express TRPV1 did not display morphological
changes upon NADA application. To confirm that the NADA-
induced spine elongation is indeed due to TRPV1 activation, we
preincubated cortical neurons with the TRPV1-specific inhibitor
5�-iodoresiniferatoxin (5�I-RTX; 1 M, 10 minutes) before
applying NADA (0.2 M, 3 minutes). Neither elongation nor
morphological alteration of spines was observed in TRPV1-
expressing neurons (Fig. 2B). We quantified and plotted the spine
lengths in ascending order from neurons that express TRPV1
after the different drug treatments. A clear shift in the spine
length after NADA treatment was observed when compared with
the spines in control conditions or after NADA treatment in the
presence of 5�I-RTX (Fig. 2C). The spines from NADA-treated
neurons were longer (average length 3.37 m, s.e.m. 0.16 m)
than those in non-stimulated control neurons (average length
1.84 m, s.e.m. 0.1137 m) (Fig. 2D). By contrast, spines from
neurons that were pretreated with 5�I-RTX prior to NADA
application were even shorter than the spines from non-stimulated
control neurons (average length 1.28 m, s.e.m. 0.626 m).
Interestingly, the significant (P~0.05) shortening of spines in this
latter condition suggests that spontaneous tonic activation of
TRPV1 might play a role in the regulation of spine length
(discussed below). Our results suggest that activation of TRPV1
by NADA promotes spine elongation and that this can be blocked
by 5�I-RTX.
TRPV1 and synaptic proteins are enriched at the tip of the
elongated filopodial structures
Previously, we observed that the majority of the TRPV1-induced
filopodial tips of F11 cells are club-shaped and therefore
morphologically resemble the elongated dendritic spines developed
from primary neurons (Goswami and Hucho, 2007). We therefore
explored whether these club-shaped filopodial structures might
indeed constitute synaptic sites. For further experiments, we used
F11 cells as a test system (Platika et al., 1985). These cells reflect
several properties of primary DRG neurons, but do not express
TRPV1 endogenously. Thus, this cell line offers the possibility to
study the effects of heterologously expressed TRPV1 on
synaptogenesis.
As observed in our earlier experiments (Goswami and Hucho,
2007), expression of TRPV1-GFP induced massive filopodia
development. In the current study, even when using low expression
levels of TRPV1-GFP, we observed fluorescence along neurites
and enrichment at the tip of filopodial structures within 6 hours.
By contrast, when overexpressing soluble mCherry, we failed to
observe such an enrichment of mCherry in filopodial structures
(Fig. 3A; supplementary material Fig. S1), indicating the specificity
of the TRPV1-GFP enrichment at the filopodial tips.
Furthermore, we tested whether TRPV1-positive filopodial
tips contain synaptic proteins. To this end we expressed Bassoon-
GFP (a presynaptic marker) or PSD-Zip45-GFP and GFP-PSD95
(markers for postsynaptic side) along with TRPV1. All three
markers were highly enriched in TRPV1-induced filopodial tips
(Fig. 3B), suggesting that TRPV1 accumulates in structures
resembling both pre- and postsynapses. Previously, two
presynaptic vesicular proteins, namely Snapin and synaptotagmin
have been reported to interact with TRPV1 (Morenilla-Palao et
al., 2004). Therefore, we assessed whether synaptic-vesicle
markers also colocalize with TRPV1. Indeed, both Snapin-GFP
and synaptophysin-GFP were found to colocalize with TRPV1 in
2046 Journal of Cell Science 123 (12)
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filopodial tips (Fig. 3B). The tips of spines as well as presynaptic
terminals contain endocytic zones at which clathrin-mediated
endocytosis takes place (Blanpied et al., 2002; Gundelfinger et
al., 2003). Thus, we coexpressed TRPV1 and clathrin-GFP in
F11 cells, which revealed that both recombinant proteins
colocalize in filopodial tips (Fig. 3B). These results indicate that
TRPV1-induced filopodial tips contain synaptic scaffold and
vesicular proteins, suggesting that these tips can act as synaptic
terminals.
To confirm that localization of TRPV1-GFP in filopodia is an
active process, we performed fluorescence recovery after
photobleaching (FRAP) experiments. We observed that TRPV1-
GFP fluorescence recovers rapidly (within 3 minutes) in filopodia.
Within individual filopodia, we noted both anterograde (Fig. 3C)
2047TRPV1 in the synaptic structure
Fig. 1. TRPV1 is present at synaptic sites. (A)Images of cortical neurons transiently expressing TRPV1 at a low level. Neurons were immunostained for TRPV1
(red) and for GluR2 (green, top panel), ProSAP1 (Shank2; green, middle panel) or Bassoon (green, lower panel). The immunofluorescence images were
superimposed to the phase-contrast images. TRPV1 localizes as distinct punctuate spots that colocalize with GluR2, ProSAP1 and Bassoon. Arrows indicate
TRPV1-positive spots that colocalize with the other marker proteins. Scale bars: 10m. (B)TRPV1 colocalizes with both pre- and postsynaptic proteins. Enlarged
areas of neurite mesh (as indicated by a white box in A) immunostained for TRPV1 (red) and different synaptic proteins (green) are shown. Merged images are
superimposed to the phase-contrast images (left panel). Enrichment of TRPV1 in spines is shown in pseudocolor (‘Inten’; right most panel). Arrows indicate the
presence of TRPV1 in these spots. Scale bars: 2m. (C,D)TRPV1 localizes both in the axon and dendrites. Shown are the images of cortical neurons transiently
expressing TRPV1. Neurons were immunostained for TRPV1 (red) and for MAP2 (green). Arrows indicate the TRPV1-positive neurites. Note that TRPV1
localizes both in MAP2-positive neurites (C) and TRPV1-negative neurites (D). Scale bars: 10m. (E)TRPV1 is present in synaptosomal fractions. Tissue
homogenates from adult rat spinal cord (lane 1), membrane fraction (lane 2), synaptosomal fraction (lane 3) and synaptic junctional protein fraction (lane 4) were
probed for TRPV1, PSD95 and synaptophysin. Arrows indicate monomeric and dimeric TRPV1. TRPV1 is enriched in synaptosomes and in synaptic junctions.
Approximately 25-g protein samples were loaded in each lane. The 37-kDa band at lane 4 is most probably a degradation product.
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and retrograde (not shown) movements of TRPV1-GFP in discrete
packets after photobleaching, suggesting that TRPV1-GFP
localization within filopodia is not due to passive diffusion within
the membrane. Taken together, these results indicate that the
enrichment of TRPV1 at the filopodial tips is specific and most
probably is an active process.
TRPV1-enriched filopodial tips at sites of cell-cell contact
contain synaptic proteins
Filopodia can establish cell-cell contacts that can either mature
and become consolidated or can disappear again. Previously, we
reported that TRPV1-enriched filopodial structures are found at
established cell-cell contacts (Goswami and Hucho, 2007). To
test whether these filopodia-based cell-cell contacts can contain
synaptic marker proteins and TRPV1, we coexpressed TRPV1
with postsynaptic, presynaptic and synaptic vesicular proteins in
F11 cells. Reconstructed three-dimensional (3D) images of these
filopodia confirmed that cell-contact-forming filopodial tips
contained TRPV1 as well as the postsynaptic proteins GFP-
PSD95, PSD-Zip45-GFP or the presynaptic protein Bassoon-GFP
(Fig. 4). Moreover, clathrin-GFP, Snapin-GFP, synaptophysin-
GFP and synapsin-GFP were present at these contacts (Fig. 4),
suggesting that filopodial tips can form consolidated synapse-
like structures.
TRPV1 is transported to neurite endings and at filopodial
bases by transport packets
If TRPV1 is a synaptic protein, then it will be transported within
neurites by transport packets. We observed that, apart from some
diffuse staining, TRPV1-GFP mainly appears as distinct puncta all
along the neurites. These fluorescent structures containing TRPV1-
GFP were variable in size and shape and were much larger than
common transport vesicles. Therefore, we explored whether these
structures represent the ‘synaptic transport packets’ (Ahmari et al.,
2000), which are characterized by the presence of synaptic proteins
as they transport synaptic components to their site of destination.
To test this hypothesis, we expressed TRPV1 along with the
synaptic-vesicle protein Snapin-GFP and performed
immunostaining. We observed that TRPV1 and Snapin-GFP
colocalize in punctate spots within long neurites (Fig. 5A). Similar
co-distribution of postsynaptic scaffold proteins such as GFP-
PSD95 or PSD-Zip45-GFP along with TRPV1 was also observed
in such punctate spots (data not shown).
Because transport packets are mobile within neurites, we tested
whether these TRPV1-GFP-containing spots are also mobile. To
achieve these objectives, we coexpressed TRPV1-GFP and
synaptophysin-mCherry in F11 cells and performed live-cell
imaging (Fig. 5B). TRPV1-GFP colocalizes with synaptophysin-
mCherry in neurites as distinct punctate spots. These spots also
move fast and travel a long distance at a stretch. It was observed
that these spots can move in both directions along neurites. The co-
migration of TRPV1-GFP and synaptophysin-mCherry within the
neurite corroborates the notion that these structures are indeed
transport packets (Fig. 5B). Remarkably, these transport packets
were frequently located at the base of short filopodia (Fig. 5C).
There, they were observed to be stationary for several minutes to
hours, especially the bigger units (~0.5 m), which remained stable
and static (either did not move at all or moved very little in both
the directions). However, if moving and in close proximity to each
other, then these bigger units often fused. For the movement of
TRPV1-positive units, we determined a maximum velocity of 0.45
m/second.
TRPV1-expressing cells reveal fast uptake of FM4-64 dye
Several reports suggest that agonists of TRPV1 stimulate
neurotransmitter release (Li and Eisenach, 2001; Schmid et al.,
1998; Xing and Li, 2007; Sikand and Premkumar, 2007;
Medvedeva et al., 2008; Gibson et al., 2008; Alter and Gereau,
2008; Marinelli et al., 2007; Starowicz et al., 2007a). It is a well
known fact that neurotransmitter release is associated with vesicle
recycling. Therefore, we studied uptake of FM4-64 dye at TRPV1-
positive sites.
We decided to test whether TRPV1-GFP-expressing F11 cells
can incorporate the dye. Indeed, the uptake of FM4-64 dye was
very fast. If used at the normally reported concentration of 0.1
g/ml of final dye solution, the cells showed almost instant loading
(often within 30 seconds). However, also after lowering the dye
concentration to as low as 0.5 ng/ml, loading of FM4-64 in TRPV1-
GFP-expressing populations was observed within a few (1-2)
minutes (supplementary material Fig. S2A). At this concentration,
mock-transfected control cells showed nearly no incorporation
(supplementary material Fig. S2B).
FM4-64 is a lipophilic dye and the fast incorporation of this dye
even in the absence of Ca2+ was quite unexpected. Therefore, we
tested the specificity of the uptake. If the uptake is specific and
actively driven it would have been possible to block this dye
2048 Journal of Cell Science 123 (12)
Fig. 2. TRPV1 affects spine length after activation. (A)Activation of
TRPV1 alters the spine morphology of cortical neurons. Examples of cortical
neurons expressing TRPV1 were stained for TRPV1 (red) and actin (green;
stained with phalloidin). Neurons stimulated (3 minutes) by NADA (0.2M)
or by NADA in the presence of the antagonist 5�I-RTX (1M) are shown. The
arrows indicate elongated spines. Scale bars: 10m. (B)Spine lengths from
cortical neurons expressing TRPV1 and treated with NADA (blue), NADA in
the presence of 5�I-RTX (red) or unstimulated (black) were arranged in
ascending order. Each spot indicate length of individual spines in y-axis.
(C)The average length of spines from TRPV1-expressing neurons treated with
NADA (blue; average 3.37m, s.e.m. 0.16m, n223, five neurons), NADA
in presence of 5�I-RTX (red; average 1.28m, s.e.m. 0.626m, n193, seven
neurons) or unstimulated (black; average 1.84m, s.e.m. 0.1137m, n232,
seven neurons) are shown (P<0.0001, unpaired one-tailed Student’s t-test).
Significant (*) differences are indicated.Jo
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uptake by pharmacological manipulation of the actin cytoskeleton,
which plays an important role in membrane trafficking processes
(Gundelfinger et al., 2003; Girao et al., 2008). Therefore, we
explored whether disruption of the actin cytoskeleton can block
uptake of the FM4-64 dye. Loading of FM4-64 was reduced upon
pre-treatment of TRPV1-GFP-expressing F11 cells with
cytochalasin D (supplementary material Fig. S3A), indicating that
the uptake was indeed actin dependent. To further confirm the
specificity, we monitored dye uptake at lower temperatures, which
also blocks active endocytosis and therefore interferes with dye
uptake. When added to TRPV1-GFP-expressing F11 cells at 4°C,
the uptake of FM4-64 was almost undetectable (supplementary
material Fig. S3B). Fluorescence-activated cell sorting (FACS)
measurements were employed to quantify the effects. As shown in
Fig. 6, a clear shift in FM4-64 uptake was observed in unstimulated
TRPV1-GFP-expressing F11 cells when compared with the F11
cells that were either non-transfected or express GFP only. TRPV1-
GFP-expressing cells incorporated 2.35-fold (P0.0001) or 1.69-
fold (P0.00053) more FM4-64 as compared with F11 populations
that were either non-transfected or expressed only GFP, respectively
(Fig. 6B). Similarly, TRPV1-GFP-expressing cells showed a
significant reduction (0.33-fold, P0.0004) of dye uptake if these
cells were treated with cytochalasin D. Cold treatment of TRPV1-
GFP-expressing F11 cells also resulted in a 0.48-fold reduction
(P0.0014) in dye uptake as compared with unstimulated TRPV1-
GFP cells. These cold-treated cells did not show a significant
difference in comparison with the non-transfected cells (0.22-fold
increase, P0.2259). Uptake of FM4-64 dye varies in different
other conditions also (supplementary material Fig. S4). From these
results we conclude that the fast and differential uptake of FM4-
64 dye in TRPV1-expressing cells reflects an active endocytotic
process that is much enhanced by the presence of TRPV1.
TRPV1-transfected F11 cells take up FM4-64 at distinct
sites enriched for TRPV1
In this study we also tried to identify the subcellular site of FM4-
64 uptake in TRPV1-transfected F11 cells. Specific and rapid
labeling (within 1-2 minutes) with FM4-64 was observed as
distinct spots along neurites (Fig. 6C). Often these spots also
contained TRPV1-GFP and appeared as donut-like structures
resembling the topology of synaptic-vesicle clusters (see
Discussion). These spots were mostly juxtaposed to the base of
small filopodia and were clearly visible as distinct bright spots
(Fig. 6C). In addition, FM4-64 loading was observed at elongated
filopodia (Fig. 6D). We also explored whether the club-shaped
filopodial tips can incorporate this dye. Indeed, a few (but not all)
TRPV1-GFP-induced club-shaped filopodial tips contained FM4-
64 (Fig. 6E; supplementary material Fig. S5A). Specific FM4-64
labeling was also observed at some areas of the cell body and
growth cones (data not shown). The fast labeling of FM4-64 to
limited cellular regions suggests that these specific structures
harbor fast-recycling vesicles.
2049TRPV1 in the synaptic structure
Fig. 3. TRPV1 localizes at filopodial tips. (A)Shown
are the enlarged views of filopodia developed from a
live F11 cell expressing TRPV1-GFP (green) or
mCherry (red) at low levels. Specific enrichment of
TRPV1-GFP (green) at filopodial tips (indicated by
arrows) is observed, whereas mCherry is mainly
restricted to the neurites. Merged fluorescence image
superimposed with phase-contrast image is shown on
the right side. The corresponding intensity profiles are
provided in the lower part. Scale bars: 2m.
(B)TRPV1 colocalizes with presynaptic, postsynaptic
and synaptic vesicular proteins at filopodial tips. Images
represent enlarged views of filopodial structures.
TRPV1 (red) and different GFP-tagged synaptic
markers (green) were expressed in F11 cells. In the left
panel, fluorescence images are superimposed to the
phase-contrast images. Arrows indicate the presence of
synaptic-marker proteins at filopodial tips. Scale bars:
5m. (C)Fluorescence recovery after photobleaching
(FRAP) of TRPV1-GFP within individual filopodia. In
the left panel, fluorescence images are superimposed to
the phase-contrast images. Corresponding intensities are
provided in the right side. Arrows indicate the
movement of discrete TRPV1-GFP particles within the
filopodia (an enlarged view is provided in the 30-
second inset). Scale bars: 2m.
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