Rapid disassembly of dynamic microtubules upon activation of thecapsaicin receptor TRPV1
C. Goswami,* M. Dreger,� H. Otto,* B. Schwappach� and F. Hucho*
*Freie Universitat Berlin, Institut fur Chemie/Biochemie, Berlin, Germany
�University Laboratory of Physiology, Oxford, UK
�ZMBH, Heidelberg, Germany
Abstract
The transmission of pain signalling involves the cytoskeleton,
but mechanistically this is poorly understood. We recently
demonstrated that the capsaicin receptor TRPV1, a non-
selective cation channel expressed by nociceptors that is
capable of detecting multiple pain-producing stimuli, directly
interacts with the tubulin cytoskeleton. We hypothesized that
the tubulin cytoskeleton is a downstream effector of TRPV1
activation. Here we show that activation of TRPV1 results in
the rapid disassembly of microtubules, but not of the actin or
neurofilament cytoskeletons. TRPV1 activation mainly affects
dynamic microtubules that contain tyrosinated tubulins,
whereas stable microtubules are apparently unaffected. The
C-terminal fragment of TRPV1 exerts a stabilizing effect on
microtubules when over-expressed in F11 cells. These find-
ings suggest that TRPV1 activation may contribute to cyto-
skeleton remodelling and so influence nociception.
Keywords: capsaicin, cytoskeleton, dynamic microtubules,
pain, TRPV1, vanilloid receptor 1.
J. Neurochem. (2006) 96, 254–266.
The capsaicin receptor TRPV1 is a member of the transientreceptor potential channel family and the founding member ofthe TRPV subfamily (Caterina et al. 1997). TRPV1 acts as anon-selective cation channel and is activated by capsaicin,various vanilloids, low pH and also by heat (Caterina et al.1997; Jordt et al. 2000). Some endogenous compounds suchas n-acyl-dopamines and lipid-derived compounds of thelipoxygenase pathway also activate TRPV1 (Hwang et al.2000; Huang et al. 2002). Furthermore, TRPV1 can act as anintegrator of polymodal stimuli (Tominaga et al. 1998), and itsparticipation in pain transmission has been established usingknockout mice (Caterina et al. 2000; Davis et al. 2000).
TRPV1 forms a homotetramer with six transmembranesequences per monomer. Both the N- and the C-terminus ofTRPV1 are located in the cytoplasm. They are subject toregulatory post-translational modifications (reviewed byCortright and Szallasi 2004), and supposedly interact witha number of proteins. Several reports suggest that cytoskel-etal structures play an important role in pain pathways(Bhave and Gereau 2003; Dina et al. 2003). Microtubule-active reagents, such as vinca alkaloids and taxol, are capableof changing nociceptor morphology and responsiveness(Topp et al. 2000; Alessandri Haber et al. 2004).
We recently reported a direct interaction of the C-terminaldomain of TRPV1 with tubulins and hypothesized that the
microtubule cytoskeleton is a downstream effector of TRPV1activation (Goswami et al. 2004). We now aim to furthersubstantiate this hypothesis. We report immunocytochemicaland biochemical data indicating that activation of TRPV1results in selective destabilization of dynamic microtubules,whereas the C-terminal fragment of TRPV1 alone clearlyexerts a stabilizing effect on the microtubules. The actin orneurofilament cytoskeleton, however, is not affected byTRPV1 activation.
Materials and methods
Antibodies and reagents
Digitonin was purchased from Calbiochem (San Diego, CA,
USA). The TRPV1 agonist resiniferatoxin (RTX), antagonist
Received July 7, 2005; revised manuscript received September 13, 2005;accepted September 13, 2005.Address correspondence and reprint requests to F. Hucho, Freie
Universitat Berlin, Institut fur Chemie/Biochemie, Thielallee 63, 14195Berlin, Germany. E-mail: [email protected] used: HBSS, Hank’s balanced salt solution; HEK,
human embryonic kidney; I-RTX, 5¢-iodo-resiniferatoxin; MBP, Maltosebinding protein; MTOC, microtubule organizing centre; PBS, phosphate-buffered saline; PFA, Paraformaldehyde; RTX, resiniferatoxin; TRPV1-Ct, C-terminal sequence of TRPV1.
Journal of Neurochemistry, 2006, 96, 254–266 doi:10.1111/j.1471-4159.2005.03551.x
� 2005 The Authors254 Journal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
5¢-iodo-resiniferatoxin (I-RTX) and the microtubule-depolymerizing
drug nocodazole were purchased from Sigma (Deisenhofen,
Germany). Mouse monoclonal antibodies anti-a-tubulin (clone
DM1A), anti-b-tubulin (clone D66), anti-b-tubulin class III (clone
SDL.3D10), anti-acetylated tubulin (clone 6-11B1), anti-tyrosinated
tubulin (clone TUB1A2), anti-polyglutamylated tubulin (clone B3),
anti-c-tubulin (clone GTU-88) and anti-160-Kd neurofilament
(clone NN18) were all purchased from Sigma. The rat monoclonal
antibody YL1/2 was purchased from AbCam Ltd (Cambridge, UK).
Mouse monoclonal anti-actin antibody (clone JLA20) was purchased
from Oncogene (Cambridge, MA, USA). Affinity-purified rabbit
polyclonal antibody against de-tyrosinated tubulin (glu tubulin),
mouse monoclonal antibody against the 200-kDa neurofilament
(clone RT97) and mouse monoclonal antibody against tau (clone
Tau-1 PC1C6)were purchased fromChemicon (Chandlers Ford,UK).
Rabbit polyclonal anti-N-terminal TRPV1 antibody and the respect-
ive blocking peptide (sequence M1EQRASLDSEESESPPQENSC21,
corresponding to the first 21 amino acid residues of TRPV1, were
from Affinity Bio Reagents (Golden, CO, USA) and from Alexis
Biochemicals (San Diego, CA, USA) respectively. Goat polyclonal
anti-TRPV1 antibody raised against the C-terminus of TRPV1 was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Alexa-594-labelled phalloidin, alexa-594-labelled anti-rat IgG
secondary antibody and alexa-594-labelled anti-mouse IgG secondary
antibody were purchased from Molecular Probes (Invitrogen,
Karlsruhe, Germany). Cy2-labelled anti-goat and Cy2-labelled
anti-rabbit IgG were purchased from ; (Hamburg, Germany).
TRPV1 constructs
For mammalian expression, the full-length rat TRPV1 cDNA
subcloned in pcDNA3.1 vector was used (Jahnel et al. 2001). Forthe expression of the C-terminus only, a cDNA fragment representing
only the C-terminal cytoplasmic domain (amino acids 681–838) from
rat TRPV1 was amplified by PCR using the primers 5¢-AT-GGGTGAGACCGTCAACAA-3¢ and 5¢-TTATTTCTCCCCTGG-GACCA-3¢, and subcloned into the vector pcDNA3.1 (Jahnel 2005).
Generation of a stable TRPV1-expressing F11 cell line
The cDNA encoding TRPV1 was subcloned into pBICD4 (Liu et al.2000), employing the EcoRI and NotI restriction sites of the vector.
F11 cells were transduced with retroviral particles obtained from a
triple transfection of human embryonic kidney (HEK)293T cells with
plasmids BICD4-TRPV1, pVPack-GP and pVPack-eco (Stratagene,
La Jolla, CA, USA). CD4-positive cells were stained using a
phycoerythrin-conjugated CD4 antibody (clone EDU-2; Dianova)
and isolated by flow cytometry on a Becton Dickinson FACS Vantage
cell sorter (Heidelberg, Germany). Expression of TRPV1 in this cell
line was confirmed by western blot analysis and immunofluorescence
analysis. This cell line is subsequently referred to as TRPV1-F11 cells.
Cell culture and transfection
F11 cells and TRPV1-F11 cells were cultured in Ham’s F12 medium
(Invitrogen) supplemented with 20% fetal calf serum (Invitrogen).
HEK cells were maintained in Dulbecco’s modified Eagle’s medium
with 10% fetal calf serum. Cells were maintained in a humidified
atmosphere that contained 5% CO2 at 37�C. For transient
transfection, lipofectamine (Invitrogen) was used according to the
manufacturer’s instructions.
Immunocytochemistry
F11, TRPV1-F11 and HEK cells were grown and transfected on
glass coverslips. Two days after seeding or transfection, the cells
were fixed with either 2% paraformaldehyde at room temperature
(25�C) or 80% methanol in phosphate-buffered saline (PBS) at
) 20�C for 10 min, permeabilized with 0.4% Triton X-100 in PBS
for 5 min, followed by incubation with 100 mM glycine dissolved
in PBS for 1 h. The cells were blocked with 5% normal goat
serum or bovine serum albumin. After incubating the cells with the
primary antibody for 1 h at room temperature, the cells were
washed three times with PBS containing 0.1% Tween 20 (PBST)
and incubated with secondary antibody diluted in PBST. The
coverslips were mounted on to glass slides with fluromount G
(Southern biotech, Eching, Germany). All anti-tubulin staining
reported here was done with the YL1/2 antibody unless stated
otherwise. The mouse monoclonal antibody against b-tubulin was
used to study the effect of C-terminal sequence of TRPV1
(TRPV1-Ct) on microtubule stabilization. Alexa-594-labelled phal-
loidin was used to visualize the actin cytoskeleton. Images were
taken on a confocal laser scanning microscope (Axiovert 100M;
Zeiss, Berlin, Germany) with a 63 · objective and analysed using
Zeiss LSM image examiner software.
TRPV1 activation assay
In order to visualize the effect of TRPV1 activation on the
cytoskeleton, F11 cells expressing TRPV1 transiently or TRPV1-
F11 cells were grown on glass coverslips for 2 days. Cells were
washed gently with Hank’s balanced salt solution (HBSS) Invitro-
gen) at room temperature, incubated with HBSS buffer supplemen-
ted with 1 mM CaCl2 and RTX (100 nM) for 1 min, and either fixed
immediately or further extracted with membrane permeabilization
buffer for 1 min before fixation. Membrane permeabilization buffer
contained 50 mM PIPES, pH 6.8, 1 mM EGTA, 0.2 mM MgCl2,
10% glycerol, 50 lg/mL digitonin and completeTM protease
inhibitor cocktail (Roche, Indianapolis, IN, USA). Quick extraction
of cells in this buffer permeabilized the membrane, but cell
morphology essentially retained intact; this method was thus
suitable for observation of the stable cytoskeleton (Lieuvin et al.1994). For blocking the TRPV1, cells were incubated with 1 lMI-RTX for 10 min and activation of TRPV1 by RTX was done in
presence of I-RTX.
For biochemical analysis of the activated cells by western blot
analysis, TRPV1-F11 cells were scraped from the culture vessels,
collected by a brief centrifugation and resuspended in HBSS. An
equal volume of suspension was distributed into different tubes.
Cells were activated by addition of an equal volume of HBSS
supplemented with RTX and CaCl2. In control experiments, an equal
volume of HBSS only was added. To examine the cytoskeleton of the
cells after TRPV1 activation, an equal volume of 2 · membrane
permeabilization buffer was added and mixed gently without
homogenization for 1 min, followed by centrifugal separation of
supernatants and pellets at 150 g for 5 min at room temperature.
Depolymerization of microtubules in cell culture
F11 cells expressing TRPV1-Ct after transfection were incubated
with 1 lM nocodazole for 15 min at 37�C, washed with HBSS at
room temperature, extracted with membrane permeabilization buffer
and fixed with Paraformaldehyde (PFA).
Modulation of microtubules by TRPV1 255
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
Western blot analysis
The amount of protein present in the extracts was determined by the
bicinchoninic acid method (Pierce, Rockford, IL, USA). For
analysis of the proteins, extracts were resolved by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis on 10% gels according
to the method of Laemmli (1970). For western blot analysis,
proteins were transferred to a nitrocellulose membrane by the
semidry method, and membranes were blocked with 5% fat-free dry
milk powder suspended in Tris-buffered saline (TBST; 20 mM Tris-
HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20). Blocked membranes
were incubated with primary antibody in TBST buffer for 1 h,
washed three times with TBST buffer, then incubated with
secondary antibody in TBST buffer for 1 h. Finally, the membranes
were washed with TBST and developed with an enhanced
chemiluminescence kit (Amersham Biosciences/GE Healthcare,
Freiburg, Germany).
Results
Activation of TRPV1 alters microtubule cytoskeleton
morphology
We recently reported that the C-terminal cytoplasmicsequence of TRPV1 interacts with tubulin dimers andpolymerized microtubules. It thereby alters some physico-chemical properties of microtubules (Goswami et al. 2004).
To understand the effect of TRPV1 activation on the tubulincytoskeleton in a cellular context, we treated TRPV1-transfected cells with the selective agonist RTX, andmonitored the integrity of cytoskeletal structures after RTXactivation of TRPV1 by indirect immunofluorescence(Fig. 1). We expressed TRPV1 in F11 cells by transienttransfection. The bright immunostaining of TRPV1 in somecells allowed us to distinguish TRPV1-expressing cells fromthe non-transfected cells which showed no immunoreactivityfor TRPV1.
TRPV1 localized to the plasma membrane as distinctpatches as well as to intracellular membranes, in line withprevious reports (Jahnel et al. 2001; Goswami et al. 2004).Both filamentous microtubules and actin fibres were clearlyvisible in non-activated F11 cells over-expressing TRPV1(Fig. S1). Accumulation of some tubulin in the TRPV1-enriched patches and significant co-localization in theseareas were observed in contrast to findings in non-transfectedcells (Fig. S1a). TRPV1-enriched patches were never foundto contain actin (Fig. S1b).
To observe the effect of TRPV1 activation, the receptorwas activated by RTX. Upon activation, the microtubulecytoskeleton was dispersed within 1 min in TRPV1-expres-sing F11 cells (Fig. 1). In these cells, the microtubule
TRPV1i ii iii
NT
NT
T
T
iv v vi
Tubulin Merge
Fig. 1 Activation of TRPV1 affects microtubule structures. Confocal
images of F11 cells (upper panel, i–iii) transiently expressing TRPV1
(indicated by T) or an enlarged area of the same cells (indicated by
white dashed line; lower panel, iv–vi). Non-transfected cells are
indicated as NT. Cells were treated with RTX to activate the TRPV1
channel and subsequently immunostained for TRPV1 (green) and
tubulin (red). TRPV1-expressing cells show a dispersed pattern of
tubulin staining whereas non-expressing cells show normal micro-
tubule staining. Scale bar 10 lm (upper panel) and 5 lm (lower
panel).
256 C. Goswami et al.
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
structure was lost and tubulin immunoreactivity was dis-persed all over the cell body. Non-transfected cells, however,showed normal microtubule structures after RTX treatment(Fig. 1). Microtubules in TRPV1-expressing cells had nor-mal fibrous structure even after RTX activation when thecells had been preincubated with I-RTX, an antagonist ofTRPV1 (data not shown). In contrast, we observed nosignificant change in the actin and neurofilament cytoskel-etons of TRPV1-expressing F11 cells after the sametreatment with RTX (data not shown). When we performedthe same experiment in HEK293 cells transfected withTRPV1, we observed a similar effect on microtubules (datanot shown). These results indicate that it is the activation, butnot the over-expression, of TRPV1 that affects the finemicrotubular structures.
Activation of TRPV1 results in increased tubulin
solubility owing to disassembly of microtubules
The apparent dispersal of microtubules subsequent toactivation of TRPV1 detected by indirect immunofluores-cence suggests that the microtubules may have beendisassembled into small tubulin oligomers and dimers. Toanalyse whether activation of TRPV1 renders an increasedfraction of tubulin soluble, we extracted cells with digitoninin an isotonic buffer immediately after activation by RTX.Under these conditions, elements of the stable cytoskeletonand associated proteins are expected to remain unaffectedwhile all soluble cytoplasmic proteins are extracted. Afterextraction, all cells that expressed TRPV1 showed asignificantly reduced tubulin content (Fig. 2a). Some resid-ual immunostaining for tubulin, which was observed in thecell body, appeared diffuse (Fig. 2a,iv). In some instances,it could be assigned as fragmented microtubules (Fig. S2).However, a perinuclear anti-tubulin immunoreactive struc-ture was retained even after RTX activation (Fig. 2a; seealso Fig. S2). No effect of RTX was observed in non-transfected cells (Fig. 2a). To prove further that theobserved disassembly of microtubules indeed results fromTRPV1 activation, we incubated the cells with antagonist I-RTX for 10 min before activation with RTX and extractionwith digitonin. As expected, I-RTX-treated TRPV1-expres-sing F11 cells did not show loss of microtubules from thecell body after RTX activation and the microtubules had anormal fibrous pattern (Fig. 2b). In addition, TRPV1-transfected, but unstimulated cells displayed a normalmicrotubule structure comparable to that of non-transfectedcells (Fig. 2c). These results indicate that the loss ofmicrotubule structures in the cell body is rapid and occurswithin a minute of TRPV1 activation. After RTX treatmentand digitonin extraction, the actin and neurofilamentcytoskeleton remained unchanged, and visible all over theTRPV1-expressing cells, comparable to that in controlcells (Fig. 2d). This indicates that activation of TRPV1has no effect on actin and neurofilament cytoskeletons.
TRPV1-mediated microtubule dispersal in stably
transfected cells
In order to achieve a homogeneous TRPV1-expressing cellpopulation for biochemical studies, we stably transfected F11cells with TRPV1 cDNA. We obtained stably transfected F11cells by a viral transduction method and subsequent fluores-cence-activated cell sorting, according to a method describedpreviously (Liu et al. 2000). Expression of TRPV1 in this cellline was confirmed by western blot analysis with anti-TRPV1antibody (data not shown) and immunofluorescence analysis.Expression of TRPV1 was detected in all cells by indirectimmunofluorescence analysis with anti-TRPV1 antibody, andthe expression level was much lower than that in transientlytransfected cells (data not shown). Low and homogeneousexpression of TRPV1 in TRPV1-F11 cells allowed us to studybiochemically the effect of TRPV1 activation.
We observed by immunofluorescence analysis that activa-tion of TRPV1 after RTX treatment of TRPV1-F11 cellsresulted in the selective disassembly of microtubules but notof the actin cytoskeleton in the majority of cells (data notshown). This effect was similar to that observed in transientlytransfected cells. Co-staining of actin and microtubules inTRPV1-F11 cells confirmed that the cells that showed adrastic reduction in microtubules in the cell body retained anormal actin cytoskeleton, even after activation of TRPV1followed by detergent extraction (Fig. 3).
To confirm the apparent increase in soluble tubulin subse-quent to activation of TRPV1 by an independent method, weapplied the detergent extraction treatment to the TRPV1-F11cells, but then separated the soluble and insoluble fractions bycentrifugation. We further analysed the soluble and insolublefractions by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis, and determined the abundance of cytoskeletalcompounds across the fractions by western blot analysis (seeflow chart in Fig. 4a). The amount of tubulin increasedsignificantly in the soluble fraction and decreased in theinsoluble fraction after activation of TRPV1 (Fig. 4b). Incontrast, only a small increase in actin and almost no change inneurofilament protein were observed in the soluble andinsoluble fractions following TRPV1 activation comparedwith levels in non-stimulated cells. These results suggest thatthe predominant disassembly of the tubulin cytoskeleton is adownstream effect of TRPV1 activation.
Dynamic microtubule structures are affected by TRPV1
activation
We always observed an area near the perinuclear zone thatretained strong tubulin immunoreactivity even after activa-tion of TRPV1 and extraction with detergent. This findingsuggested that this structure was made of mainly stablemicrotubules that are resistant to Ca2+ influx and detergentextraction. One candidate structure for this is the microtubuleorganizing centre (MTOC), which characteristically containsc-tubulin (Joshi 1993; Oakley and Akkari 1999). To
Modulation of microtubules by TRPV1 257
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
TRPV1(a)
(b)
(c)
(d)
RTX+
Digitonin
i ii iii iv
i ii iii
i ii iii
i ii iii iv
5 µm Non-transfected cell20 µm
20 µm
IRTX +RTX +
Digitonin
Buffer+
Digitonin
RTX+
Digitonin
Tubulin Tubulin Tubulin
TRPV1 Phallodin TRPV1 NF
Fig. 2 Activation, and not expression, of TRPV1 results in the rapid
disassembly of microtubules at specific cell sites. (a) Confocal indirect
immunofluorescence images of F11 cells transiently expressing
TRPV1. Cells were treated with RTX for 1 min to activate the TRPV1
channel, followed by extraction with digitonin in an isotonic buffer.
Cells were immunostained for TRPV1 (green) and tubulin (red) with
specific antibodies. TRPV1-expressing cells (indicated by an arrow)
can be distinguished from non-expressing cells (indicated by an
arrowhead) by means of anti-TRPV1 immunoreactivity (i). Anti-tubulin
immunoreactivity in the TRPV1-expressing F11 cells was much re-
duced compared with that in non-expressing cells (ii). ‘Smeary’ pres-
ence of tubulin all over the cell body except at the perinuclear region of
the MTOC, and complete absence of normal microtubular structures,
are shown in the enlarged area (iii) of TRPV1-expressing cells (indi-
cated by dashed white line in ii). Under the same conditions, RTX had
no effect on non-expressing cells, and normal microtubular structures
were retained (iv). Scale bar 20 lm for (i–ii) and 5 lm for (iii–iv). (b)
Cells were incubated with I-RTX for 10 min before RTX was added to
activate the TRPV1 channel. The cells were further extracted with
digitonin in an isotonic buffer. Immunostaining for TRPV1 (green; i)
and tubulin (red; ii) revealed no change in microtubular structure in
TRPV1-expressing cells (indicated by an arrow). Tubulin immunore-
activity of an enlarged area (indicated by white dashed lines) is shown
on the right (iii). Scale bar 20 lm (for i–ii) and 10 lm for (iii). (c)
Indirect immunofluorescence confocal images of F11 cells expressing
TRPV1 after transient transfection (indicated by arrow) or non-trans-
fected cells (indicated by arrowhead). Cells were treated with buffer
only instead of RTX, extracted with digitonin in isotonic buffer and
immunostained for TRPV1 (green; i) and tubulin (red; ii). Both TRPV1-
expressing as well as non-expressing cells retained normal microtu-
bular structures all over the cell including the neurites. This indicates
that microtubule disassembly did not occur in absence of TRPV1
activation. An enlarged area of a TRPV1-expressing cell (indicated by
dashed white lines in ii) is provided (iii). Scale bar 20 lm (for i–iii) and
5 lm for (iv). (d) Confocal indirect immunofluorescence images of F11
cells transiently expressing TRPV1 and treated with RTX, followed by
extraction with digitonin in an isotonic buffer. Cells were immuno-
stained for TRPV1 (green) and actin (red) with alexa-594-labelled
phalloidin (i–ii) or TRPV1 and neurofilament (NF) (red) with specific
antibodies (iii–iv). The patterns of the actin (ii) and the neurofilament
(iv) cytoskeleton remained unchanged in the TRPV1-expressing F11
cells (indicated by white arrows) even after RTX activation and
detergent extraction compared with those in non-transfected cells
(indicated by white arrow heads). Scale bar 20 lm.
258 C. Goswami et al.
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
determine whether the TRPV1 activation-resistant structurescontain c-tubulin, RTX-activated and detergent-extractedTRPV1-F11 cells were co-immunostained with antibodiesagainst tubulin and c-tubulin. We observed that the centre ofthis anti-tubulin-immunoreactive region indeed contained c-tubulin and thus represents the MTOC Fig. S3a). Tubulinstaining was retained in some cell extensions even after RTXtreatment and digitonin extraction (Fig. S3b), suggesting thatthe effect of TRPV1 activation on the microtubule cytoske-leton is cell-site specific and that the peripheral dynamicmicrotubules in the cell body are most affected, whereasmicrotubules at the MTOC and in neurite-like processes aremore stable.
To analyse this effect of TRPV1 activation on microtubulesubpopulations in more detail, we applied the fractionationscheme according to the flow chart (Fig. 4a), but probed theinsoluble and the soluble fractions with antibodies specificfor various post-translationally modified tubulins, as post-translational modifications of tubulin may alter the physico-chemical properties of microtubules (MacRae 1997;Westermann and Weber 2003). We also probed the samesamples for other microtubule cytoskeleton proteins, namelyc-tubulin (as a component of MTOC), the neurone- specificb-tubulin subtype III, and tau. Under conditions of RTXactivation of TRPV1-F11 cells, we observed no change in thedistribution of c-tubulins in comparison to that in non-activated cells, and the majority of the c-tubulin remained inthe insoluble fraction (Fig. 5). This is in line with data fromour immunofluorescence studies (Fig. S3a). It suggests that
the MTOC, a rigid and stable structure composed mainly ofstable microtubules, c-tubulin and other modified tubulins,remains unaffected upon TRPV1 activation. In contrast,levels of tyrosinated tubulin, a marker for dynamic micro-tubules (Gundersen et al. 1984; Kreis 1987; Wehland andWeber 1987) increased significantly in the soluble fractionafter TRPV1 activation compared with levels under controlconditions (Fig. 5). A certain amount of tyrosinated tubulin,however, remained in the insoluble fraction even afterTRPV1 activation and detergent extraction. This is in fullagreement with our immunostaining results showing thatstable microtubules at the MTOC and neurites also contain aconsiderable amount of tyrosinated tubulin (Figs S3a and b).In contrast to the tyrosinated tubulin, the proportion ofde-tyrosinated tubulin (glu tubulin), polyglutamylated tubu-lin, and acetylated tubulin was not significantly altered in thesoluble fraction after TRPV1 activation. Nor did thedistribution of neurone-specific b-tubulin subtype III changeupon TRPV1 activation. It is important to note thatde-tyrosinated tubulin, acetylated tubulin, polyglutamylatedtubulin and neurone-specific b-tubulin subtype III are allassociated with stable microtubules. After TRPV1 activation,no significant change in the soluble fraction was observed forthe neurone-specific tau, which stabilizes microtubules(Weingarten et al. 1975).
In summary, these results suggest that the dynamicmicrotubules (enriched with tyrosinated tubulin and/or non-modified tubulins) were affected by TRPV1 activation,whereas stable microtubules were not.
Tubulin
RTX+
Digitonin
i ii iii iv
viiiviivivBuffer
+Digitonin
Phase + TubPhalloidin Merge
Fig. 3 Low levels of TRPV1 are sufficient to destabilize microtubule
fibres. Confocal immunofluorescence images of stably transfected
TRPV1-F11 cells extracted with digitonin in isotonic buffer after acti-
vation with RTX (upper panel, i–iv) or after mock activation with buffer
only (lower panel, v–viii). Cells were immunostained for tubulin (green;
i and v) using the rat monoclonal antibody YL1/2, and for actin (red; ii
and vi) with alexa-594-labelled phalloidin. Tubulin staining revealed
normal microtubular structures even after detergent extraction after
mock activation (v), but tubulin was lost from most parts of the cell
body when TRPV1 was activated (i). In contrast, activated TRPV1-F11
cells, which showed reduced microtubular structures after detergent
extraction, still contained a normal actin cytoskeleton (ii). The border
of the cells is indicated by dashed white lines. Phase-contrast images
superimposed with the tubulin fluorescence (iv and viii) are shown on
the right. Scale bar 10 lm.
Modulation of microtubules by TRPV1 259
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
TRPV1-enriched membrane patches contain
unextractable tubulin
We observed that TRPV1, when over-expressed in F11 cells,was localized at the plasma membrane as distinct patches(Fig. S1a). This is in line with our previous report thatTRPV1 directly interacts with tubulin dimers and polymers
via its C-terminal domain (Goswami et al. 2004). In order tounderstand whether this tubulin forms part of a stablestructure in such TRPV1-enriched patches, F11 cells transi-ently transfected with TRPV1 were extracted with digitoninor Triton X-100 subsequent to RTX activation of TRPV1,and then subjected to indirect immunofluorescence analysis.
(b)
(a)
Total cells (T)
+/– (RTX)
+/– (Isotonic bufferwith detergent)
Incubate 1 minute
Centrifugation
Supernatant (S)
RTX +Digitonin –
1 2 3 4 5 6 7 8 9 10T S P S P T S P S P
+ – ++–
–
1 2 3 4 5 6 7 8 9 10T S P S P T S P S P
1 2 3 4 5 6 7 8 9 10T S P S P T S P S P kD
20511497
67
45
30
+ – ++–
– + – ++–
WB: anti-β-tubulin anti-actin anti-NF-200kD
Pellet (P)
Fig. 4 Activation of TRPV1 results in disassembly of the microtubule
cytoskeleton. (a) Flow chart depicting methods for TRPV1 activation
and assessment of tubulin solubility by detergent extraction. TRPV1-
F11 cells (T) were suspended in HBSS buffer and either activated with
RTX (+) or with buffer only (–) for 1 min. Thereafter, the cells were
extracted with an isotonic buffer with (+) or without (–) detergent and
fractionated into a soluble (S) and a pellet (P) fraction by centrifuga-
tion. In the absence of detergent, all cellular proteins are expected to
appear in the pellet fraction. (b) Western blot (WB) analysis of total
TRPV1-F11 cell extracts (T), the supernatant (S) and the pellet (P)
fractions probed for tubulin, actin and neurofilament. TRPV1-F11 cells
were either activated with RTX (lanes 6–10) or incubated with buffer
only (lanes 1–5). Cells were further extracted with detergent (lanes 4
and 5, 9 and 10) or with buffer only (lanes 2 and 3, 7 and 8). The
immunoreactivity for tubulin in the detergent-extracted supernatant
from activated cells (lane 9) increased significantly compared with that
in the same detergent-extracted supernatant from a non-activated
cells (lane 4). The pellet fraction derived from RTX-activated and
detergent-extracted cells (lane 10) had a much lower immunoreactivity
for tubulin than the detergent-extracted pellets from non-activated
cells (lane 5). Little increase in soluble actin and virtually no 200-kDa
neurofilament (NF) in the supernatant of RTX-activated and detergent-
extracted TRPV1-F11 cells (lane 9) were observed, indicating that the
actin and neurofilament cytoskeletons were not as affected by TRPV1
activation as the microtubule cytoskeleton.
260 C. Goswami et al.
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
Although activation of TRPV1 followed by detergentextraction resulted in the loss of microtubule structures frommost of the cell body, TRPV1-enriched patches at the plasmamembrane still retained some tubulin immunoreactivity(Fig. 6; data for Triton X-100 extraction is shown), indica-ting that the tubulin in the TRPV1-enriched patches formspart of stable microtubule structures.
Over-expression of TRPV1-Ct results in the formation of
bundled microtubules and stabilizes them
As reported before, we observed that the C-terminus, but notthe N-terminus, of TRPV1 provides stability to microtubulesin vitro (Goswami et al. 2004). Therefore we next askedwhether it is the C-terminal domain of TRPV1 that confersstability to microtubules localized in TRPV1 patches. Inorder to assess this, we transiently expressed only the C-terminal cytoplasmic domain of TRPV1 in F11 cells andperformed immunostaining of the C-terminal fragment aswell as of tubulin. TRPV1-Ct immunofluorescence appeared
as distinct spots throughout the cytoplasm (Fig. 7). Thesespots were much bigger than the expected size of vesicles.Interestingly, we observed an uneven distribution andbundling of microtubules in TRPV1-Ct-expressing cells(Fig. 7a). This bundling occurred especially in regions thatcontained clusters of TRPV1-Ct. In contrast to the transfect-ed cells, non-transfected cells did not show uneven distribu-tion or bundling, and a normal microtubule structure wasvisible (Fig. 7a). This result supports a stabilizing effect ofTRPV1-Ct on the microtubules in vivo. No changes in theactin cytoskeleton of TRPV1-Ct-expressing cells wereobserved (Fig. S4). We next extracted the cells with deter-gent before fixing them, and performed a similar immuno-staining. We observed that the cluster-like spots thatcontained TRPV1-Ct were not extractable (Fig. 7b). Afterdetergent extraction, the cells lose soluble tubulin, but retainmicrotubules. In TRPV1-Ct-expressing cells, unevenly dis-tributed microtubules all over the cell body and bundledmicrotubules in areas enriched with TRPV1-Ct spots became
TRPV1i
RTX+
TX100
ii iii iv v
1 µm
Tubulin Merge Zoom Zoom
Fig. 6 TRPV1-enriched areas contain unextractable tubulin. Confocal
indirect immunofluorescence images of TRPV1-expressing F11 cells
(i–iii) and enlarged areas (indicated by white dashed boxes) of the
same cells (iv–v). Cells were activated with RTX and subsequently
extracted with isotonic buffer containing Triton X-100. TRPV1 (green)
and tubulin (red) were immunostained with anti-TRPV1 and with
anti-tubulin (clone YL1/2). Strong anti-tubulin immunoreactivity was
associated only with the MTOC. However, membrane regions en-
riched in TRPV1 retained significant anti-tubulin immunoreactivity.
Distinct co-localization was visible in these areas (indicated by arrow).
Scale bar 20 lm (for i–iii), 5 lm (for iv) and 1 lm (for v).
RTXDigitonin
Gamma Tubulin
Tyrosinated tubulin
De-tyrosinated tubulin
Acetylated tubulin
Polyglutamylated tubulin
Beta III tubulin
TAU
TRPV1
– +– +–
T1 2 3 4 5 6 7 8 9 10
S P S P T S P S P+
Fig. 5 TRPV1 activation affects dynamic
microtubules. Samples produced as des-
cribed in Fig. 4 were probed for c-tubulin,
tyrosinated tubulin, de-tyrosinated tubu-
lin, acetylated tubulin, polyglutamylated
tubulin, neurone-specific b-tubulin subtype
III, tau and TRPV1. Only the amount of
tyrosinated tubulin increased significantly in
the supernatant (lane 9, indicated by an
asterisk), and decreased in the corres-
ponding pellet fraction (lane 10) after RTX
activation compared with levels in the same
fractions from non-activated cells (lanes 4
and 5). The distribution of other modified
tubulins and components of the microtubule
cytoskeleton remained unchanged uopn
activation. Most TRPV1 remained associ-
ated with the insoluble pellets after activa-
tion with RTX.
Modulation of microtubules by TRPV1 261
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
prominent after detergent extraction. Apart from these spots,some TRPV1-Ct immunoreactivity was also observed alongwith the microtubules after detergent extraction, especially inareas with enriched microtubule structures (Fig. 7b). Incontrast, non-transfected cells showed uniform distributionof microtubules all over the cell body and no bundling ofmicrotubules (Fig. 7b).
In order to further substantiate our conclusion that theC-terminus of TRPV1 provides stability to the microtubulesin cultured cells, we incubated cells over-expressingTRPV1-Ct with nocodazole, a microtubule-destabilizingdrug. We then extracted the cells with an isotonic buffercontaining digitonin. The TRPV1-Ct clusters remainedvisible even after nocodazole treatment and digitonin
TRPV1-Ct β-tubulin β-tubulin
Non-transfectedTransfected
Con
trol
Dig
iton
in
Zoo
mZ
oom
(a)
i ii iii iv
v vi vii viii
i ii iii iv
v vi vii viii
(b)
Merge
Fig. 7 Over-expression of the C-terminus of TRPV1 results in bundled
microtubules. (a) Confocal immunofluorescence images of an F11 cell
(i–iii) or an enlarged area (indicated by a white box) of the same cell (v–
vii) transiently expressing TRPV1-Ct. Cells were immunostained for the
C-terminus of TRPV1 (green) with a goat anti-TRPV1 antibody (spe-
cifically recognizing the C-terminus of TRPV1) and for b-tubulin (red)
with a mouse monoclonal anti-b-tubulin antibody. TRPV1-Ct immuno-
reactivity mostly appeared as distinct spots or clusters, and regions of
co-localization appeared as yellow (indicated by an arrow). TRPV1-Ct-
expressing cells showed an abnormal microtubule structure, mainly
aggregated microtubules in the area where most of the TRPV1-Ct
clusters were present (indicated by an arrowhead). The pattern of
microtubule immunoreactivity in a non-transfected cell (iv) and an en-
larged area of the same cell (viii) are shown on the right. No aggregation
or bundling of microtubules was observed in non-transfected cells.
Scale bar 10 lm (i–iv) and 2 lm (v–viii). (b) Confocal immunofluores-
cence images of an F11 cell (i–iii) and an enlarged area of the same cell
(v–vii) depicting the pattern of microtubules after extraction of the cells
with detergent in an isotonic buffer. Cells were immunostained for the
C-terminus of TRPV1 (green; i and v) and for b-tubulin (red; ii and vi).
TRPV1-Ct clusters were visible (i and v) even after detergent extraction
and areas of co-localization with anti-b-tubulin are indicated by an ar-
row. The appearance of anti-TRPV1-Ct immunoreactivity along the
microtubule-enriched areas became prominent after detergent extrac-
tion (indicated by an asterisk). Unextractable microtubules appeared
aggregated and bundled, especially in areas that co-localized with the
TRPV1-Ct clusters (indicated by an arrowhead). The pattern of im-
munoreactivity of microtubules in a detergent-extracted non-transfect-
ed cell (iv) and an enlarged area of the same cell (viii) are shown on the
right. Scale bar 10 lm (i–iv) and 5 lm (v–viii).
262 C. Goswami et al.
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
extraction (Fig. 8). We observed by immunofluorescenceanalysis that the amount of tubulin immunoreactivity leftafter nocodazole treatment and detergent extraction (noco-dazole-resistant microtubules) in TRPV1-Ct-expressing cellswas significantly higher than that in non-transfected cells(Fig. 8). We also noted a correlation between the amount ofnocodazole-resistant microtubules left after detergent extrac-tion and the level of expression of TRPV1-Ct. This wasconfirmed by a comparative analysis of the intensity oftubulin immunoreactivity of nocodazole-resistant microtu-bules in TRPV1-Ct-expressing cells and non-expressingcells (Fig. 8). Similar results were obtained when weprobed the cells with anti-a-tubulin antibody (data notshown). More often we observed the presence of nocodaz-ole-resistant microtubules all over the cell body, includingthe distal peripheral areas in the TRPV1-Ct-expressingcells, whereas the distribution of the same nocodazole-resistant microtubules in non-transfected cells was limitedonly to the presumed MTOC and nearby areas (data not
shown). These results accord well with our earlier obser-vation that MBP-TRPV1-Ct, a recombinant Maltose bind-ing protein (MBP) fusion protein with the TRPV1C-terminus, interacts with polymerized microtubules in vitroand provides stabilization against nocodazole (Goswamiet al. 2004).
In summary, our results indicate that TRPV1 activationresults in destabilization of dynamic microtubules whereasthe C-terminus of the channel has a microtubule-stabilizingeffect. Such an interplay between stabilizing and destabil-izing effects could provide the basis for the participation ofTRPV1 in microtubule cytoskeletal remodelling during paintransmission.
Discussion
Many membrane receptors, such as different isoforms ofmetabotropic glutamate receptors mGluR1 and mGluR7(Ciruela et al. 1999; Ciruela and McIlhinney 2001; Saugstad
TRPV1-Ct β-tubulinIntensity ofβ-tubulin
Box
1B
ox 2
Noc
odaz
ole
+ D
igit
onin
i ii iii 2
1
iv
v vi vii viii
ix x xi xii
Merge
Fig. 8 Over-expression of TRPV1-Ct stabilizes microtubules against
nocodazole. Indirect immunofluorescence confocal images of noco-
dazole-treated and detergent-extracted F11 cells (i–iii) or enlarged
areas depicting either a cell transiently expressing TRPV1-Ct (indi-
cated by white box 1) or a non-transfected cell (indicated by white
box 2). Cells were incubated with nocodazole, extracted with deter-
gent in an isotonic buffer and immunostained for the C-terminus of
TRPV1 (green; i, v and ix) and for b-tubulin (red; ii, vi and x). Cells
expressing high and moderate amounts of TRPV1-Ct are indicated
by an arrow and a dashed arrow respectively (i). Non-transfected
cells are indicated by arrowheads (ii). TRPV1-Ct clusters were visible
even after nocodazole treatment and detergent extraction (v). The
amount of nocodazole-resistant and unextractable microtubules was
significantly higher in the TRPV1-Ct-expressing cells and appeared
to correlate with the expression level of TRPV1-Ct. In the image on
the right, the intensities of b-tubulin immunoreactivities are shown on
a rainbow scale (iv, viii and xii). Scale bar 20 lm (i–iv) and 5 lm
(v–xii).
Modulation of microtubules by TRPV1 263
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
et al. 2002), opioid receptors and subunits of the NMDAreceptor (Panagiotou et al. 1999; van Rossum et al. 1999)have recently been shown to interact specifically with eithera- or b-tubulin. A number of studies have also established awide range of interactions between tubulin and different Gproteins, and pointed to functional implications of theseinteractions (Popova et al. 1997; Roychowdhury and Rase-nick 1997; Roychowdhury et al. 1999; Sarma et al. 2003).All these reports suggest that the cytoskeleton and itscomponents form part of complex signal transductionsystems. Furthermore, there is increasing evidence that thecytoskeleton plays a significant role in the transmission ofnociceptive information (Topp et al. 2000; Dina et al. 2003;Alessandri Haber et al. 2004).
We recently reported a Ca2+-sensitive interaction ofTRPV1 with the tubulin cytoskeleton (Goswami et al.2004), and hypothesized that the tubulin cytoskeleton mightbe a downstream effector of TRPV1 activation. Extendingthe results of our previous study, we now provide evidence ina cellular context that the integrity of the tubulin cytoskeletonis indeed affected by TRPV1 activation. Activation ofTRPV1 results in a selective depolymerization of microtu-bules into soluble tubulin. This depolymerization affectsprimarily unmodified and tyrosinated tubulins, which areknown to be part of dynamic microtubules (Gundersen et al.1984; Kreis 1987; Wehland and Weber 1987). Disassemblyof microtubules resulting from activation of TRPV1 is notdependent on the level of TRPV1 expression as stablytransfected TRPV1-F11 cells, which express a much lowerlevel of TRPV1, also exhibit the same effect. We found, onthe other hand, that some microtubules remain attached toTRPV1 after activation of the ion channel, and provide datato support our conclusion that this is due to a stabilizingeffect of the C-terminal portion of TRPV1 on the microtu-bules that interact with the channel. This is supported by ourfinding that over-expression of the C-terminal fragment ofTRPV1 alone in F11 cells leads to bundling and stabilizationof microtubules, which makes them resistant to detergent andnocodazole. The occurrence of destabilization of microtu-bules by TRPV1 activation and stabilization of microtubulesby the C-terminus of TRPV1 indicates that a fine balanceexists between TRPV1 and the microtubule cytoskeleton.TRPV1 displays characteristics that make it a good candidateas an effector of remodelling of the microtubule cytoskeletonin pain transmission.
The mechanism of tubulin cytoskeleton disruption uponTRPV1 activation, which was not the subject of this study,still remains unclear. It is known that Ca2+ has adepolymerizing effect on microtubules in vitro (Karr et al.1980; Job et al. 1981). Depolymerizing effects of Ca2+ onmicrotubules were demonstrated in vivo and it has beenshown that Ca2+ leads to two distinct processes, dynamicdestabilization and signal cascade-induced fragmentation ofmicrotubules (Lieuvin et al. 1994). Enzymatic pathways
might also be involved as RTX activation does not result inthe loss of microtubules when the enzymatic activities ofthe cells are reduced by lowering the temperature (data notshown). Activation of Ca2+-dependent proteases, for exam-ple, may occur upon TRPV1 activation, which triggersproteolysis of structural proteins as a downstream effect(Chard et al. 1995).
Prolonged stimulation of responsive neurones with cap-saicin has been known for a long time to induce neuronal celldeath (Jancso et al. 1984). In fact, retraction and degener-ation of sensory neurones, which may well involve eventsthat affect cytoskeletal integrity, may be the basis for theanalgesic effect of topical capsaicin treatment (McMahonet al. 1991). Cytotoxicity due to TRPV1 activation andsubsequent deletion of TRPV expressing neurones was alsoreported for other parts of the TRPV1-expressing nervoussystem (Karai et al. 2004; Kim et al. 2005).
In the experiments reported here, activation of TRPV1did not affect the stability of the neurofilament cytoskele-ton, but affected the actin cytoskeleton to a certain extent assome actin appeared in the soluble fraction after TRPV1activation. This increasement in actin in soluble fraction isnot surprising as actin and microtubule filaments areinterconnected within the cell (Griffith and Pollard 1978,1982). Immunofluorescence analysis of the TRPV1-activa-ted and detergent-extracted cells revealed that both actinand neurofilament cytoskeletons remained as intact poly-mers (Fig. 2d). Moreover, the N- and C-terminus of TRPV1do not interact with purified actin or enriched neurofila-ments (data not shown), whereas the C-terminus of TRPV1interacts with tubulin in a Ca2+-sensitive manner (Goswamiet al. 2004). This apparent specificity for the microtubulecytoskeleton is remarkable in the light of the wellestablished role of microtubule-active drugs in neuropathicpain. Vincristine and paclitaxel, two agents used aschemotherapeutics in the treatment of cancer, can producea painful peripheral neuropathy (Polomano and Bennett2001; Quasthoff and Hartung 2002). Notably, both vincr-istine and paclitaxel regulate microtubule stability. It hasalso been reported that TRPV1 is involved in bone cancerpain (Ghilardi et al. 2005). However, the precise mechan-ism that links the actions of vincristine and paclitaxel toperipheral neuropathy is currently unclear. On the otherhand, there are reports that the integrity of the cytoskeletonplays an important role in the development of persistentpain states in certain experimental paradigms (Dina et al.2003). Furthermore, TRPV4, another member of the TRPVfamily of ion channels, has been reported to be essential forthe development of pain in a rat model of paclitaxel-induced neuropathy (Alessandri Haber et al. 2004). Insummary, all these reports suggest that the effect of TRPV1on the microtubule cytoskeleton may well be relevant toTRPV1-mediated pain transmission, particularly in physio-pathological situations.
264 C. Goswami et al.
� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 254–266
Acknowledgements
The technical assistance of Doris Kruck and Jutta Metz is gratefully
acknowledged. We thank Dr R. Jahnel for preparing the TRPV1-Ct
construct. We acknowledge Oliver Bogen, who provided the
enriched neurofilament fraction and engaged in useful discussions.
This work was supported by grant no. 01 GG 9818/0 from
Molecular Pain Research, by the Deutsche Forschungsgemeinschaft,
Sfb 515, and by the Fonds der Chemischen Industrie. MD is
supported by the Wellcome Trust.
Supplementary material
The following material is available for this article online.
Fig. S1 Over-expression of TRVP1 results in the accumulation of
tubulin at TVRP1-enriched membrane structure
Fig. S2 Destabilization and fragmentation of microtubules due
to TVRP1 activation is cell site specific
Fig. S3 Stable microtubules at distinct cell sites are not affected
by TVRP1 activation
Fig. S4 Over-expression of TVRP1-Ct does not alter the actin
cytoskeleton
This material is available as part of the online article from http://
www.blackwell-synergy.com
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