Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1

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.


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



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.



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.



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.



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.



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).



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).



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.



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

References

Alessandri Haber N., Dina O. A., Yeh J. J., Parada C. A., Reichling D. B.and Levine J. D. (2004). Transient receptor potential vanilloid 4 isessential in chemotherapy-induced neuropathic pain in the rat.J. Neurosci. 24, 4444–4452.

Bhave G. and Gereau R. W. IV (2003) Growing pains: the cyto-skeleton as a critical regulator of pain plasticity. Neuron 39,577–579.

Caterina M. J., Schumacher M. A., Tominaga M., Rosen T. A., LevineJ. D. and Julius D. (1997) The capsaicin receptor: a heat-activatedion channel in the pain pathway. Nature 389, 816–824.

Caterina M. J., Leffler A., Malmberg A. B., Martin W. J., Trafton J.,Petersen-Zeitz K. R., Koltzenburg M., Basbaum A. I. and Julius D.(2000) Impaired nociception and pain sensation in mice lacking thecapsaicin receptor. Science 288, 306–313.

Chard P. S., Bleakman D., Savidge J. R. and Miller R. J. (1995) Cap-saicin-induced neurotoxicity in cultured dorsal root ganglion neu-rons: involvement of calcium-activated proteases. Neuroscience65, 1099–1108.

Ciruela F. and McIlhinney R. A. (2001) Metabotropic glutamate receptortype 1a and tubulin assemble into dynamic interacting complexes.J. Neurochem. 76, 750–757.

Ciruela F., Robbins M. J., Willis A. C. and McIlhinney R. A. (1999)Interactions of the C terminus of metabotropic glutamate receptortype 1a with rat brain proteins: evidence for a direct interactionwith tubulin. J. Neurochem. 72, 346–354.

Cortright D. N. and Szallasi A. (2004) Biochemical pharmacology of thevanilloid receptor. TRPV1. An update. Eur. J. Biochem. 271,1814–1819.

Davis J. B., Gray J., Gunthorpe M. J. et al. (2000) Vanilloid receptor-1 isessential for inflammatory thermal hyperalgesia. Nature 405, 183–187.

Dina O. A., McCarter G. C., de Coupade C. and Levine J. D. (2003)Role of the sensory neuron cytoskeleton in second messengersignaling for inflammatory pain. Neuron 39, 613–624.

Ghilardi J. R., Rohrich H., Lindsay T. H. et al. (2005) Selective blockadeof the capsaicin receptor TRPV1 attenuates bone cancer pain.J. Neurosci. 25, 3126–3131.

Goswami C., Dreger M., Jahnel R., Bogen O., Gillen C. and Hucho F.(2004) Identification and characterization of a Ca2+-sensitiveinteraction of the vanilloid receptor TRPV1 with tubulin.J. Neurochem. 91, 1092–1103.

Griffith L. M. and Pollard T. D. (1978) Evidence for actin filament–microtubule interaction mediated by microtubule-associated pro-teins. J. Cell Biol. 78, 958–965.

Griffith L. M. and Pollard T. D. (1982) The interaction of actin filamentswith microtubules and microtubule-associated proteins. J. Biol.Chem. 257, 9143–9151.

Gundersen G. G., Kalnoski M. H. and Bulinski J. C. (1984) Distinctpopulations of microtubules: tyrosinated and nontyrosinated alphatubulin are distributed differently in vivo. Cell 38, 779–789.

Huang S. M., Bisogno T., Trevisani M. et al. (2002) An endogenouscapsaicin-like substance with high potency at recombinant andnative vanilloid VR1 receptors. Proc. Natl Acad. Sci. USA 99,8400–8405.

Hwang S. W., Cho H., Kwak J. et al. (2000) Direct activation ofcapsaicin receptors by products of lipoxygenases: endogenouscapsaicin-like substances. Proc. Natl Acad. Sci. USA 97, 6155–6160.

Jahnel R. (2005) Investigations of molecular pain perception mecha-nisms, especially biochemical characterization of the thermosen-sitive vanilloid receptors TRPV1 and TRPV2. Thesis (http://www.diss.fu-berlin.de/2005/48/indexe.html ).

Jahnel R., Dreger M., Gillen C., Bender O., Kurreck J. and Hucho F.(2001) Biochemical characterization of the vanilloid receptor 1expressed in a dorsal root ganglia derived cell line. Eur. J. Bio-chem. 268, 5489–5496.

Jancso G., Karcsu S., Kiraly E., Szebeni A., Toth L., Bacsy E., Joo F.and Parducz A. (1984) Neurotoxin induced nerve cell degener-ation: possible involvement of calcium. Brain Res. 295, 211–216.

Job D., Fischer E. H. and Margolis R. L. (1981) Rapid disassembly ofcold-stable microtubules by calmodulin. Proc. Natl Acad. Sci. USA78, 4679–4682.

Jordt S. E., Tominaga M. and Julius D. (2000) Acid potentiation of thecapsaicin receptor determined by a key extracellular site. Proc.Natl Acad. Sci. USA 97, 8134–8139.

Joshi H. C. (1993) Gamma-tubulin: the hub of cellular microtubuleassemblies. Bioessays 15, 637–643.

Karai L., Brown D. C., Mannes A. J., Connelly S. T., Brown J., GandalM., Wellisch O. M., Neubert J. K., Olah Z. and Iadarola M. J.(2004) Deletion of vanilloid receptor 1-expressing primary afferentneurons for pain control. J. Clin. Invest. 113, 1344–1352.

Karr T. L., Kristofferson D. and Purich D. L. (1980) Calcium ion inducesendwise depolymerization of bovine brain microtubules. J. Biol.Chem. 255, 11 853–11 856.

Kim S. R., Lee da Y., Chung E. S., Oh U. T., Kim S. U. and Jin B. K.(2005) Transient receptor potential vanilloid subtype 1 mediatescell death of mesencephalic dopaminergic neurons in vivo andin vitro. J. Neurosci. 25, 662–671.

Kreis T. E. (1987) Microtubules containing detyrosinated tubulin are lessdynamic. EMBO J. 6, 2597–2606.

Laemmli U. K. (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227, 680–685.

Lieuvin A., Labbe J. C., Doree M. and Job D. (1994) Intrinsic micro-tubule stability in interphase cells. J. Cell Biol. 124, 985–996.

Liu X., Constantinescu S. N., Sun Y., Bogan J. S., Hirsch D., WeinbergR. A. and Lodish H. F. (2000). Generation of mammalian cellsstably expressing multiple genes at predetermined levels. Anal.Biochem. 280, 20–28.



MacRae T. H. (1997) Tubulin post-translational modifications – enzymesand their mechanisms of action. Eur. J. Biochem. 244, 265–278.

McMahon S. B., Lewin G. and Bloom S. R. (1991) The consequences oflong-term topical capsaicin application in the rat. Pain 44, 301–310.

Oakley B. R. and Akkari Y. N. (1999) Gamma-tubulin at ten: progressand prospects. Cell Struct. Funct. 24, 365–372.

Panagiotou S., Bakogeorgou E., Papakonstanti E., Hatzoglou A., WalletF., Dussert C., Stournaras C., Martin P. M. and Castanas E. (1999)Opioid agonists modify breast cancer cell proliferation by blockingcells to the G2/M phase of the cycle: involvement of cytoskeletalelements. J. Cell. Biochem. 73, 204–211.

Polomano R. C. and Bennett G. J. (2001) Chemotherapy-evoked painfulperipheral neuropathy. Pain Med. 2, 8–14.

Popova J. S., Garrison J. C., Rhee S. G. and Rasenick M. M. (1997)Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact toregulate phospholipase Cb1 signaling. J. Biol. Chem. 272, 6760–6765.

Quasthoff S. and Hartung H. P. (2002) Chemotherapy-induced peripheralneuropathy. J. Neurol. 249, 9–17.

van Rossum D., Kuhse J. and Betz H. (1999) Dynamic interaction be-tween soluble tubulin and C-terminal domains of N-methyl-D-aspartate receptor subunits. J. Neurochem. 72, 962–973.

Roychowdhury S. and Rasenick M. M. (1997) G protein b1 c2 subunitspromote microtubule assembly. J. Biol. Chem. 272, 31 576–31 581.

Roychowdhury S., Panda D., Wilson L. and Rasenick M. M. (1999) Gprotein asubunits activate tubulin GTPase and modulate microtu-bule polymerization dynamics. J. Biol. Chem. 274, 13 485–13 490.

Sarma T., Voyno-Yasenetskaya T., Hope T. J. and Rasenick M. M.(2003) Heterotrimeric G proteins associate with microtubulesduring differentiation in PC12 pheochromocytoma cells. FASEB J.17, 848–859.

Saugstad J. A., Yang S., Pohl J., Hall R. A. and Conn P. J. (2002)Interaction between metabotropic glutamate receptor 7 and atub-ulin. J. Neurochem. 80, 980–988.

Tominaga M., Caterina M. J., Malmberg A. B., Rosen T. A., Gilbert H.,Skinner K., Raumann, B. E., Basbaum A. I. and Julius D. (1998)The cloned capsaicin receptor integrates multiple pain-producingstimuli. Neuron 21, 531–543.

Topp K. S., Tanner K. D. and Levine J. D. (2000) Damage to thecytoskeleton of large diameter sensory neurons and myelinatedaxons in vincristine-induced painful peripheral neuropathy in therat. J. Comp. Neurol. 424, 563–576.

Wehland J. and Weber K. (1987) Turnover of the carboxy-terminaltyrosine of alpha-tubulin and means of reaching elevated levels ofdetyrosination in living cells. J Cell Sci. 88, 185–203.

Weingarten M. D., Lockwood A. H., Hwo S. Y. and Kirschner M. W.(1975) A protein factor essential for microtubule assembly. Proc.Natl Acad. Sci. USA 72, 1858–1862.

Westermann S. and Weber K. (2003) Post-translational modificationsregulate microtubule function. Nat. Rev. Mol. Cell Biol. 4, 938–947.



Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1

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