Submembraneous microtubule cytoskeleton: biochemical and functional interplay of TRP channels with the cytoskeleton.

MINIREVIEW
Submembraneous microtubule cytoskeleton: biochemical
and functional interplay of TRP channels with the
cytoskeleton
Chandan Goswami and Tim Hucho
Department for Molecular Human Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany
The microtubule cytoskeleton plays a role in a variety
of cellular aspects such as division, morphology and
motility, as well as the transport of molecules and
organelles toward and from the cell membrane.
Although all these phenomena affect the plasma mem-
brane, however, most of the microtubule filaments do
not reach to the lipid membrane region, partially due
to a thick hindering cortical actin network. However,
recent studies indicate that a small number of dynamic
microtubules can extend rapidly to the cell membrane.
Although most contacts are established only tran-
siently, there are membranous regions in which the
plus end of these pioneering microtubules is stabilized.
Stabilization appears to be mediated by the interaction
with various membrane proteins, which often are part
of large protein complexes. The dynamic properties
and the complexity of tubulin as an interacting protein
in large complexes at the membrane just are beginning
to be unravelled. One apparent function is to serve as
a scaffold protein and modulator of transmembrane
signalling.
Cytoskeletal components in signalling
complexes at membranes
The cytoplasmic domains of transient receptor pot-
ential (TRP) channels recruit large complexes of
proteins, lipids and small molecules. Depending on the
Keywords
actin; axonal guidence; cytoskeleton; growth
cone; myosin; pain; signalling complex;
transient receptor potential channels;
tubulin; varicosity
Correspondence
C. Goswami, Department for Molecular
Human Genetics, Max Planck Institute for
Molecular Genetics, Ihnestrasse 73, 14195
Berlin, Germany
Fax: +49 30 8413 1383
Tel: +49 30 8413 1243
E-mail: goswami@molgen.mpg.de
(Received 15 April 2008, revised 23 June
2008, accepted 30 July 2008)
doi:10.1111/j.1742-4658.2008.06617.x
Much work has focused on the electrophysiological properties of transient
receptor potential channels. Recently, a novel aspect of importance
emerged: the interplay of transient receptor potential channels with the
cytoskeleton. Recent data suggest a direct interaction and functional reper-
cussion for both binding partners. The bi-directionality of physical and
functional interaction renders therefore, the cytoskeleton a potent integra-
tion point of complex biological signalling events, from both the cytoplasm
and the extracellular space. In this minireview, we focus mostly on the
interaction of the cytoskeleton with transient receptor potential vanilloid
channels. Thereby, we point out the functional importance of cytoskeleton
components both as modulator and as modulated downstream effector.
The resulting implications for patho-biological situations are discussed.
Abbreviations
FHIT, fragile histidine triad protein; MAP, microtubule-associated protein; RTX, resinferatoxin; TRP, transient receptor potential; TRPV,
transient receptor potential vanilloid; TRPV1, transient receptor potential vanilloid subtype 1; TRPV1-Ct, C-terminal of transient receptor
potential vanilloid subtype 1; TRPV1-Nt, N-terminal of transient receptor potential vanilloid subtype 1; TRPV2, transient receptor potential
vanilloid subtype 2; TRPV4, transient receptor potential vanilloid subtype 4.
4684 FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS

preparation method, these structures have been
referred to as ‘signalplex’, i.e. complexes involved in
signalling events [1], or ‘channelosome’, i.e. complexes
formed around functional ion channels [2], and ⁄or as
‘lipid raft complexes’, i.e. complexes localized to this
membranous subdomain [3]. Proteomic studies of
‘signalplexes’ or ‘channelosomes’ purified from cell
lines, as well as from brain, give both direct and indi-
rect evidence for the presence of the cytoskeleton as
well as ion channels. Scaffolding adaptors like inacti-
vation-no-afterpotential D [4–6], Na+ ⁄H+ exchanger
regulatory factor [7] and ezrin ⁄moesin ⁄ radixin-binding
phosphoprotein 50 [8] are also found, some of which
interact directly with ion channels, e.g. TRP channels
[9], but also contain binding motifs for cytoskeletal
proteins [8,10]. Accordingly, cytoskeletal proteins such
as spectrin, myosin, drebrin and neurabin, as well as
tubulin and actin [1,2,6,11] are confirmed components
in signalplexes and channelosomes.
Complementary proteomic studies of purified lipid
rafts reveal the presence of several cytoskeletal proteins
[12,13] such as a- and b-tubulin, tubulin-specific chaper-
one A (a folding protein involved in tubulin dimer
assembly), KIF13 (a kinesin) [12], actin, nonconven-
tional myosin II and nonconventional myosin V [14].
Similarly, proteomics studies of the ‘membrane cyto-
skeleton’, a submembranous fraction, which is attached
to the cytoskeleton [15], and of cytoskeleton-associated
proteins in general [16], indicate the presence of lipid
raft membrane proteins as well as cytoskeletal proteins.
Together, these varying studies give strong evidence
that cytoskeletal proteins are part of signalling com-
plexes including transmembrane proteins and are
involved in their organization at membrane.
Structural features of TRP channels
The TRP family of ion channels is named after the
Drosophila melanogaster trp mutant, which is charac-
terized by a transient receptor potential in the photore-
ceptors in response to light [17]. In the meantime,
orthologues and paralogues of TRP channels have
been described in organisms ranging from simple
eukaryotes to human. They share a high degree of
homology in their amino acid sequence. TRP channels
are formed by monomers with six transmembrane
regions that assemble into tetramers, which form the
functional cation-permeable pore. The most conserved
region is the sixth transmembrane domain, which con-
stitutes most of the inner lining of the ion channel
pore. The N- and C-termini of TRP channels are
located in the cytoplasm and, depending on the respec-
tive TRP channel, consist of various functional
domains like ankyrin repeats, Ca2+-sensing EF hands,
phosphorylation sites, calmodulin-binding sites and a
so-called ‘TRP box’. Based on their sequence, the
mammalian TRP family is differentiated into six
subfamilies, namely TRP canonical (TRPC), TRP
vanilloid (TRPV), TRP melastatin (TRPM), TRP
polycystin (TRPP), TRP mucolipin (TRPML) and
TRP ankayrin (TRPA) ion channels [18]. All TRP
channels investigated to date are involved in the detec-
tion and ⁄or transduction of physical and chemical
stimuli.
TRPV1 and the cytoskeleton
Physical interaction of TRPV1 with the
cytoskeleton
TRPV1 is the founding member of the vanilloid sub-
family of TRP channels and detects several endo-
genous agonists (e.g. N-arachidonoyl-dopamine) and
noxious exogenous stimuli, such as capsaicin (the main
pungent ingredient of hot chilly) and high temperature
(> 42 �C) [19,20]. TRPV1 is a nonselective cation
channel with high permeability for Ca2+. In recent
years, TRPV1 has gained extensive attention for its
involvement in signalling events in the context of pain
and other pathophysiological conditions including
cancer [21–27].
The interaction of TRPV1 with tubulin was first iden-
tified through a proteomic analysis of endogenous inter-
actors enriched from neuronal tissue [28]. The
interaction was then confirmed by biochemical
approaches including co-immunoprecipitation, micro-
tubule co-sedimentation, pull-down and cross-linking
experiments. In contrast to the tubulin cytoskeleton, the
physical interaction of TRPV1 with actin or neurofila-
ment cytoskeleton has not been observed to date [28,29].
The C-terminus of TRPV1 (TRPV1-Ct) is sufficient
for the interaction with tubulin while the N-terminus
of TRPV1 (TRPV1-Nt) apparently does not interact
[28]. Using deletion constructs and biotinylated pep-
tides, the tubulin-binding region located within
TRPV1-Ct was mapped to two short, highly basic
regions (amino acids 710–730 and 770–797) [29]. If an
a-helical conformation is assumed, these two regions
project all their basic amino acids to one side, thus
potentially enabling interactions with negatively
charged residues (Fig. 1). Indeed, correspondingly, the
C-terminal over-hanging region of tubulin contains a
large number of negatively charged glutamate (E) resi-
dues in a stretch characterized as unstructured region
of the tubulin and referred as E-hook. These E-hooks
are known to be essential for the interaction of tubulin
C. Goswami and T. Hucho TRP channels and cytoskeleton regulate each other
FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS 4685

with various microtubule-associated proteins such as
MAPs, Tau, as well as others. Indeed, binding of
TRPV1-Ct with tubulin was abolished when the
E-hooks containing over-hangs were removed by prote-
ase treatment [29]. The tubulin-binding region of
TRPV1 apparently is under high evolutionary pressure
as its sequence is highly conserved in all TRPV1 ortho-
logues [29]. Also between homologues, the distribution
of basic amino acids composing the tubulin-binding
regions is conserved even though the overall amino acid
conservation is rather limited. Based on these data an
interaction of tubulin with TRPV2, TRPV3 and TRPV4
(Fig. 2) can also be predicted. These TRPV1 homo-
logues have the highest conservation of basic charge
distribution within the tubulin-binding sequences.
Indeed, in the meantime we could confirm this for
TRPV2 and TRPV4 (unpublished observation).
TRPV1 preferably interacts through its C-terminal
domain with b-tubulin and to a lesser extend also with
a-tubulin thereby forming a high-molecular weight
complex [29]. This suggests stronger binding of
TRPV1 to the plus end rather than the minus end of
A
B
Fig. 1. Characteristic of the tubulin-binding motifs located at the
C-terminus of TRPV1. (A) The extreme C-terminus of both a- and
b-tubulin contains highly negatively charged amino acids (indicated
in red) and is mostly unstructured. (B) The basic amino acids (indi-
cated in blue) that are located within the tubulin-binding regions of
TRPV1 are located at one side of the putative helical wheel, where
it can interact with the acidic C-terminus of tubulin.
A
B
Fig. 2. Conservation of the tubulin-binding regions in TRPV1 orthologues and homologues. (A) The tubulin-binding region is conserved in
mammals. The conserved basic amino acids are shown in blue and are indicated by an asterisk (*). NCBI accession numbers: rat
(NP-114188), mouse (CAF05661), dog (AAT71314), human (NP_542437), guinea pig (AAU43730), rabbit (AAR34458), chicken (NP_989903)
and pig (CAD37814). (B) TRPV1 homologues (based on sequences from rat species only) were aligned using CLUSTAL. The distribution of
basic amino acids (in blue) located within the first tubulin-binding motif is partially conserved. NCBI accession numbers: TRPV1 (NP-114188),
TRPV2 (AAH89215), TRPV3 (NP-001020928), TRPV4 (NP-076460), TRPV5 (AAV31121) and TRPV6 (Q9R186).
TRP channels and cytoskeleton regulate each other C. Goswami and T. Hucho
4686 FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS

microtubules as the plus ends of microtubule protofila-
ments are decorated with b-tubulin. It is therefore
tempting to speculate that TRPV1 may act as a micro-
tubule plus-end-tracking protein (+TIP) [30]. This
speculation is corroborated by the recent observation
that despite their differences in primary amino acid
sequences, the crystal structures of microtubule-bind-
ing regions of different classes of +TIP proteins such
as Stu2p, EB1 and Bim1p contain a common motif of
at least two a helices with positively charged residues
at the surface [31]. The tubulin-binding ability of
TRPV1-Ct is supported by the predicted structural
models also [32,33]. This is particularly due to the fact
that the tubulin-binding regions are predicted to con-
tain a helices. Fragile histidine triad protein (FHIT), a
tumour suppressor gene product has high sequence
homology with TRPV1-Ct and the crystal structure of
FHIT was used as a template for predicting the struc-
ture of TRPV1-Ct [32]. Remarkably, FHIT also binds
to tubulin [34].
Different post-translationally modified tubulin, like
tyrosinated tubulin (a marker for dynamic microtu-
bules), detyrosinated tubulin, acetylated tubulin, poly-
glutamylated tubulin, phospho (serine) tubulin and
neurone-specific b-III tubulin (all markers for stable
microtubules) interact with TRPV1-Ct [29]. This implies
that TRPV1 interacts not only with soluble tubulin, but
also with assembled microtubules in various dynamic
states. And indeed, the interaction of TRPV1-Ct also
with polymerized microtubules could experimentally
been proven [28]. In addition to sole binding, TRPV1-
Ct exerts a strong stabilization effect on microtubules,
which becomes especially apparent under microtubules
depolymerising conditions such as presence of noco-
dazol or increased Ca2+ concentrations [28].
TRPV1 channels are nonselective cation channels.
Therefore, the role of increased concentration of Ca2+
on the properties of TRPV1–tubulin and ⁄or TRPV1–
microtubule complex is of special interest. Tubulin
binding to TRPV1-Ct is increased by increased Ca2+
concentrations [28]. Interestingly, the microtubules
formed with TRPV1-Ct in the presence of Ca2+
become ‘cold stable’ as these microtubules do not dep-
olymerise further at low temperature [28]. The exact
mechanism how Ca2+ modulates these physicochemi-
cal properties in vitro are not clear. In this regard, it is
important to mention that tubulin has been shown to
bind two Ca2+ ions to its C-terminal sequence [35–38]
and thus Ca2+-dependent conformational changes of
tubulin [39] may underlie the observed effects of Ca2+.
The biochemical data of direct interaction as well as
microtubule stabilization find their correlates in cell
biological studies. Transfection of TRPV1 in dorsal
root ganglia-derived F11 cells results in co-localization
of TRPV1 and microtubules and accumulation of
endogenous tyrosinated tubulin (a marker for dynamic
microtubules) in close vicinity to the plasma membrane
[28] (Fig. 3). As suggested by its preference to bind to
the plus-end-exposed b-tubulin, TRPV1 apparently sta-
bilizes microtubules reaching the plasma membrane
and thereby increases the number of pioneering micro-
tubules within the actin cortex (Fig. 4). But stabiliza-
tion induces even stronger changes. The overall
cellular morphology is altered dramatically by massive
induction of filopodial structures in neuronal as well as
in non-neuronal cells [40] (Fig. 4). The mechanism for
this is currently under investigation and apparently
also includes alterations in the actin cytoskeleton. But,
co-localization of TRPV1 with tubulin was observed
all along the filopodial stalk and, of note, including
the filopodial tips [40]. Tubulin and components attrib-
uted to stable microtubules (like acetylated tubulin
and MAP2ab) were also observed within these thin
filopodial structures [40].
TRPV1-activation induced microtubule
disassembly
In contrast to the stabilization of microtubules at rest-
ing state, activation of TRPV1 results in rapid disas-
sembly of microtubules irrespective of the investigated
cellular system (Fig. 3) [41,42]. Again, the underlying
mechanism of TRPV1 activation-mediated cytoskele-
ton remodelling is largely unknown. In F11 cells,
TRPV1 activation leads to an almost complete destruc-
tion of peripheral microtubules, whereas microtubules
close to the microtubule-organizing centre, a structure
composed of c-tubulin and stable microtubules at the
perinuclear region, remain intact (Fig. 3). Also, the
integrity of other cytoskeletal filaments like actin and
neurofilaments is not affected by activation of TRPV1
[41]. Potentially, TRPV1 activation may even increase
the amount of polymerized actin [43].
Effects caused by the activation of a nonselective
cation channel are suggestive of mediation by the
influx of, for example, Ca2+. Indeed, high Ca2+ con-
centrations have the potential to depolymerize micro-
tubules in vitro and in vivo [44,45] through either
‘dynamic destabilization’, i.e. a direct effect of Ca2+
on microtubules, or indirectly by a calcium-induced
but signal-cascade-dependent depolymerization [46].
Also, chelating extracellular Ca2+ with EGTA and
depletion of intracellular Ca2+ stores with thapsigargin
cannot prevent TRPV1-activation-mediated microtu-
bule disassembly [41,47]. Thus, TRPV1-activation-
induced microtubule disassembly is apparently not a
C. Goswami and T. Hucho TRP channels and cytoskeleton regulate each other
FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS 4687

direct effect of high Ca2+ concentrations. Even com-
bined EGTA and thapsigargin, treatment cannot
exclude small changes in local Ca2+ concentration.
Therefore, these small changes in Ca2+ might trigger
an enzymatic cascade leading to depolymerization.
This view is also supported by previous studies demon-
strating that a small amount of calmodulin can cause
massive microtubule depolymerization in the presence
of catalytic amounts of Ca2+, but not in the complete
absence of Ca2+ [45,48–50]. Subsequent activation of
Ca2+-dependent proteases may also trigger proteolysis
of structural proteins as a downstream effect [51].
Another potential mechanism that can lead to rapid
disassembly of microtubules might be the phosphoryla-
tion of microtubule-associated proteins (MAPs). We
observed fragmented microtubules all over the cyto-
plasm after TRPV1 activation, which suggest that
specific microtubule-severing proteins like katanin,
fidgetin and spastin are probably also involved in this
process (Fig. 3) [52–54]. Prolonged stimulation of
TRPV1 activates through high Ca2+ concentrations
among others caspase 3 and 8, which leads eventually
to cell death [55–59]. In general, extensive fragmenta-
tion of the cellular cytoskeleton and programmed cell
death correlate well. However, in response to short-
term stimulation of TRPV1 we have not observed any
fragmented tubulin bands in western blot analysis [41].
Last, but not least, TRPV1 activation-mediated inhibi-
tion of protein synthesis and endoplasmic reticulum
fragmentation may also have impact on the microtu-
bule integrity [42].
Implications of TRPV1-induced cytoskeleton
destabilization
TRPV1 affects biological functions, like cell migration
and neuritogenesis, that are largely dependent on the
cytoskeleton [42,60,61]. Indeed, rapid disassembly of
dynamic microtubules by TRPV1 activation has a
strong effect on axonal growth, morphology and
migration. TRPV1 is endogenously expressed already
at an early embryonic stage and localizes to neurites
A
B C
10 µm
5 µm
10 µm
Fig. 3. TRPV1 regulates microtubule dynamics by two opposing manners. (A) In the absence of activation, TRPV1 co-localizes and stabilizes
microtubules at the cell membrane. Confocal immunofluorescence images of a F11 cell and an enlarged area reveals the accumulation of
tubulin (red) at the plasma membrane due to the presence of TRPV1 (green). (B) Activation of TRPV1 by RTX results in rapid the disassem-
bly of polymerized microtubules. Filamentous microtubules disappear in the TRPV1 expressing cells but not in the nontransfected cells. (C)
Detergent extraction after RTX treatment of TRPV1 expressing cells reveals loss of peripheral microtubules from majority of the cell body.
The presence of microtubules is restricted only to the microtubule organizing centre region. Some fragmented microtubules near perinuclear
region are also visible.
TRP channels and cytoskeleton regulate each other C. Goswami and T. Hucho
4688 FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS