Download Submembraneous microtubule cytoskeleton: biochemical and

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
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: [email protected]
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 repercussion for both binding partners. The bi-directionality of physical and
functional interaction renders therefore, the cytoskeleton a potent integration 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.
(Received 15 April 2008, revised 23 June
2008, accepted 30 July 2008)
doi:10.1111/j.1742-4658.2008.06617.x
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 membrane, 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 transiently, 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 potential (TRP) channels recruit large complexes of
proteins, lipids and small molecules. Depending on the
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.
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C. Goswami and T. Hucho
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 indirect evidence for the presence of the cytoskeleton as
well as ion channels. Scaffolding adaptors like inactivation-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 chaperone A (a folding protein involved in tubulin dimer
assembly), KIF13 (a kinesin) [12], actin, nonconventional myosin II and nonconventional myosin V [14].
Similarly, proteomics studies of the ‘membrane cytoskeleton’, 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 complexes 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 characterized by a transient receptor potential in the photoreceptors 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 constitutes 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 respective TRP channel, consist of various functional
TRP channels and cytoskeleton regulate each other
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 detection 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 subfamily of TRP channels and detects several endogenous 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 identified through a proteomic analysis of endogenous interactors enriched from neuronal tissue [28]. The
interaction was then confirmed by biochemical
approaches including co-immunoprecipitation, microtubule co-sedimentation, pull-down and cross-linking
experiments. In contrast to the tubulin cytoskeleton, the
physical interaction of TRPV1 with actin or neurofilament 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 peptides, 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) residues 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
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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 (indicated 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.
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 protease treatment [29]. The tubulin-binding region of
TRPV1 apparently is under high evolutionary pressure
as its sequence is highly conserved in all TRPV1 orthologues [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 homologues 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. 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).
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microtubules as the plus ends of microtubule protofilaments are decorated with b-tubulin. It is therefore
tempting to speculate that TRPV1 may act as a microtubule 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-binding 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 contain 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 structure of TRPV1-Ct [32]. Remarkably, FHIT also binds
to tubulin [34].
Different post-translationally modified tubulin, like
tyrosinated tubulin (a marker for dynamic microtubules), detyrosinated tubulin, acetylated tubulin, polyglutamylated 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, TRPV1Ct exerts a strong stabilization effect on microtubules,
which becomes especially apparent under microtubules
depolymerising conditions such as presence of nocodazol 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 depolymerise further at low temperature [28]. The exact
mechanism how Ca2+ modulates these physicochemical 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
TRP channels and cytoskeleton regulate each other
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 stabilizes microtubules reaching the plasma membrane
and thereby increases the number of pioneering microtubules within the actin cortex (Fig. 4). But stabilization 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 attributed 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 resting state, activation of TRPV1 results in rapid disassembly of microtubules irrespective of the investigated
cellular system (Fig. 3) [41,42]. Again, the underlying
mechanism of TRPV1 activation-mediated cytoskeleton remodelling is largely unknown. In F11 cells,
TRPV1 activation leads to an almost complete destruction 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+ concentrations have the potential to depolymerize microtubules 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 microtubule disassembly [41,47]. Thus, TRPV1-activationinduced microtubule disassembly is apparently not a
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C. Goswami and T. Hucho
A
5 µm
10 µm
B
C
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 disassembly 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.
direct effect of high Ca2+ concentrations. Even combined 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 demonstrating 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 phosphorylation of microtubule-associated proteins (MAPs). We
observed fragmented microtubules all over the cytoplasm 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
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among others caspase 3 and 8, which leads eventually
to cell death [55–59]. In general, extensive fragmentation of the cellular cytoskeleton and programmed cell
death correlate well. However, in response to shortterm stimulation of TRPV1 we have not observed any
fragmented tubulin bands in western blot analysis [41].
Last, but not least, TRPV1 activation-mediated inhibition of protein synthesis and endoplasmic reticulum
fragmentation may also have impact on the microtubule 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
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C. Goswami and T. Hucho
TRP channels and cytoskeleton regulate each other
A
i
B
ii
iii
C
D
Fig. 4. Effect of TRPV1–cytoskeletal cross-talk on neuritis and growth cones. (A) At growth cones, TRPV1-enriched plasma membranes stabilize pioneer microtubules within the filopodial structures. (B) Such stabilization of the microtubule results in the induction of neuritogenesis
and the formation of elongated cells. (C) Time series of a growth cone developed from F11 cell expressing TRPV1–GFP. Application of RTX
results in rapid collapse and retraction of the growth cone. (D) Longer neurites develop multiple varicosities (arrow heads) after RTX application due to disassembly of microtubules. Such varicosities are not visible in TRPV1 expressing cells in absence of activation. Even in case of
neurites developed from non-TRPV1 expressing cells, RTX application remains ineffective and do not produce such varicosities.
and growth cones (Fig. 4) [47,62]. Activation of
TRPV1 results in rapid disassembly of microtubules
within neurites (and also at growth cones) while keeping the actin cytoskeleton intact and functional. This
destroys the balance between the anterograde force
(generated by microtubule cytoskeleton) and the retrograde force (generated by actin cytoskeleton) that
determines the axonal morphology and the net neurite
growth [63,64]. Sudden loss of polymerized microtubules results in retraction of growth cones and formation of varicosities all along the neurites (Fig. 5).
Long-term low-level TRPV1 activation by an endogenous ligand results in shortening of neurites in primary neurons [47]. But as endogenous expression of
TRPV1 is widespread and not restricted to neuronal
cells, activation of TRPV1 increases the motility of
non-neuronal cells like HepG2 and dendritic cells
[42,65]. In agreement with the role of TRPV1 in cell
motility, dendritic cells from trpv1 - ⁄ - animal show less
migration than wild-type [65].
Differential activation of TRPV1 complexes can
create an asymmetry in the microtubular organization.
Thus, activation of TRPV1 in a specific cellular region
may result in the disassembly of microtubules, thereby
facilitating the retraction of that part of the cell, thus
creating a trailing edge. By contrast, stabilization of
microtubules at TRPV1-enriched plasma membranes
may facilitate a cell to extend at this region, marking
the leading edge and initiating cell migration [66].
In contrast to a strong and long-term activation of
TRPV1, which affects microtubules globally, mild and
localized short-term activation may affect parts of the
cytoskeleton differently. Thus, growth cones may be
helped to avoid a repulsive guidance cue. Reciprocally,
stabilization effect of TRPV1-enriched membranes on
the plus ends of microtubules may help a growth cone
to steer towards an attractive cue (Fig. 4). A similar
mechanism by which other TRP channels can regulate
the growth cone attraction, repulsion or retraction has
been described [67,68]. Although not tested, TRPV1
may potentially regulate the sperm motility as the presence of TRPV1 at the sperm acrosome and throughout
the tail has been reported [69]. Short-term and lowlevel activation may increase sperm motility whereas
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B
C
Fig. 5. Schematic model depicting how TRPV1 regulates growth
cone and neurite movement via cytoskeletal reorganization. (A)
Presence of microtubule cytoskeleton (Mt, red) and actin cytoskeleton (blue) at the neurite and at the growth cones are shown. Both
an anterograde force from microtubule cytoskeleton (up arrow) and
a retrograde force provided by actin cytoskeleton (down arrow)
determine the net axonal growth and movement. (B) Most of the
axonal microtubules is restricted to the central zone (C-zone) of the
growth cone. Few dynamic pioneer microtubules at the peripheral
zone (P-zone) are selectively stabilized by TRPV1-enriched membrane patches (green stars). This may have implications for the
turning of the growth cone in response to a signal (green asterisk).
(C) TRPV1 activation-mediated growth cone retraction and varicosity formation is dependent on the degree of microtubule disassembly. (Stage 1) Activation of TRPV1 (indicated by arrow) results in
the partial disassembly of microtubules, leading to the retraction of
growth cone. (Stage 2) Further disassembly leads to more retraction and initiates varicosity formation. (Stage 3) Complete disassembly of microtubules results in a stage where further retraction
is no more possible. (Stage 4) The force from the functional actin
cytoskeleton and complete disassembled microtubules results in
the varicosity formation. Strong agonists like RTX result in a quick
and irreversible shift to stage 3 and 4. By contrast, transient and
mild activation by endogenous ligands like N-arachidonoyl-dopamine
results in retraction for a longer time but rarely forms varicosities,
indicating that N-arachidonoyl-dopamine most likely results in slow
and reversible shifting at stage 1 and 2.
robust activation may cause a non-motile sperm due
to complete disassembly of microtubules at the sperm
tail.
TRPV4 and the cytoskeleton
TRPV4 is a member of the TRPV subfamily and a
close homologue of TRPV1. It is activated by
endogenous endovanilloids, by temperatures of
> 37 C, and by both hypo- and hyperosmotic
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stimuli. In many studies, the synthetic ligand
4a-PDD is alternatively employed [70]. TRPV4 is
involved in mechanosensation of the normal and the
sensitized neuron [71]. To date, the evidence for the
functional, as well as physical, interaction of TRPV4
with cytoskeletal components is mostly indirect. For
example, TRPV4 has been shown to be important
for the development of taxol-induced mechanical
hyperalgesia suggesting a functional link of TRPV4
with microtubule cytoskeleton [72]. Often, activation
of TRPV4 is correlated to cellular changes, which in
turn are known to involve cytoskeletal rearrangement
such as cell volume in regulatory volume decrease
[72–74] and cell motility due to changes in lamellipodia dynamics [60]. However, the extent to which the
changes in the cytoskeleton are induced by TRPV4
directly is mostly unknown. Biochemical and cell
biological data are sparse and patchy. The distance
between actin and TRPV4 in a live cell was measured by FRET to be < 4 nm [75] assuring, that
these two components have the potentiality to interact with each other. In addition, TRPV4 has been
identified by yeast two-hybrid screen to interact with
MAP7, an interaction confirmed by immunoprecipitation as well as pull-down experiments [76]. This
interaction is dependent on the C-terminal amino
acids 798–809. Interestingly, the C-terminal cytoplasmic domain of TRPV4 also contains a partially
conserved putative tubulin-binding site [29].
Physical and functional interaction of
other TRP channels with cytoskeletal
components
Physical links of several TRP channels other than
TRPV1 and TRPV4 with the cytoskeleton have been
established. For example, b-tubulin interacts directly
with TRPC1 [77]. Two other members of the TRPC
family, TRPC5 and TRPC6, are also interacting with
cytoskeletal proteins [1]. These also include actin and
tubulin as they are confirmed components of the purified ‘signalplex’ [1]. TRPC5 interacts with stathmin 2,
a microtubule cytoskeleton-binding protein [78]. Direct
physical and functional interplay with both the microtubule and actin cytoskeleton has also been described
for TRPP channels (see minireview by Chan et al. in
this series).
Apart from the direct interaction, many of these
TRP channels are localized to the microtubule and
actin cytoskeleton-enriched structures like filopodia,
cilia and growth cones, indicating a potential association and complex signalling with the cytoskeleton.
Again, activation of these TRP channels correlates
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C. Goswami and T. Hucho
with cytoskeleton-dependent morphological changes.
For example, both rat and human pulmonary arterial
endothelial cells express TRPP1, the activation of
which leads to a change in cell shape due to reorganization of cortical actin network [79]. Likewise, Xenopus TRPN1 (NOMPC) localizes to microtubule-based
cilia in epithelial cells, including inner ear hair cells
[80].
Almost all of the TRPC channels have been reported
to localize to growth cones [67,77,81–84]. In all cases,
activation of TRPC channels regulates growth cone
morphology and motility in response to chemical guidance cues. TRPC1 modulates the actin cytoskeleton by
modulating ADF ⁄ cofilin activity via LIM kinase [85].
It is involved in growth cone turning in response to
Netrin 1 [82,83]. TRPC4 is upregulated after nerve
injury and is important for neurite outgrowth [81].
TRPC3 and TRPC6 are important for growth cone
turning in response to brain-derived neurotrophic
factor [84]. Likewise, TRPC5 expression results in
increased length of neurites and filopodia [78].
In addition to data on the interaction of TRP channels with the cytoskeleton, there are few examples suggesting that the activity of the TRP channel is
influenced by the cytoskeleton. Mostly, alteration of
the cytoskeleton results in inhibition of TRP channel
opening. For example, the requirement for a functional
cytoskeleton in the activation of TRP channels in
store-mediated Ca2+ entry and ⁄ or store-operated
Ca2+ entry has been reported [86–88]. In human platelets, physical coupling of hTRP1 with inositol 1,4,5-triphosphate receptor (IP3R), a prerequisite step for
store-mediated Ca2+ entry, depends on the degree of
polymerized actin [86,87]. Disruption of the actin cytoskeleton by cytochalasin D also prevents phosphatidylinositol 4,5-bisphosphate (PIP2)-mediated inhibition of
TRPC4a [89]. Another example has been reported
from primary human polymorphonuclear neutrophil
cells, in which reorganization of actin results in the
internalization of endogenous TRPC1, TRPC3 and
TRPC4 from plasma membrane to the cytosol, which
correlates well with the loss of store-operated calcium
entry [88]. Accordingly, pre-disruption of the actin
cytoskeleton by cytochalasin D rescues the loss of
store-operated calcium entry, indicating that the actin
dynamics are important for this TRPC-mediated storeoperated calcium entry. Apparently, an intact actin
cytoskeleton is also essential for strong agonist-mediated activation of TRPC7. Thus, pharmacological disruption of the actin cytoskeleton results in reduced
agonist-induced activation [90]. All these examples suggest that the cytoskeleton can indeed act as modulators
of TRP channel function.
TRP channels and cytoskeleton regulate each other
TRP channels and myosin motors
Nonconventional myosin motors and TRP channels are
often localized within specific subcellular regions such
as filopodia or ciliary tips. These two groups of proteins
also share a special genotype–phenotype correlation as
abnormal expression ⁄ function of these myosins or
TRPs gives rise to similar pathophysiological conditions
like deafness, blindness, and syndromes affecting the
function of other tissues and ⁄ or organs. For example,
in case of deafness, several nonconventional myosin
motors (myosin I, IIA, IIIA, VI, VIIA and XV) are
important for either development of the stereocilia of
hair cells in the inner ear or proper localization of TRP
channels at the tip of these stereocilia, which is crucial
for the activity of these cells [91,92]. Reciprocally, mutations and abnormal expression ⁄ function of several TRP
channels (TRPML1, TRPML2, TRPML3, TRPV4,
TRPV5 and TRPV6) also lead to deafness [93–97]. In a
similar manner, both myosins and TRP channels are
causally involved in blindness. Recently it has been
reported that translocation of eGFP-tagged TRP-like
channels to the rhabdomeral membrane in Drosophila
photoreceptors is myosin III dependent [98]. Apart
from the above genetic interactions, TRP channels
interact directly with myosins. Using a proteomic
screen, myosin was identified to bind to TRPC5 and
TRPC6 [1]. Another study showed that myosin IIa is
directly phosphorylated by TRPM7, a cation channel
fused to an alpha-kinase [99]. This phosphorylation in
turn regulates cell contractility and adhesion. Notably,
TRPM7 phosphorylates positively charged coiled-coil
domain of myosin II [100].
In some cases, similar cellular phenotypes also
suggest a functional link between TRP channels and
nonconventional myosin motors. For example, we
observed that ectopic expression of TRPV1 induces
extensive filopodial and neurite-like structures in neuron-derived F11 cells as well as in non-neuronal cells
(e.g. HeLa, Cos and HEK 293 cells) [40]. Interestingly,
TRPV1 expression induces club-shaped filopodia with
a bulbous head structure that contains negligible
amount of F-actin but accumulates TRPV1 [40]. This
phenotype resembles the dominant negative effect of
the expression of the non-conventional myosin II, III,
V, X and XV [101–112]. This renders the observation
that TRPV1 expression induces drastic upregulation of
endogenous myosin IIa and IIIa temptingly suggestive
[40]. In addition, the subcellular distribution of myosins is markedly changed from a uniformly cytoplasmic
to a strongly clustered localization mostly at the
cell periphery [40]. In another study, cardiac-specific
overexpression of TRPC6 in transgenic mice resulted
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C. Goswami and T. Hucho
in an increase expression of beta-myosin heavy chain
[113]. Such phenomena strongly suggest the cooperative role of myosins and TRP channels in development
as well as proper function of ciliary and filopodial
structures.
The molecular mechanisms behind the increased
expression of these myosins are poorly understood. In
some cases, TRP channels apparently increase myosin
expression by regulating transcription factors [113]. As
myosins are also susceptible to protease-mediated degradation; a higher level of endogenous myosin might
also imply less proteolytic degradation. It is therefore
worth exploring how TRP channels affect the distribution, function and endogenous level of myosins.
Modulation of TRP ion channels
activities by the cytoskeleton
TRP channels can be modulated by Ca2+-dependent
or Ca2+-independent mechanisms. Desensitization can
be initiated by the Ca2+-influx through the channel
itself and is manifested through phosphorylation–
dephosphorylation of the TRP channel and ⁄ or by the
Ca2+-dependent interaction with calmodulin at the
C-termini of, for example, TRPV1 and TRPV4 [114–
121]. As an example of Ca2+-independent mechanisms,
the channel inactivation through physical interaction
of the TRPC channel with the cytoplasmic protein
homer has been described. TRPC mutants lacking the
homer-binding site become constitutively active [9].
This latter example spurs one to hypothesize whether
other scaffolding proteins than homer, such as actin
and ⁄ or tubulin, can regulate TRP channel properties
though to date the experimental evidence is only circumstantial. Modulation of TRPV4 by a cytosolic
component is suggested, as the channel can be activated by heat only if analysed using whole-cell recordings and not in excised patches of cell-free membranes
[122,123]. In turn, the involvement of cytoskeletal components in the regulation of TRPV4 channel activity
has been demonstrated experimentally by the addition
of cytoskeletal regulating drugs. The microtubule stabilizer taxol reduces TRPV4-dependent currents while
the microtubule-disrupting agents colchicine and vincristine as well as actin cytoskeleton regulating drugs
like phalloidin (a stabilizer) or cytochalasin B (a destabilizer) do not alter the TRPV4-mediated current [76].
In the same manner, mechanosensitive ion channel
activity in cultured sensory neurons appears to depend
largely on the status of the cytoskeleton. Thus, disruption of actin or microtubule cytoskeleton by pharmacological agents greatly reduces the activity of
mechanosensitive channels [124]. However, if the
4692
modulation of TRP channels occurs through direct
interaction with the cytoskeleton remains to be proven.
In addition to purely circumstantial evidence, few
studies attempted the establishment of a direct modulatory role of the cytoskeleton, the best of which was
performed on TRPP channels [125,126]. Montalbetti
and co-workers isolated syncytiotrophoblast apical
membrane vesicles from human placenta, and performed single-channel electrophysiological experiments
of polycystin channel 2 (PC2) on reconstituted lipid
bilayers. This system eliminates all factors except the
channel-associated complex. Biochemical analysis
revealed the presence of actin, the actin-related components a-actinin and gelsolin, tubulin including acetylated a-tubulin, and the kinesin motor proteins KIF3A
and KIF3B in these membranes [125,126]. PC2 channels interact directly with KIF3. Disruption of actin
filaments with cytochalasin D or with the actin-severing protein gelsolin activates the channel. This activation can be inhibited by the addition of soluble
monomeric G-actin with ATP, which induces actin
polymerization. This indicates that actin filaments, but
not soluble actin, are an endogenous negative regulator
of PC2 channels. Also microtubules regulate PC2
channel function only in opposing manner. Depolymerization of microtubules with colchicine rapidly
inhibits the basal level of PC2 channel activity,
whereas polymerization and ⁄ or stabilization of microtubules from soluble tubulin with GTP and taxol stimulates the PC2 channel activity [125]. Involvement of
the microtubule cytoskeleton in the regulation of PC2
channel has also been described in vivo in primary cilia
of renal epithelial cells [127]. In that system, addition
of microtubule destabilizer (colchicine) rapidly abolished channel activity, whereas the addition of microtubule stabilizers (taxol) increased channel activity
[127]. Similar results were obtained using reconstituted
lipid bilayer system, which reveals that both spontaneous activity and the opening probability of TRPP3 ion
channels is increased by the addition of a-actinin, demonstrating that this channel can be indeed modulated
by cytoskeleton [128].
TRPV1, TRPV4 and the cytoskeleton in
pathophysiological conditions
The importance of TRP channels in the development of
disease becomes increasingly evident. In particular,
TRPV channels are involved in various aspects of pain
such as inflammatory pain, cancer pain and neuropathic
pain, as well as other diseases including allergy, diabetes
and cough [129]. Indeed, data tie TRPV1 and TRPV4
to the status of the cytoskeleton in models of pain. For
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C. Goswami and T. Hucho
example, rapidly dividing cancer cells are pharmacologically targeted by modulators of the microtubule cytoskeleton such as taxol, vincristin and their derivatives.
But in addition to the deleterious effect of these agents
on the cancer, if applied systemically over the long-term
they are highly potent inducers of strong neuropathic
pain [130–132]. Systemic vincristine treatment strongly
alters the cytoskeletal architecture [133,134]. On a
shorter timescale, inflammatory signalling pathways
leading to sensitization in a healthy animal are dependent on both the ‘integrity’ and the ‘dynamics’ of the
microtubule cytoskeleton [135,136]. Short-term modulation of the cytoskeleton abolishes inflammatory mediator-induced sensitization [135]. The precise mechanism
by which vinca-drugs and taxoid-group-containing molecules influence pain is not clear. Vincristine by itself is
known to form tubulin paracrystals [137]. Taxol can
also form crystals, which can masquerade as stabilized
microtubules and can rapidly incorporate tubulin
dimers [138]. In addition, a unique type of straight
GDP–tubulin protofilament forms in the presence of
taxol [139]. Whether these uncommon altered physical
forms of tubulins ⁄ microtubules are important for pain
development remains a central question. However,
more subtle effects like differential binding properties
to other proteins might play a role.
In addition, the involvement of several TRP channels in the development of cancer and cancer pain is
increasingly prominent [22,140–142]. Endogenous
expressions of some TRP channels are either upregulated or downregulated in different tumours, cancerous
tissue and also in different cancerous cell lines. For
example, TRPV1 is overexpressed in bone cancer,
prostate cancer and pancreatic cancer [143–146]. Along
the same lines, TRPV4, which is involved in mechanosensation, has been shown to be essential for the development of chemotherapy-induced neuropathic pain in
the rat [72].
TRP channels also share functional links with the
cytoskeleton by other means, namely cytotoxicity and
cell death. For example, activation of TRPV1 leads to
the inhibition of protein synthesis and endoplasmic
reticulum fragmentation [42]. Prolonged stimulation
with capsaicin induces apoptosis in TRPV1 expressing
neurons by activating different caspase pathways
(mainly caspase 8, caspase 3) [55–58,147–153]. However, whether the cytotoxicity and cell death described
above is due to disassembly of microtubule has not
been tested. Loss of TRPV1-expressing neurons ⁄ cells
from specific tissues are functionally linked with the
development of patho-physiological conditions. For
example, loss of TRPV1 positive neurons in liver is
linked with diabetes [154].
TRP channels and cytoskeleton regulate each other
However, the deleterious effect of TRP channels on
the specific subtype of neurons or cells has some clinical advantages. In fact, retraction and degeneration of
a subset of sensory neurons (specifically TRPV1expressing neurons), which involve events that affect
the integrity of the cytoskeleton, forms the basis for
the analgesic effect of topical capsaicin-cream treatment [155]. Resinferatoxin (RTX), a potent agonist of
TRPV1 has been used successfully to eliminate pancreatic cancerous cells because TRPV1 is highly expressed
in this pancreatic cancer conditions [143–146]. Such
clinical application of agonists specific for different
TRP channels may, therefore, turn out to be effective
and has full potential as chemotherapeutics. Based on
this approach, recently use of TRP agonists as therapeutics is becoming popular in ‘TRPpathies’ or ‘channelopathies’ [156].
Concluding remarks
The last few years have seen rapid progress in the
study of TRP channels as well as other ion channels in
the context of both actin and the microtubule cytoskeleton. The presence of the microtubule cytoskeleton at
the membrane is now beyond doubt [157]. The role of
microtubule plus-end-binding proteins for specific sorting and targeting of different ion channels, receptors
to specific regions of the membrane is well established
[158]. However, the functional implications of this
remain one of the current challenges. We find the role
of the cytoskeleton to be both direct and indirect. In
particular, the inside-out modulation of ion channels
emerges as a peculiarly novel aspect with wide ranging
consequences for both the pathological and the general
homeostatic state. Because the large extended structure
of the cytoskeleton is able to potentially integrate
signalling events from very distant sides, single signals
as well as associative stimuli will have to be investigated. In addition, the cytoskeleton has been proven
to be both a target of signalling cascades and to initiate them itself. Thus it has the potential to recruit
feedback and feed-forward regulation of a number of
cellular effects. This renders the cytoskeleton as an
interesting target for therapeutic approaches with
respect to the TRP channels with much to discover.
Acknowledgements
We thank Julia Kuhn for critically reading this minireview and for her suggestions. We thank Prof.
H. H. Ropers for supporting this work. Funding from
Max Planck Institute for Molecular Genetics (Berlin,
Germany) is gratefully acknowledged. We regret for
FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS
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C. Goswami and T. Hucho
not been able to mention many of the relevant citations due to space limitation.
13
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