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, Ca 2+ -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 Ca 2+ . 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 Ca 2+ concentrations [28]. TRPV1 channels are nonselective cation channels. Therefore, the role of increased concentration of Ca 2+ on the properties of TRPV1–tubulin and ⁄ or TRPV1– microtubule complex is of special interest. Tubulin binding to TRPV1-Ct is increased by increased Ca 2+ concentrations [28]. Interestingly, the microtubules formed with TRPV1-Ct in the presence of Ca 2+ become ‘cold stable’ as these microtubules do not dep- olymerise further at low temperature [28]. The exact mechanism how Ca 2+ 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 Ca 2+ ions to its C-terminal sequence [35–38] and thus Ca 2+ -dependent conformational changes of tubulin [39] may underlie the observed effects of Ca 2+ . 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, Ca 2+ . Indeed, high Ca 2+ 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 Ca 2+ on microtubules, or indirectly by a calcium-induced but signal-cascade-dependent depolymerization [46]. Also, chelating extracellular Ca 2+ with EGTA and depletion of intracellular Ca 2+ 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 Ca 2+ concentrations. Even com- bined EGTA and thapsigargin, treatment cannot exclude small changes in local Ca 2+ concentration. Therefore, these small changes in Ca 2+ 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 Ca 2+ , but not in the complete absence of Ca 2+ [45,48–50]. Subsequent activation of Ca 2+ -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 Ca 2+ 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 and growth cones (Fig. 4) [47,62]. Activation of TRPV1 results in rapid disassembly of microtubules within neurites (and also at growth cones) while keep- ing the actin cytoskeleton intact and functional. This destroys the balance between the anterograde force (generated by microtubule cytoskeleton) and the retro- grade force (generated by actin cytoskeleton) that determines the axonal morphology and the net neurite growth [63,64]. Sudden loss of polymerized micro- tubules results in retraction of growth cones and for- mation of varicosities all along the neurites (Fig. 5). Long-term low-level TRPV1 activation by an endo- genous ligand results in shortening of neurites in pri- mary 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 pres- ence of TRPV1 at the sperm acrosome and throughout the tail has been reported [69]. Short-term and low- level activation may increase sperm motility whereas i A C D B ii iii Fig. 4. Effect of TRPV1–cytoskeletal cross-talk on neuritis and growth cones. (A) At growth cones, TRPV1-enriched plasma membranes sta- bilize 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 applica- tion 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. 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 4689 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 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 lamellipo- dia 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 meas- ured by FRET to be < 4 nm [75] assuring, that these two components have the potentiality to inter- act with each other. In addition, TRPV4 has been identified by yeast two-hybrid screen to interact with MAP7, an interaction confirmed by immunoprecipi- tation as well as pull-down experiments [76]. This interaction is dependent on the C-terminal amino acids 798–809. Interestingly, the C-terminal cyto- plasmic 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 puri- fied ‘signalplex’ [1]. TRPC5 interacts with stathmin 2, a microtubule cytoskeleton-binding protein [78]. Direct physical and functional interplay with both the micro- tubule 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 associa- tion and complex signalling with the cytoskeleton. Again, activation of these TRP channels correlates A C B 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 cytoskele- ton (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 mem- brane 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 varicos- ity formation is dependent on the degree of microtubule disassem- bly. (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 retrac- tion and initiates varicosity formation. (Stage 3) Complete disas- sembly 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. TRP channels and cytoskeleton regulate each other C. Goswami and T. Hucho 4690 FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 reorgani- zation of cortical actin network [79]. Likewise, Xeno- pus 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 guid- ance 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 chan- nels with the cytoskeleton, there are few examples sug- gesting 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 Ca 2+ entry and⁄ or store-operated Ca 2+ entry has been reported [86–88]. In human plate- lets, physical coupling of hTRP1 with inositol 1,4,5-tri- phosphate receptor (IP3R), a prerequisite step for store-mediated Ca 2+ entry, depends on the degree of polymerized actin [86,87]. Disruption of the actin cyto- skeleton by cytochalasin D also prevents phosphatidyl- inositol 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 store- operated calcium entry. Apparently, an intact actin cytoskeleton is also essential for strong agonist-medi- ated activation of TRPC7. Thus, pharmacological dis- ruption of the actin cytoskeleton results in reduced agonist-induced activation [90]. All these examples sug- gest that the cytoskeleton can indeed act as modulators of TRP channel function. 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, muta- tions 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 neu- ron-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 myo- sins 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 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 4691 in an increase expression of beta-myosin heavy chain [113]. Such phenomena strongly suggest the coopera- tive 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 deg- radation; a higher level of endogenous myosin might also imply less proteolytic degradation. It is therefore worth exploring how TRP channels affect the distribu- tion, function and endogenous level of myosins. Modulation of TRP ion channels activities by the cytoskeleton TRP channels can be modulated by Ca 2+ -dependent or Ca 2+ -independent mechanisms. Desensitization can be initiated by the Ca 2+ -influx through the channel itself and is manifested through phosphorylation– dephosphorylation of the TRP channel and ⁄ or by the Ca 2+ -dependent interaction with calmodulin at the C-termini of, for example, TRPV1 and TRPV4 [114– 121]. As an example of Ca 2+ -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 cir- cumstantial. Modulation of TRPV4 by a cytosolic component is suggested, as the channel can be acti- vated by heat only if analysed using whole-cell record- ings and not in excised patches of cell-free membranes [122,123]. In turn, the involvement of cytoskeletal com- ponents in the regulation of TRPV4 channel activity has been demonstrated experimentally by the addition of cytoskeletal regulating drugs. The microtubule stabi- lizer taxol reduces TRPV4-dependent currents while the microtubule-disrupting agents colchicine and vin- cristine as well as actin cytoskeleton regulating drugs like phalloidin (a stabilizer) or cytochalasin B (a desta- bilizer) 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, disrup- tion of actin or microtubule cytoskeleton by pharma- cological agents greatly reduces the activity of mechanosensitive channels [124]. However, if the 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 modu- latory 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 per- formed 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 compo- nents a-actinin and gelsolin, tubulin including acety- lated a-tubulin, and the kinesin motor proteins KIF3A and KIF3B in these membranes [125,126]. PC2 chan- nels interact directly with KIF3. Disruption of actin filaments with cytochalasin D or with the actin-sever- ing protein gelsolin activates the channel. This activa- tion 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. Depoly- merization of microtubules with colchicine rapidly inhibits the basal level of PC2 channel activity, whereas polymerization and ⁄ or stabilization of micro- tubules from soluble tubulin with GTP and taxol stim- ulates 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 abol- ished channel activity, whereas the addition of micro- tubule stabilizers (taxol) increased channel activity [127]. Similar results were obtained using reconstituted lipid bilayer system, which reveals that both spontane- ous activity and the opening probability of TRPP3 ion channels is increased by the addition of a-actinin, dem- onstrating 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 TRP channels and cytoskeleton regulate each other C. Goswami and T. Hucho 4692 FEBS Journal 275 (2008) 4684–4699 ª 2008 The Authors Journal compilation ª 2008 FEBS example, rapidly dividing cancer cells are pharmacolog- ically targeted by modulators of the microtubule cyto- skeleton 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 depen- dent on both the ‘integrity’ and the ‘dynamics’ of the microtubule cytoskeleton [135,136]. Short-term modula- tion of the cytoskeleton abolishes inflammatory media- tor-induced sensitization [135]. The precise mechanism by which vinca-drugs and taxoid-group-containing mol- ecules 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 chan- nels in the development of cancer and cancer pain is increasingly prominent [22,140–142]. Endogenous expressions of some TRP channels are either upregu- lated 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 mechano- sensation, has been shown to be essential for the devel- opment 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]. How- ever, 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]. However, the deleterious effect of TRP channels on the specific subtype of neurons or cells has some clini- cal advantages. In fact, retraction and degeneration of a subset of sensory neurons (specifically TRPV1- expressing neurons), which involve events that affect the integrity of the cytoskeleton, forms the basis for the analgesic effect of topical capsaicin-cream treat- ment [155]. Resinferatoxin (RTX), a potent agonist of TRPV1 has been used successfully to eliminate pancre- atic 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 thera- peutics is becoming popular in ‘TRPpathies’ or ‘chan- nelopathies’ [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 cytoskel- eton. The presence of the microtubule cytoskeleton at the membrane is now beyond doubt [157]. The role of microtubule plus-end-binding proteins for specific sort- ing 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 investi- gated. In addition, the cytoskeleton has been proven to be both a target of signalling cascades and to initi- ate 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 mini- review 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 C. Goswami and T. 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TRPV1 and the cytoskeleton Physical interaction of TRPV1 with the cytoskeleton TRPV1 is the founding member of the. progress in the study of TRP channels as well as other ion channels in the context of both actin and the microtubule cytoskel- eton. The presence of the microtubule cytoskeleton at the membrane. MINIREVIEW Submembraneous microtubule cytoskeleton: biochemical and functional interplay of TRP channels with the cytoskeleton Chandan Goswami and Tim Hucho Department for Molecular