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 . 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 . 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) . If an α-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 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 . The tubulin-binding region of TRPV1 apparently is under high evolutionary pressure as its sequence is highly conserved in all TRPV1 orthologues . 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).
Figure 1. Characteristic of the tubulin-binding motifs located at the C-terminus of TRPV1. (A) The extreme C-terminus of both α- and β-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.
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Figure 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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TRPV1 preferably interacts through its C-terminal domain with β-tubulin and to a lesser extend also with α-tubulin thereby forming a high-molecular weight complex . This suggests stronger binding of TRPV1 to the plus end rather than the minus end of microtubules as the plus ends of microtubule protofilaments are decorated with β-tubulin. It is therefore tempting to speculate that TRPV1 may act as a microtubule plus-end-tracking protein (+TIP) . 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 α helices with positively charged residues at the surface . 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 α 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 . Remarkably, FHIT also binds to tubulin .
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 β-III tubulin (all markers for stable microtubules) interact with TRPV1-Ct . 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 . 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 nocodazol or increased Ca2+ concentrations .
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 . 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 . 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  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  (Fig. 3). As suggested by its preference to bind to the plus-end-exposed β-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  (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 . Tubulin and components attributed to stable microtubules (like acetylated tubulin and MAP2ab) were also observed within these thin filopodial structures .
Figure 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.
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Figure 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.
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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 γ-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 . Potentially, TRPV1 activation may even increase the amount of polymerized actin .
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 . 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-activation-induced microtubule disassembly is apparently not a 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 .
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 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 short-term stimulation of TRPV1 we have not observed any fragmented tubulin bands in western blot analysis . Last, but not least, TRPV1 activation-mediated inhibition of protein synthesis and endoplasmic reticulum fragmentation may also have impact on the microtubule integrity .
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 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 . 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 .
Figure 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.
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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 .
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 . Short-term and low-level activation may increase sperm motility whereas robust activation may cause a non-motile sperm due to complete disassembly of microtubules at the sperm tail.