TRPV1 expression-dependent initiation and regulation of filopodia C. Goswami and T. Hucho Signal Transduction in Pain and Mental Retardation, Department of Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany Abstract Transient receptor potential vanilloid subtype 1 (TRPV1), a non-selective cation channel, is present endogenously in dorsal root ganglia (DRG) neurons. It is involved in the rec- ognition of various pain producing physical and chemical stimuli. In this work, we demonstrate that expression of TRPV1 induces neurite-like structures and filopodia and that the expressed protein is localized at the filopodial tips. Exogenous expression of TRPV1 induces filopodia both in DRG neuron-derived F11 cells and in non-neuronal cells, such as HeLa and human embryonic kidney (HEK) cells. We find that some of the TRPV1 expression-induced filopodia contain microtubules and microtubule-associated components, and establish cell-to-cell extensions. Using live cell microscopy, we demonstrate that the filopodia are responsive to TRPV1- specific ligands. But both, initiation and subsequent cell-to-cell extension formation, is independent of TRPV1 channel activ- ity. The N-terminal intracellular domain of TRPV1 is sufficient for filopodial structure initiation while the C-terminal cyto- plasmic domain is involved in the stabilization of microtubules within these structures. In addition, exogenous expression of TRPV1 results in altered cellular distribution and in enhanced endogenous expression of non-conventional myosin motors, namely myosin IIA and myosin IIIA. These data indicate a novel role of TRPV1 in the regulation of cellular morphology and cellular contact formation. Keywords: cytoskeletal rearrangement, filopodia, micro- tubules, myosin, neurite, TRPV. J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04846.x Neurons are distinct from most other cells by their extreme morphology. Axonal and dendritic structures show particular cytoskeletal composition, morphology, and function. Through these structures neurons are able to establish complex functional networks by connecting a large number of cells. The precise formation of cell-to-cell connections depends on many factors in a multi-step process, namely: ‘protrusion,’ i.e., initiation of small membranous structures (initiation of filopodia), ‘engorgement,’ i.e., advancement of microtubules within the protrusion thereby facilitating trans- port of membranous organelles (Burmeister et al. 1991), and finally ‘consolidation,’ i.e., disassembly of actin and shrink- ing of membranes around microtubules [reviewed in Dent and Gertler (2003); Jontes and Smith (2000), Song and Poo (2001)]. After extension toward neighboring cells, contact formation including the establishment of synapses is initi- ated. Filopodial structures extending from axonal growth cones, from dendrites and from the cell body are similar in many aspects. All are formed by rearrangement of the actin cytoskeleton and are involved in ‘chemotaxis’ leading to the formation of cellular contacts [reviewed in Faix and Rottner (2006); Small and Resch (2005); Mitchison and Cramer (1996); Small et al. (2002); Wood and Martin (2002); da Silva and Dotti (2002)]. Although several cytoskeletal and regulatory proteins are known to play a role in filopodial initiation and regulation, a large number of filopodial proteins remain unidentified. The complex process of cell–cell contact formation by developing filopodia requires the involvement of structural proteins and regulatory factors including intracellular Ca 2+ . It has been shown that filopodial Ca 2+ -transients and Ca 2+ - spikes determine the nature and function of these structures (Goldberg and Grabham 1999; Gomez and Spitzer 2000; Conklin et al. 2005). Though the participation of a variety of Ca 2+ -binding proteins, and Ca 2+ channels in the regulation of filopodial dynamics has been established, the identity of individual calcium channels and their contribution in this complex signaling event remains to be elucidated (Gomez et al. 2001; Robles et al. 2003; Henley and Poo 2004; Lohmann et al. 2005; Gomez and Zheng 2006). Previously we showed that TRPV1, a Ca 2+ -permeable non-selective cation channel, interacts with tubulin via its C-terminal cytoplasmic domain (Goswami et al. 2004). We demonstrated that the C-terminal cytoplasmic domain of Received April 19, 2007; revised manuscript received June 25, 2007; accepted June 27, 2007. Address correspondence and reprint requests to C. Goswami, Signal Transduction in Pain and Mental Retardation, Department of Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Ihnestraße. 73, 14195, Berlin, Germany. E-mail: goswami@molgen.mpg.de Abbreviations used: DRG, dorsal root ganglia; GFP, green flourescent protein; HEK, human embryonic kidney; PBS, phosphate-buffered saline; PFA, paraformaldehyde; TRPV1, Transient receptor potential vanilloid subtype 1. Journal of Neurochemistry , 2007 doi:10.1111/j.1471-4159.2007.04846.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04846.x 1