Primary cilia are not calcium-responsive mechanosensors.

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium. This Ca(2+)-responsive mechanosensor hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers. Here we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. We developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca(2+) influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signalling.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Novel mutation in the TOR1A (DYT1) gene in atypical early onset dystonia and polymorphisms in dystonia and early onset parkinsonism.

Dystonia is a movement disorder involving sustained muscle contractions and abnormal posturing with a strong hereditary predisposition and without a distinct neuropathology. In this study the TOR1A (DYT1) gene was screened for mutations in cases of early onset dystonia and early onset parkinsonism (EOP), which frequently presents with dystonic symptoms. In a screen of 40 patients, we identified three variations, none of which occurred in EOP patients. Two infrequent intronic single base pair (bp) changes of unknown consequences were found in a dystonia patient and the mother of an EOP patient. An 18-bp deletion (Phe323_Tyr328del) in the TOR1A gene was found in a patient with early onset dystonia and myoclonic features. This deletion would remove 6 amino acids close to the carboxy terminus, including a putative phosphorylation site of torsinA. This 18-bp deletion is the first additional mutation, beyond the GAG-deletion (Glu302/303del), to be found in the TOR1A gene, and is associated with a distinct type of early onset dystonia.

Myosin-VIIb, a novel unconventional myosin, is a constituent of microvilli in transporting epithelia.

Mouse myosin-VIIb, a novel unconventional myosin, was cloned from the inner ear and kidney. The human myosin-VIIb (HGMW-approved symbol MYO7B) sequence and exon structure were then deduced from a human BAC clone. The mouse gene was mapped to chromosome 18, approximately 0.5 cM proximal to D18Mit12. The human gene location at 2q21.1 was deduced from the map location of the BAC and confirmed by fluorescence in situ hybridization. Myosin-VIIb has a conserved myosin head domain, five IQ domains, two MyTH4 domains coupled to two FERM domains, and an SH3 domain. A phylogenetic analysis based on the MyTH4 domains suggests that the coupled MyTH and FERM domains were duplicated in myosin evolution before separation into different classes. Myosin-VIIb is expressed primarily in kidney and intestine, as shown by Northern and immunoblot analyses. An antibody to myosin-VIIb labeled proximal tubule cells of the kidney and enterocytes of the intestine, specifically the distal tips of apical microvilli on these transporting epithelial cells.

Transport and localization of the DEG/ENaC ion channel BNaC1alpha to peripheral mechanosensory terminals of dorsal root ganglia neurons.

Mammalian brain sodium channel (BNaC, also known as BNC/ASIC) proteins form acid-sensitive and amiloride-blockable sodium channels that are related to putative mechanosensory channels. Certain BNaC isoforms are expressed exclusively in dorsal root ganglia (DRG) and have been proposed to form the ion channels mediating tissue acidosis-induced pain. With antibody labeling, we find that the BNaC1alpha isoform is expressed by most large DRG neurons (low-threshold mechanosensors not involved in acid-induced nociception) and few small nociceptor neurons (which include high-threshold mechanoreceptors). BNaC1alpha is transported from DRG cell bodies to sensory terminals in the periphery, but not to the spinal cord, and is located specifically at specialized cutaneous mechanosensory terminals, including Meissner, Merkel, penicillate, reticular, lanceolate, and hair follicle palisades as well as some intraepidermal and free myelinated nerve endings. Accordingly, BNaC1alpha channels might participate in the transduction of touch and painful mechanical stimuli.

Two mechanisms for transducer adaptation in vertebrate hair cells.

Deflection of the hair bundle atop a sensory hair cell modulates the open probability of mechanosensitive ion channels. In response to sustained deflections, hair cells adapt. Two fundamentally distinct models have been proposed to explain transducer adaptation. Both models support the notion that channel open probability is modulated by calcium that enters via the transduction channels. Both also suggest that the primary effect of adaptation is to shift the deflection-response [I(X)] relationship in the direction of the applied stimulus, thus maintaining hair bundle sensitivity. The models differ in several respects. They operate on different time scales: the faster on the order of a few milliseconds or less and the slower on the order of 10 ms or more. The model proposed to explain fast adaptation suggests that calcium enters and binds at or near the transduction channels to stabilize a closed conformation. The model proposed to explain the slower adaptation suggests that adaptation is mediated by an active, force-generating process that regulates the effective stimulus applied to the transduction channels. Here we discuss the evidence in support of each model and consider the possibility that both may function to varying degrees in hair cells of different species and sensory organs.

Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin.

Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.

Insect mechanoreception: what a long, strange TRP it's been.

Insect bristles are model mechanosensory organs. An ion channel of the TRP superfamily has recently been identified which is required for production of mechanoreceptor currents by insect bristles, and seems likely to represent a new kind of mechanically gated channel.

Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors.

The introduction of foreign genes into cells has become an effective means of achieving intracellular expression of foreign proteins, both for therapeutic purposes and for experimental manipulation. Gene delivery to the nervous system has been extensively studied, primarily using viral vectors. However, to date less work has focused on gene delivery to the inner ear, and existing studies have primarily used adenovirus and adeno-associated virus. Using two recombinant viral vectors, herpes simplex type 1 (HSV-1), and vaccinia virus, bearing the Escherichia coli lacZ gene, we tested gene delivery to the guinea pig cochlea in vivo with beta-galactosidase staining as an assay. The HSV-1 and vaccinia virus vectors were both found to infect and elicit transgene expression successfully in many cells in the guinea pig cochlea, including cells in the organ of Corti. These data demonstrate the feasibility of gene delivery to the inner ear using these two viral vectors. Such techniques may facilitate study of the auditory systems, and might be used to develop gene therapy strategies for some forms of hearing loss.

TRP2: a candidate transduction channel for mammalian pheromone sensory signaling.

The vomeronasal organ (VNO) of terrestrial vertebrates plays a key role in the detection of pheromones, chemicals released by animals that elicit stereotyped sexual and aggressive behaviors among conspecifics. Sensory transduction in the VNO appears unrelated to that in the vertebrate olfactory and visual systems: the putative pheromone receptors of the VNO are evolutionarily independent from the odorant receptors and, in contrast to vertebrate visual and olfactory transduction, vomeronasal transduction is unlikely to be mediated by cyclic-nucleotide-gated channels. We hypothesized that sensory transduction in the VNO might instead involve an ion channel of the transient receptor potential (TRP) family, members of which mediate cyclic-nucleotide-independent sensory responses in Drosophila melanogaster and Caenorhabditis elegans and play unknown functions in mammals. We have isolated a cDNA (rTRP2) from rat VNO encoding a protein of 885 amino acids that is equally distant from vertebrate and invertebrate TRP channels (10-30% amino acid identity). rTRP2 mRNA is exclusively expressed in VNO neurons, and the protein is highly localized to VNO sensory microvilli, the proposed site of pheromone sensory transduction. The absence of Ca2+ stores in sensory microvilli suggests that, in contrast to a proposed mechanism of activation of mammalian TRP channels, but in accord with analysis of TRP function in Drosophila phototransduction, the gating of TRP2 is independent from the depletion of internal Ca2+ stores. Thus, TRP2 is likely to participate in vomeronasal sensory transduction, which may share additional similarities with light-induced signaling in the Drosophila eye.

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.

To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.

The TOR1A (DYT1) gene family and its role in early onset torsion dystonia.

Most cases of early onset torsion dystonia are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A gene (alias DYT1, DQ2), resulting in loss of a glutamic acid in the carboxy terminal of the encoded protein, torsin A. TOR1A and its homologue TOR1B (alias DQ1) are located adjacent to each other on human chromosome 9q34. Both genes comprise five similar exons; each gene spans a 10-kb region. Mutational analysis of most of the coding region and splice junctions of TOR1A and TOR1B did not reveal additional mutations in typical early onset cases lacking the GAG deletion (N = 17), in dystonic individuals with apparent homozygosity in the 9q34 chromosomal region (N = 5), or in a representative Ashkenazic Jewish individual with late onset dystonia, who shared a common haplotype in the 9q34 region with other late onset individuals in this ethnic group. A database search revealed a family of nine related genes (50-70% similarity) and their orthologues in species including human, mouse, rat, pig, zebrafish, fruitfly, and nematode. At least four of these genes occur in the human genome. Proteins encoded by this gene family share functional domains with the AAA/HSP/Clp-ATPase superfamily of chaperone-like proteins, but appear to represent a distinct evolutionary branch.

The usher syndromes.

Mutations in the gene (MYO7A) encoding myosin-VIIa, a member of the large superfamily of myosin motor proteins that move on cytoplasmic actin filaments, and in the USH2A gene, which encodes a novel protein resembling an extracellular matrix protein or a cell adhesion molecule, both cause Usher syndrome (USH), a clinically heterogeneous autosomal recessive disorder comprising hearing and visual impairment. Patients with USH1 have severe to profound congenital hearing impairment, vestibular dysfunction, and retinal degeneration beginning in childhood, while those with USH2 have moderate to severe hearing impairment, normal vestibular function, and later onset of retinal degeneration. USH3 is characterized by progressive hearing loss and variable age of onset of retinal degeneration. The phenotype resulting from MYO7A and USH2A mutations is variable. While most MYO7A mutations cause USH1, some cause nonsyndromic hearing impairment, and one USH3 phenotype has been described. USH2A mutations cause atypical USH as well as USH2. MYO7A is on chromosome region 11q13 and USH2A is on 1q41. Seven other USH genes have been mapped but have not yet been identified. USH1A, USH1C, USH1D, USH1E, and USH1F have been assigned to chromosome bands 14q32, 11p15.1, 10q, 21q21, and 10, respectively, while USH2B is on 5q, and USH3 is at 3q21-q25. Myosin VIIa mutations also result in the shaker-1 (sh1) mouse, providing a model for functional studies. One possibility is that myosin-VIIa is required for linking stereocilia in the sensory hair bundle; another is that it may be needed for membrane trafficking. The ongoing studies of myosin-VIIa, the USH2A protein, and the yet to be identified proteins encoded by the other USH genes will advance understanding of the Usher syndromes and contribute to the development of effective therapies. Am. J. Med. Genet. (Semin. Med. Genet.) 89:158-166, 1999.

Localization of myosin-Ibeta near both ends of tip links in frog saccular hair cells.

Current evidence suggests that the adaptation motor of mechanoelectrical transduction in vertebrate hair cells is myosin-Ibeta. Previously, confocal and electron microscopy of bullfrog saccular hair cells using an anti-myosin-Ibeta antibody labeled the tips of stereocilia. We have now done quantitative immunoelectron microscopy to test whether myosin-Ibeta is enriched at or near the side plaques of tip links, the proposed sites of adaptation, using hair bundles that were serially sectioned parallel to the macular surface. The highest particle density occurred at stereocilia bases, close to the cuticular plate. Also, stereocilia of differing lengths had approximately the same number of total particles, suggesting equal targeting of myosin-Ibeta to all stereocilia. Finally, particles tended to clump in clusters of two to five particles in the distal two-thirds of stereocilia, suggesting a tendency for self-assembly of myosin-Ibeta. As expected from fluorescence microscopy, particle density was high in the distal 1 micrometer of stereocilia. If myosin-Ibeta is the adaptation motor, a difference should exist in particle density between regions containing the side plaque and those excluding it. Averaging of particle distributions revealed two regions with approximately twice the average density: at the upper ends of tip links in a 700-nm-long region centered approximately 100 nm above the side plaque, and at the lower ends of tip links within the tip plaques. Controls demonstrated no such increase. The shortest stereocilia, which lack side plaques, showed no concentration rise on their sides. Thus, the specific localization of myosin-Ibeta at both ends of tip links supports its role as the adaptation motor.

The nematode degenerin UNC-105 forms ion channels that are activated by degeneration- or hypercontraction-causing mutations.

Nematode degenerins have been implicated in touch sensitivity and other forms of mechanosensation. Certain mutations in several degenerin genes cause the swelling, vacuolation, and death of neurons, and other mutations in the muscle degenerin gene unc-105 cause hypercontraction. Here, we confirm that unc-105 encodes an ion channel and show that it is constitutively active when mutated. These mutations disrupt different regions of the channel and have different effects on its gating. The UNC-105 channels are permeable to small monovalent cations but show voltage-dependent block by Ca2+ and Mg2+. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field. Mammalian cells expressing the mutant channels accumulate membranous whorls and multicompartment vacuoles, hallmarks of degenerin-induced cell death across species.

Mechanoelectrical transduction and adaptation in hair cells of the mouse utricle, a low-frequency vestibular organ.

Hair cells of inner ear organs sensitive to frequencies above 10 Hz adapt to maintained hair bundle deflections at rates that reduce their responses to lower frequencies. Mammalian vestibular organs detect head movements at frequencies well below 10 Hz. We asked whether hair cells of the mouse utricle adapt, and if so, whether the adaptation was similar to that in higher frequency organs such as the frog saccule. Whole-cell transduction currents were recorded from hair cells in the epithelium of the mouse utricle. Hair bundles were deflected by a fluid jet or a stiff probe. The transduction currents evoked by step deflections adapted over 10-100 msec. The mean operating range was 1.5 micron (deflection of the tip of the bundle), approximately threefold larger than in frog saccule. Taller and more compact bundles of the mouse utricle account for this difference. As in frog saccular hair cells, adaptation shifted the current-deflection (I(X)) relation along the deflection axis. These adaptive shifts had time constants of 10-20 msec and reached 60-80% of stimulus amplitude. The adaptive shift and voltage-dependent bundle movement are consistent with the motor model of adaptation. When the fluid jet was used, adaptation also broadened the I(X) relation and reduced the maximum current. Adaptation attenuated the transduction currents evoked by sinusoidal bundle deflections below 5 Hz, within the frequency range of the utricle, but because it was incomplete, substantial responses remained. Moreover, the adaptive shift mechanism preserves sensitivity even in the presence of large stimuli that would otherwise saturate transduction.

The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein.

Early-onset torsion dystonia is a movement disorder, characterized by twisting muscle contractures, that begins in childhood. Symptoms are believed to result from altered neuronal communication in the basal ganglia. This study identifies the DYT1 gene on human chromosome 9q34 as being responsible for this dominant disease. Almost all cases of early-onset dystonia have a unique 3-bp deletion that appears to have arisen idependently in different ethnic populations. This deletion results in loss of one of a pair of glutamic-acid residues in a conserved region of a novel ATP-binding protein, termed torsinA. This protein has homologues in nematode, rat, mouse and humans, with some resemblance to the family of heat-shock proteins and Clp proteases.