We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions.There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample but a true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been yet achieved. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, that allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . However, until now, SICM was restricted to imaging relatively flat surfaces, like all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen (Fig. 1a). Here we report a novel mode of SICM that allows imaging of even the most convoluted surface structures at the nanoscale. SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM a nanopipette is mounted on a three-dimensional piezo and automatic feedback control moves the pipette up or down to keep the pipette current at a constant level (the set point), while the sample is scanned in X-Y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In our hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features (Fig. 1b). The reference current is measured while the pipette is well away from the surface. The pipette then approaches until the current is reduced by a predefined amount, usually 0.25-1%. The Z-position when the current achieves this reduction is recorded as the height of the sample at this imaging point. Typically, even at a 1% reduction of the current, the pipette is still at a distance of about one inner pipette radius from the surface. Therefore, the probe never touches the surface of the cell. The pipette is then withdrawn away from the surface and the sample moved laterally to determine the next imaging point. By continuously upd...
Stereocilia are microvilli-derived mechanosensory organelles that are arranged in rows of graded heights on the apical surface of inner-ear hair cells. The 'staircase'-like architecture of stereocilia bundles is necessary to detect sound and head movement, and is achieved through differential elongation of the actin core of each stereocilium to a predetermined length. Abnormally short stereocilia bundles that have a diminished staircase are characteristic of the shaker 2 (Myo15a(sh2)) and whirler (Whrn(wi)) strains of deaf mice. We show that myosin-XVa is a motor protein that, in vivo, interacts with the third PDZ domain of whirlin through its carboxy-terminal PDZ-ligand. Myosin-XVa then delivers whirlin to the tips of stereocilia. Moreover, if green fluorescent protein (GFP)-Myo15a is transfected into hair cells of Myo15a(sh2) mice, the wild-type pattern of hair bundles is restored by recruitment of endogenous whirlin to the tips of stereocilia. The interaction of myosin-XVa and whirlin is therefore a key event in hair-bundle morphogenesis.
Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation.
Sensorineural hearing loss is genetically heterogeneous. Here we report that mutations in CIB2, encoding a Ca2+- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). There is one mutation of CIB2 that is a prevalent cause of DFNB48 deafness in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In rodents, CIB2 is localized in the mechanosensory stereocilia of inner ear hair cells and in retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of Ca2+ binding, CIB2 significantly decreased the ATP-induced Ca2+ responses in heterologous cells, while DFNB48 mutations altered CIB2 effects on Ca2+ responses. Furthermore, in zebrafish and Drosophila, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We show that CIB2 is a new member of the vertebrate Usher interactome.
SUMMARY Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets, but distinct from bundles formed by espin, an actin cross-linker in stereocilia. We generated mutant Triobp mice (TriobpΔex8/Δex8) that are profoundly deaf. Stereocilia of TriobpΔex8/Δex8 mice develop normally, but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.
cyto-Actin and ␥cyto-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that ␥cyto-actin null mice (Actg1 ؊/؊ ) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of cyto-actin. The surprising viability and normal hearing of young Actg1 ؊/؊ mice means that cyto-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although ␥cyto-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, ␥cyto-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1 ؊/؊ stereocilia similar disruptions are observed even without noise exposure. We conclude that ␥cyto-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.actin ͉ cytoskeleton ͉ hearing T here are six genes encoding six vertebrate actins that are classified according to where they are predominately expressed. ␣ skeletal -Actin, ␣ smooth -actin, ␣ cardiac -actin, and ␥ smoothactin are primarily found in muscle cells, whereas cytoplasmic  cyto -actin and ␥ cyto -actin are ubiquitously and highly expressed in non-muscle cells, as reviewed elsewhere (1). Athough  cytoactin and ␥ cyto -actin differ at only four biochemically similar amino acid residues in their N-termini, several lines of evidence suggest that each protein is functionally distinct. The amino acid sequences of  cyto -and ␥ cyto -actin are each exactly conserved among avian and mammalian species. In addition,  cyto -and ␥ cyto -actin proteins are differentially localized (2-5) and posttranslationally modified (6). Finally, although dominant missense mutations in ACTB encoding  cyto -actin are associated with syndromic phenotypes including severe developmental malformations and bilateral deafness (7), humans carrying a variety of dominant missense mutations in ACTG1 develop postlingual nonsyndromic progressive hearing loss (DFNA20, OMIM 604717) (8-11).␥ cyto -Actin is widely expressed in the inner ear sensory epithelial cells on which mammalian hearing depends. These cells are organized in rows along the cochlea length: one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) (Fig. 2A). IHCs function as auditory receptors, converting sound energy into neuronal signals, whereas OHCs enhance sensitivity to sound stimuli, as reviewed elsewhere (12). The apical surface of a hair cell is topped with actin-rich microvilli-derived protrusions termed stereocilia, which deflect in response to sound stimuli, initiating mechanoelectrical transduction (Fig. 2B).  cyto -and ␥ cyto -Actin are both thou...
Generation of a strong electrical potential in the cochlea is uniquely mammalian and may reflect recent evolutionary advances in cellular voltage-dependent amplifiers. This endocochlear potential is hypothesized to dramatically improve hearing sensitivity, a concept that is difficult to explore experimentally, because manipulating cochlear function frequently causes rapid degenerative changes early in development. Here, we examine the deafness phenotype in adult Claudin 11-null mice, which lack the basal cell tight junctions that give rise to the intrastrial compartment and find little evidence of cochlear pathology. Potassium ion recycling is normal in these mutants, but endocochlear potentials were below 30 mV and hearing thresholds were elevated 50 dB sound pressure level across the frequency spectrum. Together, these data demonstrate the central importance of basal cell tight junctions in the stria vascularis and directly verify the two-cell hypothesis for generation of endocochlear potential. Furthermore, these data indicate that endocochlear potential is an essential component of the power source for the mammalian cochlear amplifier.
Do not touch! The surface of a living cell is soft and responsive and therefore high‐resolution imaging of the cell membrane has not been possible to date. Now noncontact imaging of protein complexes in the plasma membrane of living cells has been demonstrated (see picture) and has been used to follow the cells' structural reorganization. This breakthrough opens up a wealth of new experiments in membrane and cell biology.
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