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...

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...

Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear.

Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of nonselective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mocktransfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.