My oh my(osin): Insights into how auditory hair cells count,measure, and shape

The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle’s morphology and hearing.The mammalian cochleae are lined with hair cells, each with a precisely arranged, morphologically polar hair bundle that captures energy produced by sound stimuli. A fascinating problem in cell biology is how this elaborately structured mechanosensory hair bundle forms with precision to enable hearing. Each hair bundle is comprised of up to a few hundred actin-filled stereocilia, arrayed in a staircase-like pattern of increasing heights. Deflection of a hair bundle in the direction of the tallest stereocilium opens mechanically gated ion channels at the stereociliary tips, allowing an influx of cations from the endolymph bathing the stereocilia and thus depolarizing the cell. The staircase array of stereocilia forms by elongation of microvilli at the apical surface of the developing hair cell (Tilney et al., 1992). Over 20 years ago, Tilney et al. (1992) detailed the steps of stereociliogenesis as observed by electron microscopy. In this issue, Lelli et al. provide molecular insight into how hair cells count, measure, and shape stereocilia.Loss of hair cells is a major cause of human hearing loss, which is often the result of genetic mutations affecting the development of stereocilia (Raphael, 2002). Mutations in genes encoding several different myosin motor proteins in stereocilia have been associated with human hearing loss, including myosin VIIA (Weil et al., 1995), myosin VI (Avraham et al., 1995; Melchionda et al., 2001), and myosin XVA (Wang et al., 1998). Additionally, loss-of-function mutations in MYO3A, encoding myosin IIIA, are responsible for hereditary progressive hearing loss DFNB30 (Walsh et al., 2002).Myosins, the ATP-dependent motors that move along actin-based filaments, are typically composed of three functional domains: the head, the neck, and the tail domains (Krendel and Mooseker, 2005). Class III myosins are unconventional myosins that each have a kinase domain at their N terminus regulated by PKA phosphorylation and autophosphorylation (Kempler et al., 2007). The kinase activity of myosin III was shown to act on its own motor domain to reduce the motor activity. Myosin III proteins concentrate at actin-based cellular protrusions, such as stereocilia of inner ear hair cells (Dosé et al., 2003; Schneider et al., 2006). The two class III isoforms found in vertebrates, myosin IIIa and IIIb, differ toward their C termini: myosin IIIa is longer than myosin IIIb and has one additional actin-binding domain, myosin III tail homology domain II (3THDII). In cultured cells, whereas myosin IIIa localizes to the tips of filopodia on its own, previous work showed that myosin IIIb requires its interaction partner espin-1, an actin-binding protein, for localization to filopodia tips (Merritt et al., 2012). Mutagenesis studies performed on myosin IIIa revealed a relationship between the phosphorylation state of myosin IIIa and the length and density of filopodia (Quintero et al., 2013). How myosin IIIa and IIIb work individually or together to regulate stereociliogenesis remains to be investigated.In this issue, Lelli et al. (2016) use Myo3a (Myo3a^−/−), Myo3b (Myo3b^−/−), and double (Myo3a^−/−Myo3b^−/−) knockout mice to dissect the complex roles of myosin IIIa and IIIb in hearing. Mice null for myosin IIIa, Myo3a^−/−, were initially normal but showed defects in hearing quality from 2 to 4 mo of age, as detected by auditory brain stem response measurements, which monitor the electrical response of the auditory pathway to short sound stimuli, but not by distortion product otoacoustic emissions, which test outer hair cell (OHC) function. These results indicated that inner hair cells (IHCs) are deleteriously impacted but that OHCs are not, and that these mice had progressive hearing loss. Myo3b^−/− mutants had normal hearing, indicating that redundancy with myosin IIIa may obscure the role of this protein in single knockouts. Investigation of double knockouts, however, clarified the redundant functions of the two isoforms, as the mice were profoundly deaf at 1 mo of age.Interestingly, Lelli et al. (2016) investigated the potential requirement for myosin IIIa in the maintenance of hearing. They generated conditional knockout (cKO) mice, Myo3a-cKO, in which myosin IIIa is inactivated postnatally in a Myo3b^−/− background. These animals hear normally up to at least 6 mo of age. Therefore, myosin IIIa is required during a critical period in auditory system development but is not required to maintain hearing. The researchers further studied the relationship between myosin IIIa and myosin IIIb in the Myo3a-cKO mice and noted an increase in auditory brain stem response thresholds. This fascinating result likely indicates that inactivation of myosin IIIa elicits a pernicious effect of myosin IIIb at mature stages. Determining the mechanism by which myosin IIIb damages hearing in the absence of myosin IIIa and whether it is due to its lack of a single 3THDII domain may yield insight into the roles of class III myosins in hearing.Given that myosin III proteins localize to the tips of stereocilia and are required for hearing, Lelli et al. (2016) explored the morphology of hair bundles lacking these proteins. Although double knockout Myo3a^−/−Myo3b^−/− mice displayed normal positioning of the kinocilium, the cilium reflecting the initial position and polarity of the developing hair bundle, and of asymmetric cell division proteins Par-6 and Gαi₃, which constrain the shape and positioning of the hair bundle, many hair cells from these mice exhibited hair bundle abnormalities. These abnormalities were first described during embryonic auditory hair bundle morphogenesis, at which time 19% of IHCs and 81% of OHCs displayed misshapen bundles. Several of these misshapen IHC and OHC bundles also contained abnormally long, ungraded protrusions, which Lelli et al. (2016) referred to as “long amorphous” bundles. By birth, most IHCs displayed long amorphous bundles. Although most OHC hair bundles were abnormally shaped, they had developed normal staircase organization of their stereocilia heights, and the long amorphous protrusions were no longer present. Presumably, another molecular mechanism comes into play and “corrects” the protrusions to establish the staircase organization. Knockout of candidate proteins could be an interesting strategy to uncover this mechanism in the future. Furthermore, other stereociliary phenotypes of Myo3a^−/−Myo3b^−/− mutant mice are also intriguing. Do myosin III proteins serve different mechanistic roles in IHCs and OHCs to give rise to different phenotypes? Or, are there other key factors that drive stereocilia into various degrees of hair bundle defects in the absence of myosin III proteins?In Myo3a^−/−Myo3b^−/− mice, many of the OHC bundles had abnormal side rows of extra stereocilia, which sometimes closed the bundle off into a circular shape. Interestingly, the stereocilia in these abnormal bundles were also significantly taller than in controls. Although the height of the tallest row of stereocilia in OHCs normally decreases after birth (Sekerková et al., 2011), the height of these stereocilia did not change in the Myo3a^−/−Myo3b^−/− mice, leading to an increased height difference 9 d after birth. The increased height and number of stereocilia in Myo3a^−/−Myo3b^−/− hair bundles is suggestive of unstable actin dynamics. The authors suggest that class III myosins stabilize the F-actin cores of the stereocilia, thus controlling their selective elongation by limiting their growth (Fig. 1). Surprisingly, class III myosins, which presumably climb the full lengths of the stereocilia to perch themselves near the tips, act to restrict their growth. This result seems to contrast with previous work in which myosin IIIa had been proposed to promote elongation of the stereocilia by transporting espin-1 to the stereocilia tips (Salles et al., 2009). Interestingly, Lelli et al. (2016) found that espin-1 was still properly targeted to the tips of stereocilia in Myo3a^−/−Myo3b^−/− mutant mice. The researchers extended the study to another binding partner of myosin IIIa, retinophilin/MORN4. Surprisingly, MORN4 was normally targeted to stereocilia tips in Myo3a^−/−Myo3b^−/− mutant mice. This suggests that there may be redundancy in the mechanisms of transport of stereocilia tip proteins. Whether or not distinct myosin isoforms interchange cargoes so that these cargoes are properly localized is a relevant avenue of future investigation. Furthermore, identification and knockout of class III myosin binding partners could help define the mechanism by which these proteins control elongation of stereocilia.Open in a separate windowFigure 1.Myosin IIIa and myosin IIIb regulate stereociliary length. (top) Schematics of myosin IIIa and myosin IIIb. The stop signs indicate the role these proteins have in limiting the heights of stereocilia. (bottom) A single stereocilium with wild-type copies of myosin IIIa and myosin IIIb is shorter than a stereocilium from a Myo3a^−/−Myo3b^−/− mutant when stereocilia from equivalent positions between hair bundles are compared. In addition to characterizing the lengths of stereocilia, Lelli et al. (2016) noted transient defects in the numbers of stereocilia during development of hair cells within genetically altered mice. At birth, Myo3a^−/−Myo3b^−/− mice had an increased number and density of stereocilia in both IHCs and OHCs as compared with controls. However, 9 d after birth, the number and density of the stereocilia had decreased to levels similar to those seen in control animals. Their results indicate that class III myosins help to control the initial selection of microvilli to become stereocilia, but a different, dominant molecular mechanism overrides this step and is likely involved in refining the number of stereocilia at later, postnatal stages. Identification of this mechanism by knockout of candidate molecules in the Myo3a^−/−Myo3b^−/− mice would be of interest for future work. The defects, though transient, indicate that myosins are key molecular components that establish how cells count the number of protrusions to be produced.In this work, Lelli et al. (2016) determined that myosins IIIa and IIIb are required for normal hair bundle development and hearing. These motor proteins influence the number and lengths of stereocilia to be produced and the overarching shape of the hair bundle. Evaluating the importance of the kinase domains of myosin IIIa and IIIb in hair bundle development is an important next step. The phosphorylation states of proteins in cellular protrusions, such as filopodia and stereocilia, can be regulatory. For example, phosphorylation of the actin cross-linking protein fascin 2b reduces this protein’s capacity to lengthen filopodia (Chou et al., 2011). Moreover, phosphorylation of fascin 2b increases the exchange rate of this protein within zebrafish stereocilia (Hwang et al., 2015). Additionally, in other systems, PKA modulates a signaling cascade that activates the actin-severing protein cofilin to control actin-filament dynamics (Nadella et al., 2009). Future work will be needed to test whether signal transduction stemming from the kinase domains of class III myosins are consequential for hair bundle development.Lelli et al. (2016) observed that the constitutive absence of both class III myosins leads to deafness as a result of hair bundle developmental defects. However, mice deficient for only one of the isoforms did not display such a striking hearing phenotype, indicating that myosins IIIa and IIIb compensate for the loss of one another in the developing cochlea. A role for myosin IIIa in the maintenance of stereocilia had been suggested by the discovery of loss-of-function mutations in MYO3A in patients with hereditary progressive hearing loss DFNB30 (Walsh et al., 2002). Remarkably, the analyses of Myo3a^−/−Myo3b^−/− and Myo3a-cKO Myo3b^−/− mice showed that the redundancy between myosins IIIa and IIIb is critical during hair bundle formation, but not during later, mature stages. Furthermore, the hearing impairment seen in Myo3a-cKO mice suggests that myosin IIIb exerts deleterious effects on hearing past the developmental stages of hair bundle morphogenesis. Although the mechanisms at work are unclear, these findings suggest that the down-regulation of MYO3B expression might be an effective way of preventing late-onset hearing loss in patients with MYO3A mutations.     

Mating behavior and time budget of an androdioecious crustacean,Eulimnadia texana (Crustacea: Conchostraca)

The clam shrimp,Eulimnadia texana (Crustacea, Conchostraca), is found in freshwater ephemeral environments throughtout the United States. Individual clam shrimp of this species are either hermaphroditic or male, a relatively rare mating system for animals known as androdioecy. Comparison of sex ratios between four neighboring populations ofE. texana in Southern New Mexico showed wide variation in the ratio of males to hermaphrodites with males making up as much as 42% of some populations and not occurring at all within others. Since little is known about the behavior of this species, an ethogram and time budget were prepared based on observations of laboratory populations. Males attempt to clasp hermaphrodites prior to mating. Precopulatory mate guarding occurs in this species. Outcrossing generally occurs during mate guarding and after the hermaphrodite molts. Hermaphrodites, however, seem to control the mating process. Successful mating by males never occured if the hermaphrodite struggled with him; hermaphrodite will self in the presence of males.     

Assessment of the Bioaccessibility of Trace Elements in Cat’s Claw Teas by In Vitro Simulated Gastrointestinal Digestion Using FAAS

Beer is a widely consumed drink throughout the world, and because its manufacture involves the use of water, beer can be, in some cases, a source of fluorides. For this reason, the objective of this study was to determine the concentration of fluorides in 50 samples of beers from different sources sold in two different types of container (aluminum can and glass bottle). The possible significant differences between the different types of packaging and the intake of fluoride from the consumption of these beers were evaluated. The concentration of fluoride in beers has been determined using the potentiometric method of fluoride determination by standard addition. The concentration of fluoride ranged between 0.06 and 1.77 mg/L. In general, the concentration was below 1 mg/L, except for three beer samples from Ireland and the USA, whose concentration was over 1.5 mg/L. No significant differences were found between the types of packaging. The contribution of fluoride to the diet from beer consumption is not high (<27%); however, it is necessary to warn consumers whenever they are in areas of high concentrations of fluoride in the water supply.     

Vitamin A in the Vision of Insects

Acetone-methanol extracts of honeybees (Apis mellifera) were chromatographed from petroleum ether on columns of aluminum oxide and magnesium oxide:celite. Vitamin A₁ was identified by the Carr-Price (antimony chloride) reaction. These experiments provide the first demonstration of vitamin A in the tissues of an insect. Like retinene, vitamin A is confined to the heads and is not found in either thoraces or abdomens. Dark-adapted bees have very little vitamin A. During light adaptation the vitamin A increases, but at the expense of retinene, which decreases. As much as 0.1 µg of vitamin A/gm of heads has been recovered from light-adapted bees. Two methods are described for demonstrating the enzymic reduction of retinene to vitamin A, using an extract of the heads of honeybees.     

Effect of secondary metabolites associated with anaerobic soil conditions on ion fluxes and electrophysiology in barley roots

The effects of secondary metabolites produced by waterlogged soils on net K(+), H(+), and Ca(2+) fluxes were studied in the mature zone of roots of two barley (Hordeum vulgare) cultivars contrasting in their waterlogging (WL) tolerance using the noninvasive microelectrode ion flux measuring technique. In WL-sensitive variety 'Naso Nijo', all three lower monocarboxylic acids (formic, acetic, and propionic acids) and three phenolic acids (benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic acids) caused a substantial shift toward steady K(+) efflux, accompanied by an immediate net influx of H(+). Detrimental effects of secondary metabolites on K(+) homeostasis in root cells were absent in WL-tolerant 'TX' variety. Root treatment with Mn(2+) caused only a temporary K(+) loss that returned to the initial level 10 min after treatment. Phenolic acids slightly increased Ca(2+) influx immediately after treatment, while other metabolites tested resulted in transient Ca(2+) efflux from the root. In the long-term (24 h) treatment, all metabolites tested significantly reduced K(+) uptake and the adverse effects of phenolic acids were smaller than for monocarboxylic acids and Mn(2+). Treatment with monocarboxylic acids for 24 h shifted H(+) from net efflux to net influx, while all three phenolic acids did not cause significant effects compared with the control. Based on results of pharmacological experiments and membrane potential measurements, a model explaining the effects of secondary metabolites on membrane transport activity is proposed. We also suggest that plant tolerance to these secondary metabolites could be considered a useful trait in breeding programs.     

Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagle dogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.     

Isolation and characterization of the Omp-PA porin from Porphyromonas asaccharolytica, determination of the omp-PA gene sequence and prediction of Omp-PA protein structure

A single monomeric porin, Omp-PA (37kDa), was isolated from the outer membrane of the gram-negative anaerobic rod Porphyromonas asaccharolytica. Further characterization revealed that this porin consists of two different fractions: a heat-modifiable fraction which in its denatured form migrated on SDS-PAGE as a protein with a molecular weight of 41kDa and a heat-resistant fraction which did not change its migration on SDS-PAGE after boiling. A liposome swelling assay revealed that only the heat-resistant fraction was able to transport sugars after its incorporation into the liposomes, although it did not discriminate between differently sized sugars. We hypothesize that the heat-modifiable fraction corresponds to the "closed" conformer of Omp-PA, whereas the heat-resistant fraction corresponds to the "open" conformer of the protein. Cloning of the omp-PA gene revealed an open reading frame of 1161 bases, with a predicted protein sequence of 387 amino acids. The mature protein consists of 366 amino acids with a calculated MW of 41,102Da and an estimated pI of 7.24. The C-terminal domain of Omp-PA is homologous to the characteristic OmpA signature domain (71% similarity with the OmpA consensus domain). Sequence comparison with other anaerobes from the Bacteroides family demonstrated homology across the entire ORF. Digestion of the P. asaccharolytica outer membrane analysis of trypsin-digested Omp-PA yielded two proteins migrating with apparent molecular weights of 37 and 27kDa. These data fully supported our hypothesis that the C-terminal domain of the two-domain "closed" conformer of Omp-PA was digested by trypsin, whereas the single domain beta-barrel "open" conformer was inaccessible to trypsin.