Cochlear outer hair cell electromotility enhances organ of Corti motion on a cycle-by-cycle basis at high frequencies in vivo.

Mammalian hearing depends on an amplification process involving prestin, a voltage-sensitive motor protein that enables cochlear outer hair cells (OHCs) to change length and generate force. However, it has been questioned whether this prestin-based somatic electromotility can operate fast enough in vivo to amplify cochlear vibrations at the high frequencies that mammals hear. In this study, we measured sound-evoked vibrations from within the living mouse cochlea and found that the top and bottom of the OHCs move in opposite directions at frequencies exceeding 20 kHz, consistent with fast somatic length changes. These motions are physiologically vulnerable, depend on prestin, and dominate the cochlea's vibratory response to high-frequency sound. This dominance was observed despite mechanisms that clearly low-pass filter the in vivo electromotile response. Low-pass filtering therefore does not critically limit the OHC's ability to move the organ of Corti on a cycle-by-cycle basis. Our data argue that electromotility serves as the primary high-frequency amplifying mechanism within the mammalian cochlea.

Generation of mature and functional hair cells by co-expression of Gfi1, Pou4f3, and Atoh1 in the postnatal mouse cochlea.

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.

Structure and Regulation of the Movement of Human Myosin VIIA.

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca(2+). Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca(2+), and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.

Tumor necrosis factor alpha and inflammation disrupt the polarity complex in intestinal epithelial cells by a posttranslational mechanism.

Inflammatory processes disrupt the barrier function in epithelia. Increased permeability often leads to chronic of inflammation. Important among other cytokines, tumor necrosis factor alpha (TNF-α) initiates an NF-κB-mediated response that leads to upregulation of myosin light chain kinase (MLCK), a hallmark of the pathogenesis of inflammatory bowel disease. Here, we found that two components of the evolutionarily conserved organizer of tight junctions and polarity, the polarity complex (atypical protein kinase C [aPKC]-PAR6-PAR3) were downregulated by TNF-α signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Decreases in aPKC levels were due to decreased chaperoning activity of Hsp70 proteins, with failure of the aPKC rescue machinery, and these effects were rescued by NF-κB inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-α signaling, including apical nonmuscle myosin II accumulation and myosin light chain phosphorylation. These effects, including ZO-1 downregulation, were rescued by overexpression of constitutively active aPKC. We conclude that this novel mechanism is a complementary effector pathway for TNF-α signaling.

Association among polymorphisms at MYH9, environmental factors, and nonsyndromic orofacial clefts in western China.

Myosin heavy chain 9, nonmuscle (MYH9) and environmental factors have been shown to be associated with nonsyndromic cleft lip with or without cleft palate in several populations. Our study aimed to confirm the contribution of MYH9 and environmental factors to nonsyndromic orofacial cleft risk in western Han Chinese. Four single-nucleotide polymorphisms were investigated in 180 case trios and 224 normal peers in western China using transmission disequilibrium test, family-based association test analysis, and logistic regression models. Strong evidence of linkage disequilibrium was found between these markers and the disease by both single-nucleotide polymorphism analysis (G allele at rs2269529 and T allele at rs16996652) and haplotype analysis (G-T [for rs2269529 and rs16996652] and G-A-T [for rs2269529, rs3752462, and rs16996652] among others). Mothers' illness, medication, and passive smoking during the first trimester may increase the risk of nonsyndromic orofacial clefts, but mothers' vitamin (including folic acid) supplementation during the first trimester is a protective factor. Interactions between mothers' passive smoking during the first trimester and T/T genotype of rs16996652 had statistical significance. Risk factors identified in our study may provide a better understanding of the etiological role of MYH9 and influence of environmental factors in nonsyndromic orofacial cleft incidence.

A chimera analysis of prestin knock-out mice.

A chimera is a genetic composite containing a unique mix of cells derived from more than one zygote. This mouse model allows one to learn how cells of contrasting genotype functionally interact in vivo. Here, we investigate the effect that different proportions of prestin-containing outer hair cells (OHC) have on cochlear amplification. To address this issue, we developed a prestin chimeric mouse in which both ROSA26 wild-type (WT) and prestin knock-out (KO) genotypes are present in a single cochlea. The WT ROSA26 mice express a cell marker, allowing one to identify cells originating from the WT genome. Examination of cochlear tissue indicated that prestin chimeric mice demonstrate a mosaic in which mutant and normal OHCs interleave along the cochlear partition, similar to all other chimeric mouse models. The anatomical distribution of prestin-containing OHCs was compared with physiological data including thresholds and tuning curves for the compound action potential (CAP) recorded in anesthetized mice. Analysis of these measures did not reveal mixed phenotypes in which the distribution of prestin-containing OHCs impacted sensitivity and frequency selectivity to different degrees. However, by reducing the number of prestin-containing OHCs, phenotypes intermediate between WT and KO response patterns were obtained. Accordingly, we demonstrate a proportional reduction in sensitivity and in the tip length of CAP tuning curves as the number of OHCs derived from the KO genome increases; i.e., genotype ratio and phenotype are closely related.

The concerted nature between three catalytic subunits driving the F1 rotary motor.

F(1), a rotational molecular motor, shows strong cooperativity during ATP catalysis when driving the rotation of the central gamma subunit surrounded by the alpha(3)beta(3) subunits. To understand how the three catalytic beta subunits cooperate to drive rotation, we made a hybrid F(1) containing one or two mutant beta subunits with altered catalytic kinetics and observed its rotations. Analysis of the asymmetric stepwise rotations elucidated a concerted nature inside the F(1) complex where all three beta subunits participate to rotate the gamma subunit with a 120 degrees phase. In addition, observing hybrid F(1) rotations at various solution conditions, such as ADP, P(i) and the ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) provides additional information for each elementary event. This novel experimental system, which combines single molecule observations and biochemical methods, enables us to dynamically visualize the catalytic coordination inside active enzymes and shed light on how biological machines provide unidirectional functions and rectify information from stochastic reactions.

Differential expression of molecular motors in the motor cortex of sporadic ALS.

The molecular mechanisms underlying the selective neurodegeneration of motor neurons in amyotrophic lateral sclerosis (ALS) are inadequately understood. Recent breakthroughs have implicated impaired axonal transport, mediated by molecular motors, as a key element for disease onset and progression. The current work identifies the expression of 15 kinesin-like motors in healthy human motor cortex, including three novel isoforms. Our comprehensive quantitative mRNA analysis in control and sporadic ALS (SALS) motor cortex specimens detects SALS-specific down-regulation of KIF1Bbeta and novel KIF3Abeta, two isoforms we show to be enriched in the brain, and also of SOD1, a key enzyme linked to familial ALS. This is accompanied by a marked reduction of KIF3Abeta protein levels. In the motor cortex KIF3Abeta localizes in cholinergic neurons, including upper motor neurons. No mutations causing splicing defects or altering protein-coding sequences were identified in the genes of the three proteins. The present study implicates two motor proteins as possible candidates in SALS pathology.

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension.

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

Kinetic mechanism of myosin IXB and the contributions of two class IX-specific regions.

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.

A novel spermatogenesis-related factor-1 gene expressed in maturing rat testis.

A rat gene with testis-specific expression coinciding with spermatogenesis was cloned by differential display. This spermatogenesis-related factor-1 (SRF-1) gene was not expressed in other organs. Testicular expression was detected from 5 weeks of age and increased up to 15 weeks; this level of expression was maintained for 63 weeks. The 750-bp cloned gene was coded for an open reading frame of 202 amino acids. According to in situ hybridization at 7 weeks, this gene was expressed mainly in spermatocyte. The gene product may function as a molecular motor in meiosis, as the deduced amino acid sequence showed partial homology with kinesin-related proteins. The action of this gene and its product with respect to division of reproductive cells requires further investigation.

Structure and mechanism of the RuvB Holliday junction branch migration motor.

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.

A new look at the microtubule binding patterns of dimeric kinesins.

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.

Regulation of the enzymatic and motor activities of myosin I.

Myosins I were the first unconventional myosins to be purified and they remain the best characterized. They have been implicated in various motile processes, including organelle translocation, ion channel gating and cytoskeletal reorganization but their exact cellular functions are still unclear. All members of the myosin I family, from yeast to man, have three structural domains: a catalytic head domain that binds ATP and actin; a tail domain believed to be involved in targeting the myosins to specific subcellular locations and a junction or neck domain that connects them and interacts with light chains. In this review we discuss how each of these three domains contributes to the regulation of myosin I enzymatic activity, motor activity and subcellular localization.

Dictyostelium myosin II G680V suppressors exhibit overlapping spectra of biochemical phenotypes including facilitated phosphate release.

We have biochemically characterized 13 intragenic suppressors of the G680V mutation of Dictyostelium myosin II. In the absence of the G680V mutation, the suppressors result in a number of deviant behaviors, most commonly an increase in the basal (actin-independent) ATPase of the motor. This phenotype is complementary to that of the G680V mutant and supports our proposal that the latter impairs phosphate release. Different subsets of the mutants also suffer from poor ATPase enhancement by 1 mg/ml actin, failure to release from actin in the presence of ATPgammaS (or ADP and salt), and excessive release from actin in the presence of ADP. The patterns of suppressor behaviors suggest that, in general, they are facilitating P(i)-releasing state(s) of the motor, but that different individual suppressors may secondarily perturb other states or actions of the motor.