Poster Sessions BP08: Cytoskeleton and Molecular Motors. Sticking out its neck: myosin‐1c binding to hair‐cell receptors requires its IQ domains and is regulated by calmodulin

As the sensory receptor cells of the inner ear, hair cells transduce mechanical stimuli into electrical signals. While the electrophysiological properties of hair cells are well understood, little is known about the molecular basis of mechanoelectrical transduction. In particular, the identities of the transduction channel and other components of the transduction machinery are unknown. Myosin‐1c, an unconventional myosin formerly called myosin‐1 beta, is one identified component of the hair‐cell transduction apparatus where it functions in the adaptation process. Because of its role in adaptation, myosin‐1c must interact, either directly or indirectly, with other components of the transduction machinery, including the channel itself. As a result, we sought to characterize myosin‐1c receptors in hair cells. Using recombinant fragments of myosin‐1c with its associated calmodulin light chains, we examined binding of these complexes to myosin‐1c receptors in the hair bundle, the mechanosensitive organelle housing the transduction apparatus. Astonishingly, binding to hair‐cell receptors was not dependent upon the myosin's tail sequences, the domain of the protein long proposed to bind to intracellular cargo. Instead, binding to intracellular receptors depends upon the neck region of myosin‐1c which contains four calmodulin‐binding IQ domains. Calmodulin blocks myosin‐1c interaction with its receptors by binding to the myosin and obscuring the myosin‐1c/receptor interaction domain. The calcium‐sensitive binding of calmodulin to myosin‐1c may therefore regulate myosin‐1c interactions with other components of the transduction apparatus.     

Myosins and pathology: genetics and biology

This article summarizes current knowledge on the genetics and possible molecular mechanisms of Human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (beta cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the beta cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments.     

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.     

A model of stereocilia adaptation based on single molecule mechanical studies of myosin I

We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features that have not been seen using fast skeletal muscle myosin II. (i) The working stroke occurs in two, distinct phases, producing an initial 3 nm and then a further 1.5 nm of movement. (ii) Two types of binding interaction were observed: short-lived ATP-independent binding events that produced no movement and longer-lived, ATP-dependent events that produced a full working stroke. The stiffness of both types of interaction was similar. (iii) In a new type of experiment, using feedback to apply controlled displacements to a single acto-myosin cross-bridge, we found abrupt changes in force during attachment of the acto-Myo1b cross-bridge, a result that is consistent with the classical 'T2' behaviour of single muscle fibres. Given that these myosins might exhibit the classical T2 behaviour, we propose a new model to explain the slow phase of sensory adaptation of the hair cells of the inner ear.     

ATPase activity of myosin in hair bundles of the bullfrog's sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.     

The motor mechanism of myosin V: insights for muscle contraction

It is 50 years since the sliding of actin and myosin filaments was proposed as the basis of force generation and shortening in striated muscle. Although this is now generally accepted, the detailed molecular mechanism of how myosin uses adenosine triphosphate to generate force during its cyclic interaction with actin is only now being unravelled. New insights have come from the unconventional myosins, especially myosin V. Myosin V is kinetically tuned to allow movement on actin filaments as a single molecule, which has led to new kinetic, mechanical and structural data that have filled in missing pieces of the actomyosin-chemo-mechanical transduction puzzle.     

Two adaptation processes in auditory hair cells together can provide an active amplifier

The hair cells of the vertebrate inner ear convert mechanical stimuli to electrical signals. Two adaptation mechanisms are known to modify the ionic current flowing through the transduction channels of the hair bundles: a rapid process involves Ca(2+) ions binding to the channels; and a slower adaptation is associated with the movement of myosin motors. We present a mathematical model of the hair cell which demonstrates that the combination of these two mechanisms can produce "self-tuned critical oscillations", i.e., maintain the hair bundle at the threshold of an oscillatory instability. The characteristic frequency depends on the geometry of the bundle and on the Ca(2+) dynamics, but is independent of channel kinetics. Poised on the verge of vibrating, the hair bundle acts as an active amplifier. However, if the hair cell is sufficiently perturbed, other dynamical regimes can occur. These include slow relaxation oscillations which resemble the hair bundle motion observed in some experimental preparations.     

Head-head and head-tail interaction: a general mechanism for switching off myosin II activity in cells

Intramolecular interaction between myosin heads, blocking key sites involved in actin-binding and ATPase activity, appears to be a critical mechanism for switching off vertebrate smooth-muscle myosin molecules, leading to relaxation. We have tested the hypothesis that this interaction is a general mechanism for switching off myosin II-based motile activity in both muscle and nonmuscle cells. Electron microscopic images of negatively stained myosin II molecules were analyzed by single particle image processing. Molecules from invertebrate striated muscles with phosphorylation-dependent regulation showed head-head interactions in the off-state similar to those in vertebrate smooth muscle. A similar structure was observed in nonmuscle myosin II (also phosphorylation-regulated). Surprisingly, myosins from vertebrate skeletal and cardiac muscle, which are not intrinsically regulated, undergo similar head-head interactions in relaxing conditions. In all of these myosins, we also observe conserved interactions between the 'blocked' myosin head and the myosin tail, which may contribute to the switched-off state. These results suggest that intramolecular head-head and head-tail interactions are a general mechanism both for inducing muscle relaxation and for switching off myosin II-based motile activity in nonmuscle cells. These interactions are broken when myosin is activated.     

Fast adaptation in vestibular hair cells requires myosin-1c activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.     

Novel myosins

The traditional view of myosin, drawn from studies of myosins from striated muscles, is that of an elongated two-headed molecule that assembles into filaments. However, biochemical, molecular genetic and genetic studies have uncovered a host of ubiquitous single-headed nonfilamentous myosins known collectively as myosins I. All of the myosins I possess the myosin head domain, the motor portion of muscle myosins they have tail the filament-forming tail domain of muscle myosins they have tail domains that interact variously with membranes, actin and calmodulin. These alternative molecular interactions confer novel motile properties on myosins I, such as the ability to move membranes relative to actin and to move actin relative to actin without having to assemble into filaments. The numerous actin-based movements retained by cells lacking myosin II, the two-headed filamentous form of nonmuscle myosin, may be supported by myosins I.     

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail

We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.     

Enzymatic activities and ATP-induced fluorescence enhancement of myosin from fast and slow skeletal and cardiac muscles.

The maximal ATP-induced enhancement of fluorescence and the dependence of this enhancement on ATP concentration were determined for myosins from fast and slow skeletal and cardiac muscle of the rabbit. With myosins from slow and cardiac muscle modifications in the preparative procedure and chromatography on DEAE-Sephadex were required to obtain preprations which were free of actin, which exhibited the maximal fluorescence enhancement and which bound two moles of ATP per mole of myosin. Since the fluorescence enhancement of cardiac and slow muscle myosins is labile at slightly alkaline pH, it was also necessary to minimize incubation at pH greater than 7 in order to attain the maximal enhancement. With fast muscle myosin the changes in preparative procedure, together with chromatography, led to a 50 to 100% increase in the steady-state rate of ATP hydrolysis and fluorescence enhancement, without changing the maximal binding of ATP. From a comparison of the rate of steady-state hydrolysis of ATP with the rate of decay of the enhanced fluorescence, it appears that for all three myosins, both ATP binding sites have the same enzymatic activity, the steady-state rate per site being slower for cardiac and slow muscle myosins than for fast muscle myosin.     

Effectiveness of Hair Bundle Motility as the Cochlear Amplifier

The effectiveness of hair bundle motility in mammalian and avian ears is studied by examining energy balance for a small sinusoidal displacement of the hair bundle. The condition that the energy generated by a hair bundle must be greater than energy loss due to the shear in the subtectorial gap per hair bundle leads to a limiting frequency that can be supported by hair-bundle motility. Limiting frequencies are obtained for two motile mechanisms for fast adaptation, the channel re-closure model and a model that assumes that fast adaptation is an interplay between gating of the channel and the myosin motor. The limiting frequency obtained for each of these models is an increasing function of a factor that is determined by the morphology of hair bundles and the cochlea. Primarily due to the higher density of hair cells in the avian inner ear, this factor is ∼10-fold greater for the avian ear than the mammalian ear, which has much higher auditory frequency limit. This result is consistent with a much greater significance of hair bundle motility in the avian ear than that in the mammalian ear.     

Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish

Unconventional myosins have been associated with hearing loss in humans, mice, and zebrafish. Mutations in myosin VI cause both recessive and dominant forms of nonsyndromic deafness in humans and deafness in Snell's waltzer mice associated with abnormal fusion of hair cell stereocilia. Although myosin VI has been implicated in diverse cellular processes such as vesicle trafficking and epithelial morphogenesis, the role of this protein in the sensory hair cells remains unclear. To investigate the function of myosin VI in zebrafish, we cloned and examined the expression pattern of myosin VI, which is duplicated in the zebrafish genome. One duplicate, myo6a, is expressed in a ubiquitous pattern during early development and at later stages, and is highly expressed in the brain, gut, and kidney. myo6b, on the other hand, is predominantly expressed in the sensory epithelium of the ear and lateral line at all developmental stages examined. Both molecules have different splice variants expressed in these tissues. Using a candidate gene approach, we show that myo6b is satellite, a gene responsible for auditory/vestibular defects in zebrafish larvae. Examination of hair cells in satellite mutants revealed that stereociliary bundles are irregular and disorganized. At the ultrastructural level, we observed that the apical surface of satellite mutant hair cells abnormally protrudes above the epithelium and the membrane near the base of the stereocilia is raised. At later stages, stereocilia fused together. We conclude that zebrafish myo6b is required for maintaining the integrity of the apical surface of hair cells, suggesting a conserved role for myosin VI in regulation of actin-based interactions with the plasma membrane.     

Myo1c is designed for the adaptation response in the inner ear

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.     

The functional, comparative and evolutionary anatomy of myosins

Actins and myosins are generally present in all animal, plant and fungal cells, and in some, if not all, prokaryotes as well. It is proposed that, in general, myosins can carry specific loads as they move along actin filaments, thus mediating a form of active transport. Myosins exert their mechanical forces by a lever action (moklokinesis) of a part of the molecule: the S-1 "head" in the case of muscle myosins. Various portions of myosin molecules can be assigned specialized functions. Each such part can be designated by an appropriate functional name. Examples are: the enzyme portion (zymomere), the motor portion (dynamere), the lever portion (moklomere), a connecting portion (desmomere), a coupling or binding portion (haptomere), and one or more flexible portions (kamptomeres). These parts can be recognized in highly evolved and specialized muscle myosins and can be postulated in simpler, single headed myosins. A primitive myosin, represented principally by a moklomere equipped with a zymomere and a dynamere, is envisioned as an evolutionary ancestor of all myosins. This primitive myosin resembles the S-1 head of muscle myosin. I suggest that from such a primitive myosin, more elaborate single-headed myosins have evolved, equipped with specific haptomeres coupled to the moklomere by desmomeres and kamptomeres. From such general single-headed myosins have arisen the highly specialized two-headed myosins represented in muscle. It is suggested that the two-headed feature is favored in myosins capable of forming bipolar filaments.     

Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling

Myosin-1c (also known as myosin-Ibeta) has been proposed to mediate the slow component of adaptation by hair cells, the sensory cells of the inner ear. To test this hypothesis, we mutated tyrosine-61 of myosin-1c to glycine, conferring susceptibility to inhibition by N(6)-modified ADP analogs. We expressed the mutant myosin-1c in utricular hair cells of transgenic mice, delivered an ADP analog through a whole-cell recording pipette, and found that the analog rapidly blocked adaptation to positive and negative deflections in transgenic cells but not in wild-type cells. The speed and specificity of inhibition suggests that myosin-1c participates in adaptation in hair cells.     

Adaptation in auditory hair cells

The narrow stimulus limits of hair cell transduction, equivalent to a total excursion of about 100nm at the tip of the hair bundle, demand tight regulation of the mechanical input to ensure that the mechanoelectrical transducer (MET) channels operate in their linear range. This control is provided by multiple components of Ca(2+)-dependent adaptation. A slow mechanism limits the mechanical stimulus through the action of one or more unconventional myosins. There is also a fast, sub-millisecond, Ca(2+) regulation of the MET channel, which can generate resonance and confer tuning on transduction. Changing the conductance or kinetics of the MET channels can vary their resonant frequency. The tuning information conveyed in transduction may combine with the somatic motility of outer hair cells to produce an active process that supplies amplification and augments frequency selectivity in the mammalian cochlea.     

Myosin types in cultured muscle cells

Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins.