The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism.

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Myopodin isoforms alter the chemokinetic response of PC3 cells in response to different migration stimuli via differential effects on Rho-ROCK signaling pathways.

The gene encoding myopodin, an actin binding protein, is commonly deleted in invasive, but not in indolent, prostate cancers. There are conflicting reports on the effects of myopodin expression on prostate cancer cell migration and invasion. The recent recognition that myopodin is expressed as four different isoforms further complicates our understanding of how this potentially important invasive prostate cancer biomarker affects tumor cell migration and invasion. We now show that myopodin affects the chemokinetic, rather than the chemotactic, properties of PC3 prostate cancer cells. Furthermore, all myopodin isoforms can either increase or decrease PC3 cell migration in response to different chemokinetic stimuli. These migration properties were reflected by differences in cell morphology and the relative dependence on Rho-ROCK signaling pathways induced by the environmental stimuli. Truncation analysis determined that a unique 9-residue C-terminal sequence in the shortest isoform and the conserved, PDZ domain-containing N-terminal region of the long isoforms both contribute to the ability of myopodin to alter the response of PC3 cells to chemokinetic stimuli. Matrigel invasion assays also indicated that myopodin primarily affects the migration, rather than the invasion, properties of PC3 cells. The correlation between loss of myopodin expression and invasive prostate cancer therefore reflects complex myopodin interactions with pathways that regulate the cellular migration response to diverse signals that may be present in a tumor microenvironment.

Mouse class III myosins: kinase activity and phosphorylation sites.

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.

Effect of phosphorylation in the motor domain of human myosin IIIA on its ATP hydrolysis cycle.

Previous findings suggested that the motor activity of human myosin IIIA (HM3A) is influenced by phosphorylation [Kambara, T., et al. (2006) J. Biol. Chem. 281, 37291-37301]; however, how phosphorylation controls the motor activity of HM3A is obscure. In this study, we clarify the kinetic basis of the effect of phosphorylation on the ATP hydrolysis cycle of the motor domain of HM3A (huM3AMD). The affinity of human myosin IIIA for filamentous actin in the presence of ATP is more than 100-fold decreased by phosphorylation, while the maximum rate of ATP turnover is virtually unchanged. The rate of release of ADP from acto-phosphorylated huM3AMD is 6-fold greater than the overall cycle rate, and thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form is markedly increased by phosphorylation by 30-fold. The dissociation constant for dissociation of the ATP-bound form of huM3AMD from actin is greatly increased by phosphorylation, and this result agrees well with the significant increase in the K(actin) value of the steady-state ATPase reaction. The rate constant of the P(i) off step is greater than 60 s(-1), suggesting that this step does not limit the overall ATP hydrolysis cycle rate. Our kinetic model indicates that phosphorylation induces the dissociation of huM3AMD from actin during the ATP hydrolysis cycle, and this is due to the phosphorylation-dependent marked decrease in the affinity of huM3AMD.ATP for actin and the increase in the ATP hydrolysis rate of huM3AMD in the actin-dissociated state. These results suggest that the phosphorylation of myosin IIIA significantly lowers the duty ratio, which may influence the cargo transporting ability of the native form of myosin IIIA that contains the ATP-independent actin binding site in the tail.

Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium.

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin-dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE.

The kinase domain alters the kinetic properties of the myosin IIIA motor.

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.

The actin-binding interface of a myosin III is phosphorylated in vivo in response to signals from a circadian clock.

Class III unconventional myosins are critical for the normal function of auditory hair cells and the function and maintenance of photoreceptors; however, the roles of class III myosins in these sensory cells are unknown. Class III myosins are unique in that they have a kinase domain at their N-terminus; thus, they may have both signaling and motor functions. In the horseshoe crab Limulus polyphemus, enhanced phosphorylation of an abundant, photoreceptor specific class III myosin at night correlates with well-characterized circadian changes in photoreceptor structure and function. Thus, the Limulus visual system may be particularly useful for investigating the properties, modulation, and functions of a class III myosin. Previously, we showed that two sites within the actin interface of full-length Limulus myosin III expressed in baculovirus are substrates for both cyclic AMP-dependent protein kinase and autophosphorylation. In the current study, mass spectrometry was used to show that these same sites are phosphorylated in the endogenous protein extracted from Limulus lateral eye, and that enhanced phosphorylation at these sites occurs in vivo in response to natural circadian clock input to these eyes. These findings demonstrate in vivo changes in myosin III phosphorylation in response to a natural stimulus. This phosphorylation may modulate myosin III-actin interactions.

Loop 2 of limulus myosin III is phosphorylated by protein kinase A and autophosphorylation.

Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.

Human myosin III is a motor having an extremely high affinity for actin.

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.