Functional Expression of Mammalian Myosin Iβ:  Analysis of Its Motor Activity

Zhu, Tao; Sata, Masataka; Ikebe, Mitsuo

doi:10.1021/bi952053c

Cited by 70 publications

(89 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Calcium had very little effect on the ATPase activity, increasing the V max by Ͻ10% (V max ϭ 2.0 Ϯ 0.3 s Ϫ1 ; K m for actin 20 Ϯ 7 M at pCa 4.6). This low sensitivity to calcium is similar to that reported for native Myo1c under slightly different conditions [V max 0.185 and 0.189 mol of ATP per minute per miligram of protein in EGTA and calcium, respectively (16)]. …”

Section: Resultssupporting

confidence: 87%

“…Myo1c is the first member of the myosin superfamily shown to have a calcium-sensitive ATP hydrolysis step. The small effect of calcium on the native Myo1c ATPase activity is in contrast with the calcium-induced inhibition of motility in the in vitro sliding-filament assays, which is attributed to a calcium-induced conformational change in calmodulin bound to the neck, if not calmodulin dissociation itself (16,17). …”

mentioning

confidence: 82%

“…Previous work has suggested that actin-Myo1c complexes that retain bound Pi could reverse the power stroke generating the weakly bound M-ADP-Pi or M-ATP states that will detach from actin, thereby allowing the motor complex to ''slip'' relative to the actin bundles in the stereocilia (14,15). In addition, the transient increase in calcium concentration would result in a calcium-induced change in the conformation of the calmodulin/lever arm and possible calmodulin dissociation (16,17). Either could reduce the stiffness of the lever arm, thereby lowering the strain in the motor, allowing ADP to escape and leading to detachment and hence slippage, as described above.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear

Adamek

Coluccio

Geeves

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The class I myosin Myo1c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by Ca 2؉ , permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a Myo1c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle. We show that, although the steady-state ATPase activity shows little calcium sensitivity, individual molecular events of the cross-bridge cycle are calcium-sensitive. Of significance is a 7-fold inhibition of the ATP hydrolysis step and a 10-fold acceleration of ADP release in calcium. These changes result in an acceleration of detachment of the cross-bridge and a lengthening of the lifetime of the detached M-ATP state. These data support a model in which slipping adaptation, which reduces tip-link tension and allows the transduction channels to close after an excitatory stimulus, is mediated by Myo1c and modulated by the calcium transient. molecular motor ͉ myosin M yosins are actin-based molecular motors that support a variety of cellular functions including muscle contraction and cell migration. There are at least two dozen classes in the myosin superfamily based on analysis of sequences in the catalytic domain (1). Myo1c is a class I myosin that is widely expressed in vertebrate tissues (2, 3). It consists of a motor domain, a neck or lever arm domain (three or four IQ repeats each of which binds a calmodulin), and a cargo-binding domain (4-6). In adipocytes Myo1c facilitates glucose transporter recycling in response to insulin (7,8), and in amphibian oocytes Myo1c mediates exocytosis (9). In the specialized cells of the inner ear, there is considerable evidence that Myo1c acts as a mediator of adaptation of mechanoelectrical transduction in stereocilia (10, 11).Neighboring stereocilia on the hair cells of the inner ear are connected by extracellular tip links that are attached to transduction channels, which in turn are attached to an adaptation-motor complex consisting of Myo1c molecules. The prevailing model for mechanotransduction is that deflection of the stereocilia by sound or motion affects tip-link tension and causes opening or closing of transduction channels through which potassium and calcium ions pass (5, 12, 13). Myo1c sets the transducer sensitivity by moving along the core bundle of actin filaments in the stereocilia until the resting tension in the tip link is at a point where the channels are poised just below the threshold tension required to open the channels (Fig. 1). During an excitatory stimulus, movement of the stereocilia increases tip-link tension, opening the channels. Fig. 1 illustrates how in adaptation the motor/transducer complex responds to increased tension by slipping down the actin cytoskeleton thereby reducing tip-link tension. Alternatively, reduced tension causes Myo1c to ascend the ac...

show abstract

Section: Resultssupporting

confidence: 87%

mentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear

Adamek

Coluccio

Geeves

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The sample was ultracentrifuged at 100,000 ϫ g for 10 min, and then the supernatant and the pellets were analyzed by SDS-PAGE. The amount of the co-sedimented huM3AIQ2 heavy chain was determined by densitometry as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Komaba

Inoue

Maruta

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actinbased motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with K actin of 112 M and V max of 0.34 s ؊1 , indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 ؎ 0.05 m/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.Myosin III is a member of the myosin superfamily, which consists of at least 18 classes (1-5). The class III myosin represents the most divergent member of the myosin superfamily. The motor domain of myosin III shows ϳ24 -27% sequence identity to myosin Is and ϳ22-26% sequence identity to myosin-IIs (6). Of particular interest is that myosin III has an amino terminus domain that resembles to protein kinases. This domain contains the characteristic motifs for protein kinases such as a glycine-rich loop, an invariable Lys residue required for ATP binding, and a catalytic loop. Given its high degree of divergence, if myosin III evolved at the same rate as other myosins, the myosin III lineage would predate the divergence of yeast. Class III myosin was originally found in Drosophila photoreceptor cells and subsequently found in vertebrates including human myosin III (7,8).The function of Myosin III is best studied in Drosophila photoreceptor cells. Each photoreceptor cell has a specialized organelle consisting of a stack of microvilli known as a rhabdomere. The phototransduction machinery is localized in the rhabdomere (9). Drosophila photoreceptors undergo a prolonged depolarization afterpotential that persists after cessation of the light stimulus. Prolonged depolarization afterpotential results from the stable conversion of rhodopsin to the light-activated form, metarhodopsin, in response to blue light. During a prolonged depolarization afterpotential, photoreceptor cells become refractory to subsequent prolonged depolarization afterpotential-inducing stimuli and are inactivated. Mutants that are defective for both inactivation and the prolonged depolarization afterpotential are known as neither inactivation nor afterpotential (nina) mutants (10, 11), and the myosin III was identified (12) as one of eight nina complementation gr...

show abstract

“…Molecular mass markers used were smooth muscle myosin heavy chain (204 kDa), ␤-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), myosin regulatory light chain (20 kDa), and ␣-lactalbumin (14.2 kDa). The amount of the myosin VI heavy chain and calmodulin was determined by densitometry as described previously (28).…”

mentioning

confidence: 99%

Human Deafness Mutation of Myosin VI (C442Y) Accelerates the ADP Dissociation Rate

Satō

White

Inoue

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The missense mutation of Cys 442 to Tyr of myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects of the C442Y mutation on the kinetics of the actomyosin ATP hydrolysis mechanism and motor function of myosin VI. The largest changes in the kinetic mechanism of ATP hydrolysis produced by the C442Y mutation are about 10-fold increases in the rate of ADP dissociation from both myosin VI and actomyosin VI. The rates of ADP dissociation from acto-C442Y myosin VI-ADP and C442Y myosin VI-ADP are 20 -40 times more rapid than the steady state rates and cannot be the rate-limiting steps of the hydrolysis mechanism in the presence or absence of actin. The 2-fold increase in the actin gliding velocity of C442Y compared with wild type (WT) may be explained at least in part by the more rapid rate of ADP dissociation. The C442Y myosin VI has a significant increase (ϳ10-fold) in the steady state ATPase rate in the absence of actin relative to WT myosin VI. The steady state rate of actin-activated ATP hydrolysis is unchanged by the C442Y mutation at low (<10 ؊7 M) calcium but is calcium-sensitive with a 1.6-fold increase at high (ϳ10 ؊4 M) calcium that does not occur with WT. The actin gliding velocity of the C442Y mutant decreases significantly at low surface density of myosin VI, suggesting that the mutation hampers the processive movement of myosin VI.Myosin is a motor protein that interacts with actin filaments and converts energy from ATP hydrolysis into mechanical activity. Among diverse myosin superfamily proteins, class VI myosins are identified in various organisms in the animal kingdom (1-4). In mammals, myosin VI is found in various tissues, but its physiological significance is most recognized in the auditory system (5). The mouse myosin VI gene was discovered to be responsible for deafness in the Snell's waltzer mice, which show the characteristic waltzing and circling behavior (6).Myosin VI is expressed in both the inner and outer hair cells of the inner ear and is localized in the pericuticular necklace, present between the cuticular plate and circumferential actin structures. The location suggests that myosin VI may be responsible for the maintenance of sensory hair cells. In Snell's walzer mice, the cochlea shows progressive loss of hair cells soon after birth (6, 7), suggesting that myosin VI function is involved in the maintenance of sensory hair cells. These findings suggest that myosin VI may function to maintain the association of the cuticular plate with the stereocilia rootlets, which in turn stabilize the hair cells (8). Although the functional deficiency of myosin VI mutation is most pronounced in auditory function, myosin VI is expressed in a variety of cell types.The location of class VI myosin in Drosophilia (Drosophila 95F myosin) in the cytoplasmic particles that move along microfilaments suggests that myosin VI is involved in the transport processes (9). In polarized cells from the intestinal brush border, myosin VI is concentrated in the terminal web with a sma...

show abstract

Functional Expression of Mammalian Myosin Iβ: Analysis of Its Motor Activity

Cited by 70 publications

References 60 publications

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Human Deafness Mutation of Myosin VI (C442Y) Accelerates the ADP Dissociation Rate

Contact Info

Product

Resources

About