Mouse Myosin X: Molecular Architecture and Tissue Expression as Revealed by Northern Blot and in Situ Hybridization Analyses

doi:10.1006/bbrc.2000.2669

Biochemical and Biophysical Research Communications

Volume 271, Issue 2, 10 May 2000, Pages 526-533

https://doi.org/10.1006/bbrc.2000.2669 Get rights and content

Abstract

The structure of the coding region of mouse myosin X cDNA was determined. The predicted protein sequence indicated an approximately 240 kDa molecular mass with 2062 amino acids. When aligned with the structure predicted for calf myosin X (GenBank Accession No. U55042), extremely highly conserved pleckstrin homology domains and a myosin tail homology 4 domain were apparent in the tail region, suggesting their importance for myosin X's function. Northern blot analysis revealed the existence of a myosin X mRNA, 8.7 kb in size, in various mouse tissues, while a similar size of human type myosin X mRNA was recognized mainly in the testis. In addition to the adult-type transcripts in mice, a smaller embryo-specific mRNA, 4.8 kb in size, was identified in early to late embryonic stages, suggesting the presence of a shorter myosin X isoform in mouse embryos. In situ hybridization experiments with mouse testis revealed that myosin X mRNA was restricted to Sertoli cells at stages VIII–X of the spermatogenesis cycle, suggesting that myosin X is implicated in the supporting cells during the spermatid morphogenesis.

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Cited by (25)

Myosin-X: A molecular motor at the cell's fingertips
2005, Trends in Cell Biology
Research in several areas, including unconventional myosins and deafness genes, has converged recently on a group of myosins whose tails contain myosin tail homology 4 (MyTH4) and band 4.1, ezrin, radixin, moesin (FERM) domains. Although these ‘MyTH-FERM’ myosins are not present in yeast and plants, they are present in slime molds, worms, flies and mammals, where they mediate interactions between the cytoskeleton and the plasma membrane. The most broadly distributed MyTH-FERM myosin in vertebrate cells appears to be myosin-X (Myo10). This myosin can act as a link to integrins and microtubules, stimulate the formation of filopodia and undergo a novel form of motility within filopodia.
Myosin X is a high duty ratio motor
2005, Journal of Biological Chemistry
Myosin X is expressed in a variety of cell types and plays a role in cargo movement and filopodia extension, but its mechanoenzymatic characteristics are not fully understood. Here we analyzed the kinetic mechanism of the ATP hydrolysis cycle of acto-myosin X using a single-headed construct (M10IQ1). Myosin X was unique for the weak “strong actin binding state” (AMD) with a K_d of 1.6 μm attributed to the large dissociation rate constant (2.1 s^-1). V_max and K_ATPase of the actin-activated ATPase activity of M10IQ1 were 13.5 s^-1 and 17.4 μm, respectively. The ATP hydrolysis rate (>100 s^-1) and the phosphate release rate from acto-myosin X (>100 s^-1) were much faster than the entire ATPase cycle rate and, thus, not rate-limiting. The ADP off-rate from acto-myosin X was 23 s^-1, which was two times larger than the V_max. The P_i-burst size was low (0.46 mol/mol), indicating that the equilibrium is significantly shifted toward the prehydrolysis intermediate. The steady-state ATPase rate can be explained by a combination of the unfavorable equilibrium constant of the hydrolysis step and the relatively slow ADP off-rate. The duty ratio calculated from our kinetic model, 0.6, was consistent with the duty ratio, 0.7, obtained from comparison of K_{m ATPase} and K_{m motility}. Our results suggest that myosin X is a high duty ratio motor.
Myosin X transports Mena/VASP to the tip of filopodia
2004, Biochemical and Biophysical Research Communications
Myosin X (M10) is a two-headed actin based motor expressed in a variety of cell types, that is thought to play a role in cargo movement in mammalian cells, but its cellular function is unknown. Here we found that M10 binds to Mena/VASP, which facilitates actin polymerization by competing with actin capping proteins. Immunocytochemistry revealed that endogenous M10 co-localized with Mena/VASP at the tip of filopodia. Consistently, both EGFP-M10 and RFP-VASP were found at the tip of filopodia. The result raises a hypothesis that M10 transports Mena/VASP towards the tip of filopodia. Supporting this idea, the amount of VASP at the tip of filopodia was proportional to that of M10. Furthermore, we directly visualized the movement of M10 and VASP in living HeLa cells under fluorescence microscope. EGFP-M10 and RFP-VASP move together from the root to the tip of the filopodia. Interestingly, the amount of M10 at the tip of filopodia was linearly related to the length of filopodia, consistent with the actin filament extending function of VASP. These results show that M10 is a specific motor carrying Mena/VASP from the root to the tip of the filopodia where extension of actin filament takes place.
The Spatial and Temporal Dynamics of Pleckstrin Homology Domain Binding at the Plasma Membrane Measured by Imaging Single Molecules in Live Mouse Myoblasts
2004, Journal of Biological Chemistry
Citation Excerpt :
Cloning—The full-length myosin X cDNA was amplified from mouse myoblasts by long reverse transcription-PCR using primers based on the mouse sequence (GenBank™ accession number AJ249706) (14).
Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 μm^-1 μm^-2 s^-1 and a detachment rate constant of 0.05 s^-1. The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function.
Motor Function and Regulation of Myosin X
2001, Journal of Biological Chemistry
Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg²⁺-ATPase activity of myosin X was significantly activated by actin with lowK_ATP. The actin-activated ATPase activity was reduced at Ca²⁺ concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 μm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca²⁺ atpCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, theK_actin of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.
Human calmodulin-like protein is an epithelial-specific protein regulated during keratinocyte differentiation
2001, Experimental Cell Research
Human calmodulin-like protein (CLP) is a calcium-binding protein down-regulated in a cell culture model of mammary tumorigenesis as well as in a majority of breast cancers in vivo. CLP down-regulation may be a result of the poorly differentiated state of these cell lines and tumors, or CLP expression may be incompatible with the uncontrolled cell growth associated with tumorigenesis. To learn more about CLP expression and regulation, we determined the distribution of CLP in various human tissues by immunohistochemistry. CLP was expressed exclusively in the epithelium of the tissues surveyed and was most abundant in thyroid, breast, prostate, kidney, and skin. CLP expression appears to increase in stratified epithelium during differentiation, as illustrated in the skin where CLP staining intensified from the basal through the spinous to the granular layers. Using a normal human keratinocyte culture model, we examined CLP expression in response to various agents known to affect keratinocyte differentiation. Agents that inhibit (epidermal growth factor, EGF) or permit (keratinocyte growth factor) terminal differentiation correspondingly regulate CLP expression. Factors modulating the EGF receptor signaling pathway were particularly potent in regulating CLP expression. CLP expression correlated with an agent's ability to promote terminal differentiation regardless of the agent's effect on keratinocyte proliferation. These studies show that CLP expression is coordinately regulated by, and may be involved in, the program of terminal differentiation in human keratinocytes and, likely, other differentiating epithelial cell types.

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¹: To whom correspondence should be addressed at Department of Developmental Biology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan. Fax: 0568-88-0829. E-mail: [email protected].

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Regular ArticleMouse Myosin X: Molecular Architecture and Tissue Expression as Revealed by Northern Blot and in Situ Hybridization Analyses

Abstract

Trends Genet.

Am. J. Hum. Genet.

Cell

Genomics

Genomics

Am. J. Hum. Genet.

Trends Biochem. Sci.

Trends Biochem. Sci.

Trends Biochem. Sci.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Genomics

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Unconventional myosins

Annu. Rev. Cell Dev. Biol.

Unconventional myosins in cell movement, membrane traffic, and signal transduction

Science

Regular Article
Mouse Myosin X: Molecular Architecture and Tissue Expression as Revealed by Northern Blot and in Situ Hybridization Analyses