Tomonobu M. Watanabe

Myosin-X Induces Filopodia by Multiple Elongation Mechanism

Filopodia are actin-rich finger-like cytoplasmic projections extending from the leading edge of cells. Unconventional myosin-X is involved in the protrusion of filopodia. However, the underlying mechanism of myosin-X-induced filopodia formation is obscure. Here, we studied the movements of myosin-X during filopodia protrusion using a total internal reflection microscope to clarify the mechanism of myosin-X-induced filopodia formation. Myosin-X was recruited to the discrete site at the leading edge where it assembles with exponential kinetics before the filopodia extension. The myosin-X-induced filopodia showed repeated extension-retraction cycles with each extension of 2.4 μm, which was critical to produce long filopodia. Myosin-X, lacking the FERM domain, could move to the tip as does the wild type. However, it was transported toward the cell body during filopodia retraction, did not undergo multiple extension-retraction cycles, and failed to produce long filopodia. During the filopodia protrusion, the single molecules of full-length myosin-X moved within filopodia. The majority of the fluorescence spots showed two-step photobleaching, suggesting that the moving myosin-X is a dimer. Deletion of the FERM domain did not change the movement at the single molecule level with the same velocity of ∼600 nm/s as wild-type, suggesting that the myosin-X in filopodia moves without interaction with the attached membrane via the FERM domain. Based upon these results, we have proposed a model of myosin-X-induced filopodia protrusion.

Identification of Three Distinct Functional Sites of Insulin-mediated GLUT4 Trafficking in Adipocytes Using Quantitative Single Molecule Imaging

We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events.

Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor

There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. KATPase of the actin-activated ATPase was 62 μm, which is much higher than that of myosin Va (∼1 μm). The rate of ADP release from actomyosin Vc was 12.7 s-1, which was 2 times greater than the entire ATPase cycle rate, 6.5 s-1. Pi burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms.

Mechanical Characterization of One-Headed Myosin-V Using Optical Tweezers

Class V myosin (myosin-V) is a cargo transporter that moves along an actin filament with large (∼36-nm) successive steps. It consists of two heads that each includes a motor domain and a long (23 nm) neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics) of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 kBT from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.

Effect of serum on inhibition of DNA synthesis in leukemia cells by cis- and trans-[Pt(NH3)2Cl2]

We examined the inhibition of DNA synthesis by cis- and trans-diamminedichloroplatinum (II) (cis- and trans-Pt) in leukemia cells, YAC-1 and RADA1. The degree of inhibition by trans-Pt was about the same as that by cis-Pt in vitro in the absence of serum, but the former was much lower than the latter in vivo or in the presence of serum in vitro. Atomic absorption studies showed that the amount of trans-Pt trapped by the serum in vitro is much larger than that of cis-Pt. Therefore, the amount of trans-Pt bound to DNA in vivo must be considerably smaller than that of cis-Pt, which eventually results in the antitumor-inactive nature of trans-Pt.

Activated full-length myosin-X moves processively on filopodia with large steps toward diverse two-dimensional directions

Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10FullLZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M101–979HMM). M10FullLZ localizes at the tip of filopodia like myosin-X full-length (M10Full). M10FullLZ moves on actin filaments in the presence of PI(3,4,5)P3, an activator of myosin-X. Single molecule motility analysis revealed that the step sizes of both M10FullLZ and BAP-M101–979HMM are widely distributed on single actin filaments that is consistent with electron microscopy observation. M10FullLZ moves on filopodial actin bundles of cells with a mean step size (~36 nm), similar to the step size on single actin filaments (~38 nm). Cartesian plot analysis revealed that M10FullLZ meandered on filopodial actin bundles to both x- and y- directions. These results suggest that the lever-arm of full-length myosin-X is flexible enough to processively steps on different actin filaments within the actin bundles of filopodia. This characteristic of myosin-X may facilitate actin filament convergence for filopodia production.

Brillouin scattering imaging of stiffness heterogeneity in multicellular systems (Conference Presentation)

Brillouin scattering microscopy is the only potential tool to realize microscopic mapping of mechanical property in multicellular system (i.e. colony, tissue) with sub-cell resolution. We built a laser-scanning Brillouin microscope system for our biological study on spatial heterogeneity of stiffness in multicellular system. High-numerical-aperture (NA) objective lens (NA ≥ 0.7) and a dual-VIPA based spectrometer were employed to achieve high spatial resolution and high sensitivity, respectively. Addition of a spatial filter at a Fourier plane of the EMCCD detector surface effectively rejected strongly reflected excitation light without loss of Brillouin scattering signal, which accordingly allowed us to observe cells just above glass substrate surface. We performed three-dimensional imaging of Brillouin scattering to see three-dimensional stiffness distribution within a multicellular system. It was found that cells in relatively central region or in close vicinity of glass substrate have higher stiffness, which agrees to biological prediction. We also found presence of anomaly cells with much higher elasticity than surrounding cells. The stiffness imaging was applied to various kinds of multicellular systems including ES cell colonies upon differentiation and artificial tissue grown from iPS cells. The results certified the effectiveness of Brillouin scattering microscope in mechanobiology, developmental biology and regenerative medicine. Several practical issues for biomedical application will also be discussed.