Research

 

 

 

Sakamoto's laboratory is interested in (1) the mechanical function of molecular motors and (2) the mechanical properties of cytoskeletal actin. We also focus on heart failure and cancer research.

(1) Single molecule study for molecular motors:

Sakamoto's laboratory is interested in understanding the mechanical property of molecular motors. Molecular motors are biological molecular machines that convert chemical energy to mechanical force. There are three major linear motors: myosins, kinesins and dyenins. Myosins are actin based motor, the others are microtubule motors. Currently, our laboratory focuses on class V, X, and XVI myosin motors which function as cargo transporter or tethers for organelles in cells.

The cargos transported by myosin V include melanosome, granules, and mRNA, etc. How do molecular motors transport cargos? During this decade, the mechanism of myosin V motion has been intensively studied at the single molecule level. Studies have revealed that myosin V "walks" along the actin track with a step-size (stride length) of 36-nm and run length of 1 ~ 2 micro-meter along the actin filament. To visualize a single myosin V molecule, we labeled myosin V with green fluorescent protein (GFP) and are imaging those fluorescently-labeled myosin V by using total internal reflection fluorescence (TIRF) microscope, which can illuminate 100 ~ 300 nm depth from the cover-glass surface (Figure 1A green color above of blue line). A fluorescently-labeled myosin V molecule binds on the actin filament, which is immobilized on the surface via a biotin-avidin system. The myosin V molecule starts to move unidirectionally and is "processive". We observed the movement of the myosin V molecule as a spot movement and measured the velocity, run-length (processivity), and step-size (Figure 1 B). For step-size measurement, we have used FIONA analysis techniques to achieve 2 nm resolution (Figure 1 C) and analyzed the step-size and dwell time (Figure 1 D).

How do multiple myosin molecules effectively transport a cargo? Does a stiffness of the linker between the myosin molecule affect the movement? To answer these question, we imaged multiple molecules on the linker by different fluorescent dyes.

Here are sample movies

Left: Two-color TIRF imaging, Right: Deac-aminoATP attaching/dissociating from myosinVa

click here to see a movie of myosin 5a movement (left hand side) and deac-aminoATP binding/dissociation events (right hand side).

(2) Stiffness and structure of cytoskeletal actin / actin bundle with actin binding/bundling proteins

Our interest is to understand the molecular mechanism of actin bundle formation by TRIOBP. Actin is a 42 kDa protein with globular shapes. In cells, actin forms highly ordered structures including uni- or bi-polarized bundles, and mesh-works with actin binding protein. TRIOBP is an actin binding/bundling protein associated with hearing loss. Mutations in the human TRIOBP gene lead to nonsyndromic deafness DFNB28. Studies discovered that TRIOBP is localized at the rootlets of stereocilia in inner ear hair cells. Moreover, purified TRIOBP binds actin filaments and forms extremely condensed actin bundles. How does TRIOBP bind onto an actin filaments? How does TRIOBP form the condensed actin bundles? What are the stiffness and durability of the TRIOBP-actin bundles? Answering these questions will help us to understand the molecular mechanism of TRIOBP-associated hearing loss. We are currently measuring the structure of TRIOBP-actin bundles by using electron microscope, fluorescence microscope, dynamic light scattering, NMR, Small-angle X-Ray Scattering (SAXS), and circular dichroism (CD). We will directly image the dynamics of the actin bundling structure using a TIRF microscope.

TRIOBP bundles actin filaments and stabilizes the bundles. What is the function of TRIOBP for forming the bundle? How much stiffness do they need in cells? To answer the question, using optical tweezers and atomic force microscope (AFM), we are measuring the stiffness and durability of the actin bundles with TRIOBP.