Sakamoto's laboratory is interested in(1) the mechanical function of molecular motors (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.
(2) Mechanical force measurement and determine the function of TRIOBP in cancer cells
Our interests are to determine a traction force exerted by a cell. Cell motility relies on the coordination of cellular proteins and structures, involving cytoskeleton, actin, myosin and focal adhesion proteins. Cells in motion are polarized, forming a leading edge pointing in the direction of migration and a trailing edge at the other end (Figure 1). The protrusion forms a structure called lamelliopodium. As the lamellipodium protrudes and touches down on the substrate, the focal adhesion proteins on the cell membrane will anchor themselves on the substrate integrin to provide cell traction (Figure 1). To measure the traction force, acrylamide-gel (light blue) in figure 1 was used and a magnetic beads were embedded in the gel where is very closed the surface. We have also developed our own software to measure the traction force by Physon as shown in Movie 1. Light-hand-side shows two cells move, while left-hand-side shows the displacement of the beaded.