Abstract
Transport of proteins and organelles in cells is essential for survival of cells. Nature has optimized transport strategies to enhance efficiency. Generally, cargoes are transported along cytoskeleton by motor proteins including myosin, kinesin and motor. Understanding the motility mechanisms of motor proteins has shedded the light on the important cellular functions of these motor proteins. In order to study these mechanisms, real-time singleparticle tracking based on fluorescent imaging has been widely used and revealed the detailed mechanisms of stepping behaviors of motor proteins. In this thesis, I employed an advanced imaging system to investigate the stepping mechanism of single myosin proteins both in vitro and in living cells assays. My data reveal key motility features of these motor proteins, including their step size, run length, and velocity.
Myo10 is known to be the first myosin to form an antiparallel dimer and move along actin bundles processively. However, the role of this antiparallel dimer in the Myo10 stepping mechanism still remains unknown. Hence, I constructed chimeric proteins that combine domains from Myo5 and Myo10, and investigate their motility by using the single-molecule motility assays. My data reveal that a the chimera, comprising the Myo5’s motor and the Myo10’s lever arm and antiparallel coiled-coil, showed a variety of forward step sizes and processive movement, mirroring the motility behaviors observed in fulllength Myo10. Conversely, another chimera which combines the Myo10’s motor and lever arm with the Myo5’s parallel coiled-coil, showed a stepping size of approximately 40 nm at lower ATP condition, but lost its capability for processive movement at higher ATP condition. In addition, I found that a Myo10 mutant, which included four alterations in the antiparallel coiled-coil domain, was unable to form a dimer, hence exhibited nonprocessive movement. My findings indicate that the antiparallel coiled-coil domain plays a critical role in the multiple forward step sizes characteristic of Myo10 motility.
Myo6 is an only myosin that exhibits movement to the minus end of actin filaments. Its reverse directionality and larger step size on actin filaments set it apart from other myosin proteins, making its motility mechanism intriguing. While past researches have uncovered the in vitro stepping mechanisms of Myo6, its movement within living cells remains largely unexplored. Thus, I introduced a technique for single-protein labelling using HaloTag and brighter HaloTag ligand. This labelling technique allowed the selective and specific binding of Myo6 in living cells, eliminating non-specific binding. By harnessing this labelling method and the advanced imaging system, I have been able to monitor the motility of single Myo6 molecules and accurately tracked single Myo6 molecules within living cells with high temporal precision, revealing the features of Myo6 stepping mechanism in living cells.
In conclusion, real-time single-particle tracking, in combination with advanced labeling methods, provides innovative and valuable insights into the dynamics of proteins and particles within biological processes. This approach allows us to track individual proteins within complex environments, such as living cells, with high precision. Furthermore, the single-protein labeling technique employed in this study is able to be applied to a broad range of particles and molecules. This offers an useful method to investigate biological processes at the molecular level.