Excitation Light Confinement in Nano-Slits for Single-Molecule Observation of the Interaction Between Kinesin and Nucleotide
Kazuya Fujimoto
Department of Micro Engineering, Kyoto University, Kyoto, JAPAN
fujimoto.kazuya.2m@kyoto-u.ac.jp
Abstract
Single-molecule observation has been utilized to explore a mechanism of motor protein such as kinesin that moves on microtubules fueled by hydrolysis of adenosine tori-phosphate (ATP). Researchers have been observed binding and dissociation of individual fluorescently labelled nucleotides and kinesin to establish a shared understanding of how chemical reaction and mechanical displacement correlate.
Conventional experiment setups, however, suffer from low concentration available for observation because background noise from a high concentration of fluorescently labelled molecule disables to detect the signal from an individual molecule. For example, total internal reflection microscopy (TIRFM), which is the most widely used setup for single-molecule observation, permit only ~100 nM of concentration. This limitation results in low observation efficiency and discrepancy of experimental condition from in vivo, where the concentration is higher in order of magnitude.
A nano-scale device called zero-mode waveguides (ZMWs) broke the concentration limit by utilizing nano-optical effect by which excitation light for fluorescent microscopy is confined in small pits formed on a thin metal layer. They are used in various fields from an application such as gene sequencing to pure biophysical studies. However, the size of the cytoskeletal filaments on which motor proteins move has hindered them from the utilization of ZMWs.
To conquer this limitation, we developed linear-shaped ZMWs (LZMWs) compose of nano-slit structures. LZMWs can confine excitation light whose polarization direction is parallel to the slits, enabling the usage of higher concentration of dyes while allowing the introduction of filamentous objects such as microtubules. We established the experimental system to observe interactions between labelled ATP and kinesin accompanied by microtubule gliding motility assay in nano-slits. It achieved the use of 500 nM of labelled ATP concentration, way higher than past reported condition. Observation results indicated the existence of microtubule effect to the binding rate of ATP to kinesin.
Short Bio
Kazuya Fujimoto is an assistant professor at the Department of Micro Engineering, graduate school of Kyoto University. He received his B.S., in 2011, M.S., in 2013 and Ph. D. in 2016 at Kyoto University. In Ph. D course, he was selected to JSPS Research Fellowship for Young Scientists (DC1). After getting Ph. D., he worked as a postdoctoral researcher at Kyoto University. After that, he also worked for a company as a software engineer and a machine learning researcher. His research has been focused on measurement and control of motor protein systems such as kinesin and microtubules, with expertise in multiple technical fields including micro-nano fabrication technology, an optical system for microscopy, and, numerical simulation of micro–nano scale phenomenon. His research interest also includes image processing, machine learning, and automated systems, toward an overarching goal establishing autonomous artificial systems composed of highly ordered elements like organisms.