A03 NISHIZAKA, Takayuki |Proposed Research Projects (2014-2015)

Paper | Original Paper


Wonju Lee, Yoshiaki Kinosita, Youngjin Oh, Nagisa Mikami, Heejin Yang, Makoto Miyata, Takayuki Nishizaka, and *Donghyun Kim,
Three-Dimensional Superlocalization Imaging of Gliding Mycoplasma mobile by Extraordinary Light Transmission through Arrayed Nanoholes,
ACS Nano 9, 10896-10908 (2015).

[Summary] In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding Mycoplasma mobile (M. mobile). The results analyzed with multiple M. mobile bacteria show individual characters and reveal that M. mobile undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision.

Shin Yamaguchi, Kei Saito, Miki Sutoh, Takayuki Nishizaka, Yoko Y Toyoshima, Junichiro Yajima,
Torque generation by axonemal outer-arm dynein,
Biophysical Journal 108, 872-879 (2015).

[Summary] Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (alphabetagamma) as well as proteolytically generated two-headed (betagamma) and one-headed (alpha) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed alphabetagamma and one-headed alpha exhibited significantly longer pitch. In contrast, the pitch driven by two-headed betagamma did not display this sensitivity. In the assays on lawns containing mixtures of alpha and betagamma at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of alpha in the mixture increased. Even small proportions of alpha-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (alphabetagamma) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.

Jun Kurushima, Daisuke Nakane, Takayuki Nishizaka, and *Haruyoshi Tomita,
Bacteriocin protein BacL1 of Enterococcus faecalis targets cell division loci and specifically recognizes L-Ala2-crossbridged peptidoglycan,
Journal of Bacteriology 197, 286-295 (2015).

[Summary] Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis, and consists of two extracellular proteins BacL1 and BacA. We previously reported that BacL1 protein (595 amino acids; 64.5 kDa) was a bacteriolytic peptidoglycan d-isoglutamyl-l-lysine endopeptidase inducing cell lysis of E. faecalis when an accessory factor BacA is co-present. However, the target of BacL1 remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent-conjugated recombinant protein demonstrated that BacL1 specifically localized at the cell division associated site including the equatorial ring, division septum, and nascent cell wall on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the 329-590 amino acid region of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1 and BacA. The static growth state also abolished the binding and the targeting of BacL1 to the cell division associated site. Furthermore, the targeting of BacL1 was detectable among Gram-positive bacteria with l-Ala-l-Ala crossbridging peptidoglycan including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae but not among bacteria with alternate peptidoglycan structure such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL1 specifically targets the l-Ala-l-Ala-crossbridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.


Yoshiaki Kinosita, Daisuke Nakane, Mitsuhiro Sugawa, Tomoko Masaike, Kana Mizutani, Makoto Miyata, and *Takayuki Nishizaka,
Unitary step of gliding machinery in Mycoplasma mobile,
Proceedings of the National Academy of Sciences 111, 8601-8606 (2014).

[Summary] Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0-4.5 mums(-1) with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.

International Conferences



*Yoshiaki Kinosita, Nariya Uchida, Daisuke Nakane and Takayuki Nishizaka,
Real-time imaging of a polymorphism, rotation rate and stepwise rotation of archaeal flagella (archaella),
Gordon Research Conference: Sensory Transduction in Microorganisms (Jan. 17-22, 2016), Ventura, USA.



*Takayuki Nishizaka, Daisuke Nakane,
Mechanics of novel motors revealed under advanced optical microscopes,
The 45th NIPS International symposium “Cutting-edge approaches towards the functioning mechanisms of membrane proteins” (Nov. 25-28, 2014), Okazaki, Japan.

Oral (contributed)

Takayuki Nishizaka, Takanobu Kato, Yoshiaki Kinosita,
Analysis of three-dimensional motion of motor proteins and supermolecular motility machinery,
The 6th International Symposium on Aero Aqua Bio-Mechanics (Nov. 13-16, 2014), Honolulu, Hawaii, USA.

Grant-in-Aid for Scientific Research (KAKENHI) on Innovative Areas, MEXT, Japan
Synergy of Fluctuation and Structure : Quest for Universal Laws in Non-Equilibrium Systems