(

A03 KIMURA, Akatsuki |Proposed Research Projects (2016-2017)

Paper | Original Paper

2016

*Yuko Sato, Tomoya Kujirai, Ritsuko Arai, Haruhiko Asakawa, Chizuru Ohtsuki, Naoki Horikoshi, Kazuo Yamagata, Jun Ueda, Takahiro Nagase, Tokuko Haraguchi, Yasushi Hiraoka, Akatsuki Kimura, Hitoshi Kurumizaka, and *Hiroshi Kimura,
A genetically encoded probe for live-cell imaging of H4K20 monomethylation.,
Journal of Molecular Biology 428, 3885-2902 (2016).

[Summary] Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

Ritsuya Niwayama, Hiromichi Nagao, Tomoya Kitajima, Lars Hufnagel, Kyosuke Shinohara, Tomoyuki Higuchi, Takuji Ishikawa, and *Akatsuki Kimura,
Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.,
PLoS ONE 11, e0159917 (2016).

[Summary] Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

*Shigeru Matsumura, Tomoko Kojidani, Yuji Kamioka, Seiichi Uchida, Tokuko Haraguchi, Akatsuki Kimura, and Fumiko Toyoshima,
Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1.,
Nature Communications 7, 11858 (2016).

[Summary] Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1-LGN-NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry.