1. Inaki, M., Yang L. J., and Matsuno K.
Left-right asymmetric morphogenesis in Drosophila and other invertebrates: the discovery of intrinsic cell chirality and its functions.
Reviews in Cell Biology and Molecular Medicine in press (2017)

The formation of left-right (LR) asymmetry is one of the fundamental problems to be solved in developmental biology. In various vertebrate species, the LR axis forms as a result of a leftward flow of extraembryonic fluid that is generated by motile cilia. However, recent studies show that the mechanisms of LR-asymmetric development are evolutionarily divergent even among vertebrates. In snails and nematodes, the LR asymmetry of blastomeres plays a key role in the LR-asymmetric disposition of blastomeres during subsequent development, leading to LR-asymmetric cell-cell interactions among the blastomeres. Such LR asymmetry of cells can be defined as cell chirality. An object has chirality if it cannot be superimposed onto its mirror image. In some Drosophila organs, epithelial cells have intrinsic cell chirality that drives LR-asymmetric morphogenesis. Thus, the mechanisms of LR-asymmetric development in Lophotrochozoa and Ecdysozoa differ from the motile-cilia-driven mechanisms found in vertebrates. Although intrinsic cell chirality has been observed in various cultured vertebrate cells, the biological role of this chirality is unknown. Cell chirality might be a general mechanism for LR-asymmetric development across phyla.





1. Inaki, M., Yang L. J., and Matsuno K.
Cell chirality: its origin and roles in left-right asymmetric development.
Phil Trans B 371, 20150403 (2016).
DOI: 10.1098/rstb.2015.0403

An item is chiral if it cannot be superimposed on its mirror image. Most biological molecules are chiral. The homochirality of amino acids ensures that proteins are chiral, which is essential for their functions. Chirality also occurs at the whole-cell level, which was first studied mostly in ciliates, single-celled protozoans. Ciliates show chirality in their cortical structures, which is not determined by genetics, but by 'cortical inheritance'. These studies suggested that molecular chirality directs whole-cell chirality. Intriguingly, chirality in cellular structures and functions is also found in metazoans. In Drosophila, intrinsic cell chirality is observed in various left–right (LR) asymmetric tissues, and appears to be responsible for their LR asymmetric morphogenesis. In other invertebrates, such as snails and DCaenorhabditis elegans, blastomere chirality is responsible for subsequent LR asymmetric development. Various cultured cells of vertebrates also show intrinsic chirality in their cellular behaviours and intracellular structural dynamics. Thus, cell chirality may be a general property of eukaryotic cells. In Drosophila, cell chirality drives the LR asymmetric development of individual organs, without establishing the LR axis of the whole embryo. Considering that organ-intrinsic LR asymmetry is also reported in vertebrates, this mechanism may contribute to LR asymmetric development across phyla.



2. Matsumoto, K., Ayukawa, T., Ishio, A., Sasamura, T., Yamakawa, T. and Matsuno, K.
Dual roles of O-glucose glycans redundant with monosaccharide O-fucose on Notch in Notch Trafficking.
J. Biol. Chem. 291, 13743-13752 (2016).

Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions.





1. Okumura, T., Sasamura, T., Inatomi, M., Hozumi, S., Nakamura, M., Hatori, R., Taniguchi, K., Nakazawa,N., Suzuki, E., Maeda, R., Yamakawa, T., and Matsuno, K.
Class I myosins have overlapping and specialized functions in left-right asymmetric development in Drosophila.
Genetics 199 (4) 1183-1199 (2015).
DIO: 10.1534/genetics.115.174698.

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.



2. Ishio, A., Sasamura, T., Ayukawa, T., Kuroda, J., Ishikawa, H. O., Aoyama, N., Matsumoto, K., Gushiken, T., Okajima, T., Yamakawa, T., and Matsuno, K.
O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation.
J. Biol. Chem. 290, 505-519 (2015).
DIO: 10.1074/jbc.M114.616847.

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1(R245A knock-in)), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1(R245A knock-in) and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein's functions.

Notchシグナルは、ショウジョウバエの左右非対称性形成に必須である。Notchシグナルは、細胞間相互作用を介した細胞運命決定において重要な役割を担っている。膜貫通型受容体であるNotchの細胞外ドメインには、リガンドが結合するEGF様リピート存在する。これらのEGF様リピートの一部には、protein O-fucosyltransferase 1(O-fut1)によってO-フコースが、RumiによってO-グルコースが付加される。我々は、O-fut1遺伝子がNotchシグナル伝達に必須であることを明らかにした。一方、O-fut1は、酵素活性非依存的な、Notchに対するシャペロン機能をもっている。O-fut1遺伝子がNotchシグナルに必須であることは、O-fut1のシャペロン機能がNotchシグナルに不可欠であると考えても説明できることから、NotchのO-フコース単糖修飾の機能の有無については、異なった結果が報告されていた。