文部省 科学研究費補助金 新学術領域研究 植物発生ロジックの多元的開拓

PubMed ID 25118245 Pubmed 日付 2014/Aug/
タイトル Dynamics of vacuoles and H+-pyrophosphatase visualized by monomeric green fluorescent protein in Arabidopsis: artifactual bulbs and native intravacuolar spherical structures.
ジャーナル The Plant cell  2014/Aug/  26(8)  3416-34 
著者 Segami Shoji S,Makino Sachi S,Miyake Ai A,Asaoka Mariko M,Maeshima Masayoshi M
抄録 We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H(+)-pyrophosphatase (H(+)-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H(+)-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H(+)-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H(+)-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H(+)-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.

 液胞の可視化の問題点と解決策

名古屋大学 細胞ダイナミクス 前島正義  投稿日時[2016-01-26 16:46:00]

植物液胞膜を可視化することは、植物細胞のダイナミクスを理解する上で不可欠である。通常のGFPを液胞膜プロトンポンプH+-Pyrophosphatase(H+-PPase)に導入したばあいは、GFP分子そのものの二量体形成により、細胞内に人工的な構造(これまでbulbと呼ばれた構造の一部)が形成されることを実証した。その解決策として、単量体型GFP(mGFP)を、H+-PPaseの可変部位に導入することにより、酵素機能を保持したままのH+-PPaseを可視化できる。これにより、分裂後の若い細胞が成熟して行く過程での液胞形態のダイナミクスを観察することが可能となり、intravacuolar strand形成など、液胞の形態変化についての詳細な知見が得られた。