核分裂酵母で細胞動因タンパク質をグローバルとローカルにカウントする
Jian-Qiu Wu1, Thomas D Pollard
1Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.
まとめ
研究者は,黄色の光タンパク質 (YFP) 融合を用いて,分裂酵母におけるタンパク質濃度を測定した. この顕微鏡法では,グローバルおよびローカルタンパク質レベルを定量化し,重要な細胞分割を明らかにします.
科学分野:
- 細胞生物学 細胞生物学
- バイオフィジックス 生物物理学
- 分子生物学は分子生物学である.
背景:
- タンパク質濃度の理解は,細胞機能にとって極めて重要です.
- 以前の方法では,グローバルおよびローカルタンパク質レベルを同時に測定する際の制限がありました.
研究 の 目的:
- グローバルおよびローカルタンパク質濃度 in vivo を測定する方法を開発し,検証する.
- Schizosaccharomyces pombeの28の細胞骨格およびシグナリングタンパク質の濃度を定量化するために.
主な方法:
- 光顕微鏡を用いて,黄光タンパク質 (YFP) 融合タンパク質濃度を測定した.
- 機能的なYFP融合タンパク質の制御された発現のためにネイティブプロモーターを使用.
- タンパク質濃度の精度のために,定量的免疫ボルトリングに対して検証された光測定値.
主要な成果:
- 光とタンパク質濃度の間の直接的比例を確立しました.
- 全球サイトプラズマ濃度測定は,0.04から63ミクロモラーまでの28種類のタンパク質について行われました.
- 細胞サイクルの間,収縮環やスパインドルポールボディのような特定の細胞区画で,重要なタンパク質濃縮 (最大7500倍) が観察されました.
結論:
- 開発された光顕微鏡のアプローチは,グローバルおよびローカルタンパク質濃度を正確に測定します.
- 核分裂酵母におけるタンパク質の豊富さの空間的および時間的な規制が顕著であることが示されています.
- この方法は,あらゆる融合タンパク質の濃度をin vivoで定量化するために適用できます.
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