Lolodi Ogheneochukome(分子情報解析学分野、博士後期課程)さんらの研究成果が米国科学雑誌「Molecular Biology of the Cell」(電子版)に掲載されました。

細胞質-核間での分子輸送現象を速度論的に解明

yoshimura01

Lolodi Ogheneochukome

筆者からのコメント:
細胞内のタンパク質はすべて細胞質のリボソームで合成されます。核内部で機能するタンパク質は、核膜に存在する核膜孔を通過して核内へと運ばれますが、この過程を制御しているのがインポーティンなどの核輸送因子群です。核膜孔は分子選択性があるため、多くのタンパク質は通過することができません。インポーティンはターゲットとするタンパク質に結合し、核膜孔を通過しやすくするはたらきがあります。インポーティンは、その名前の通り、細胞質から核質へ向かう一方向輸送を促進しますが、ターゲットとするタンパク質を運び終わると自身はまた細胞質へと戻ります。このような一見すると非平衡的なプロセスが、いかに細胞内で制御されて定常状態を維持しているかに関するシステム的な原理は不明のままでした。今回の論文では、これを反応速度論的観点から解析し、細胞内の定常状態における分子ダイナミクスの理論的基盤に関する重要な知見を得ることに成功しました。この成果は、細胞内の分子局在を成り立たせている膨大な動的平衡の統合的理解につながるとともに、このシステムの不全が細胞異常や病態につながるメカニズムの解明や、ドラッグデリバリーにおいて人工分子の細胞内動態を制御する技術の開発に向けた理論的基盤となることが期待されます。

Summary:
Proteins that are required for efficient nuclear functions are imported into the nucleus through the nuclear pore complexes (NPCs) which are also the export gates for nuclear products that are required in the cytoplasm. These molecules are usually transported by a class of protein vehicles called karyopherins, the best-studied of which is importin . In this study, we investigated in vivo dynamics of such nuclear transport processes, and found that the process that we have known as “import” is not a unidirectional process from cytoplasm to nucleoplasm, but a steady-state established by both influx and efflux between two compartments.
We performed a series of kinetic analyses of importin  and its target molecule (cargo) in a living cell by using FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching), as well as in vitro transport analysis and binding assay to extract kinetic parameters of each step of the reaction cycle. These parameters were used to build up a kinetic model of the entire transport cycle. Our model could successfully provide various reaction parameters and information on the cycle, such as intracellular distribution and fluxes (influx and efflux) of the cargo and importin , and a rate limiting step in various conditions. This result revealed a theoretical background of how a steady-state distribution of cellular proteins is established by bidirectional fluxes between cytoplasm and nucleoplasm.

Background:
Imagine a kitchen sink filled with a certain amount of water. If you unplug the drain, it will become empty. But, if you open the tap at the same time and keep supplying an appropriate amount of water, the volume of water will be apparently “constant”, which is maintained by the continuous inflow and outflow. This point is referred to as steady-state. This is also the case in living organisms whose cells contain a nucleus connected to the cytoplasm through special drains called nuclear pore complexes (NPCs). Proteins that are required for efficient nuclear functions are imported through the NPCs which are also the export gates for nuclear products that are required in the cytoplasm. These cargoes are usually transported by a class of protein vehicles called karyopherins, the best-studied of which is importin . The dynamic events of the importin -mediated pathway at steady-state are still not well known. In this study, we used fluorescence techniques and kinetic parameters to investigate how the steady-state nuclear transport cycle is maintained.

Methods:
The cytoplasm of live HeLa cells expressing nuclear-specific cargoes was continuously photobleached and the fluorescence signal of the nucleus was simultaneously monitored in a process called fluorescence loss in photobleaching (FLIP). The entire nucleus was also photobleached and the extent and speed of recovery of fluorescence signals was recorded in a process called fluorescence recovery after photobleaching (FRAP). In addition, in vitro nuclear transport assays and surface plasmon resonance (SPR) were used to obtain kinetic parameters with which a model was established to explain the entire cargo import cycle facilitated by importin .

Results:
Our FLIP experiment showed a progressively weaker nuclear fluorescence signal. This indicates that, at steady-state, the imported cargo could also be exported out of the nucleus by importin . The FRAP experiment revealed that the flux (import-export cycles) was inversely proportional to the size of the cargoes. In addition, our kinetic model revealed various reaction parameters and information on the cycle, such as intracellular distribution and fluxes of the cargo and importin The model also revealed that the affinity of importin  for its cargo in the cytoplasm determines the rate of the import process. Taken together, our data strongly suggests that shuttling of import and export cargoes may be a general feature of living cells at steady-state.

Perspectives:
Our model serves as a basis for understanding a variety of nucleocytoplasmic molecular dynamics at steady-state, and how this balance is disturbed by endogenous/exogenous factors. We will further focus on how the abnormality in this system leads to cellular aberrations and pathological status. Precise control of artificial molecules in cells will also be achieved by predicting steady-state dynamics according to our established system, which will benefit virus suppression and drug delivery techniques.

Lolodi O, Yamazaki H, Otsuka S, Kumeta M, Yoshimura SH. “Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport.” Mol Biol Cell (2015)
http://www.molbiolcell.org/content/early/2015/11/02/mbc.E15-08-0601.abstract

GSB-HP_Summary_Figure

分子情報解析学分野
http://www.lif.kyoto-u.ac.jp/j/?post_type=labos&p=164