Optogenetic control of phosphoinositide metabolism
Optogenetic control of phosphoinositide metabolism
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DOI：
10.1073/pnas.1211305109
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发表时间：
2012-08-28
影响因子：
11.1
通讯作者：
De Camilli, Pietro
中科院分区：
文献类型：
--
作者：
Idevall-Hagren, Olof;Dickson, Eamonn J.;De Camilli, Pietro
磷脂酰肌醇(PI)是细胞膜上调节多种细胞功能的脂类成分。在这里，我们利用蓝光诱导的两种植物蛋白--隐花色素2(CRY2)和转录因子CIBN之间的二聚化，来快速、局部和可逆地控制质膜PI水平。将作用于PI(4，5)P-2和PI(3，4，5)P-3的OCRL(5-ptase，OCRL)的肌醇5-磷酸酶结构域与CRY2的光解酶同源区融合，并将CRY2结合区CIBN与质膜靶向基序融合。表达这些结构物的哺乳动物细胞在蓝光(458-488 nm)照射下，5-ptase(OCRL)几乎立即被募集到质膜上，通过各种细胞检测发现，其靶标快速(在数秒内)和可逆(在数分钟内)去磷酸化：PI(4，5)P-2和PI(3，4，5)P-3生物传感器解离，胞内cathrin包被的凹坑消失，KCNQ2/3通道电流几乎完全受到抑制，膜褶皱消失。焦点照射导致局部和瞬时的5-ptase(OCRL)募集和PI(4，5)P-2去磷酸化，不仅导致细胞边缘或突起的局部塌陷和回缩，而且还导致PI(4，5)P-2生物传感器的代偿性积累和细胞对侧的膜褶皱。用同样的方法来募集PI3K，可以诱导局部PI(3，4，5)P-3的合成和膜的褶皱，而在照明区远端相应的褶皱消失。这项技术为以高时空动力学解剖各种PI的细胞功能和可逆地控制这些信号脂质下游效应器的功能提供了有力的工具。
Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P-2 and PI(3,4,5)P-3, was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P-2 and PI (3,4,5)P-3 biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P-2 dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P-2 biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P-3 synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.