Phosphatidic acid is a pleiotropic lipid involved in lipid transport across different cellular organelles as well as major cellular signaling pathways through its metabolism. However, understanding and mapping its physiological functions require methods that can detect its rapid spatial concentration changes. Optogenetic membrane editors based on the biosynthesis of phosphatidic acid using phospholipase D (PLD) have been developed; although their activity was improved in superactive PLDs (superPLDs), these editors show background activities that limit the temporal resolution and cause cytotoxicity in cells. Now, Li et al. report a strategy to directly control the enzymatic activity of superPLDs, without toxicity.

The design involves engineering the light–oxygen–voltage 2 (LOV2) domain of phototropin 1 from Avena sativa so that it allosterically controls the activity of superPLDs by changing conformation upon blue light illumination, to create LOVPLDs. When transfected into HEK293T cells, the construct showed an increase in signal-to-noise ratio due to the difference between active and inactive states. To further lower the residual background activity, the team combined LOVPLD with blue-light-induced dimerization systems so that the light stimulus would serve as a single trigger for controlling both PLD catalytic activity and localization. Of these, improved light-induced dimer (iLID)-LOVPLD dissociated from the membrane rapidly in the absence of blue light, enabling tighter spatiotemporal control.

磷脂酸是一种多效性脂质，参与跨不同细胞器的脂质转运以及通过其代谢的主要细胞信号传导途径。然而，理解和绘制其生理功能需要能够检测其快速空间浓度变化的方法。已开发出基于使用磷脂酶 D (PLD) 生物合成磷脂酸的光遗传学膜编辑器；尽管它们的活性在超活性 PLD (superPLD) 中得到了改善，但这些编辑器显示出限制时间分辨率并导致细胞毒性的背景活性。现在，李等人。报告了一种直接控制 superPLD 酶活性且无毒性的策略。

该设计涉及对燕麦向光素1 的光氧电压 2 (LOV2) 结构域进行改造，使其通过在蓝光照射下改变构象来变构控制 superPLD 的活性，从而创建 LOVPLD。当转染到 HEK293T 细胞中时，由于活性和非活性状态之间的差异，该构建体显示出信噪比的增加。为了进一步降低残留背景活性，研究小组将 LOVPLD 与蓝光诱导二聚系统结合起来，这样光刺激就可以作为控制 PLD 催化活性和定位的单一触发器。其中，改进的光诱导二聚体 (iLID)-LOVPLD 在没有蓝光的情况下迅速从膜上解离，从而实现更严格的时空控制。