1.
Using split protein reassembly strategy to optically control PLD enzymatic activity.
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Yao, Y
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Lou, X
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Jianxu, L
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Zhu, M
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Qian, X
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Liu, J
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Zhang, L
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Zhang, P
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He, L
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Li, H
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Xu, Y
Abstract:
Phospholipase D (PLD) and phosphatidic acid (PA) play a spatio-temporal role in regulating diverse cellular activities. Although current methodologies enable optical control of the subcellular localization of PLD and by which influence local PLD enzyme activity, the overexpression of PLD elevates the basal PLD enzyme activity and further leads to increased PA levels in cells. In this study, we employed a split protein reassembly strategy and optogenetic techniques to modify superPLD (a PLDPMF variant with a high basal activity). We splited this variants into two HKD domains and fused these domains with optogenetic elements and by which we achieved light-mediated dimerization of the two HKD proteins and then restored the PLD enzymatic activity.
2.
Optogenetic control of epithelial-mesenchymal transition in cancer cells.
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Zhou, X
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Wang, J
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Chen, J
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Qi, Y
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Nan, D
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Jin, L
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Qian, X
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Wang, X
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Chen, Q
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Liu, X
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Xu, Y
Abstract:
Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the initiation and promotion of cancer cell metastasis. The phosphoinositide 3-kinase (PI3K) signaling pathway has been demonstrated to be involved in TGF-β induced EMT, but the complicated TGF-β signaling network makes it challenging to dissect the important role of PI3K on regulation of EMT process. Here, we applied optogenetic controlled PI3K module (named 'Opto-PI3K'), which based on CRY2 and the N-terminal of CIB1 (CIBN), to rapidly and reversibly control the endogenous PI3K activity in cancer cells with light. By precisely modulating the kinetics of PI3K activation, we found that E-cadherin is an important downstream target of PI3K signaling. Compared with TGF-β treatment, Opto-PI3K had more potent effect in down-regulation of E-cadherin expression, which was demonstrated to be regulated in a light dose-dependent manner. Surprisingly, sustained PI3K activation induced partial EMT state in A549 cells that is highly reversible. Furthermore, we demonstrated that Opto-PI3K only partially mimicked TGF-β effects on promotion of cell migration in vitro. These results reveal the importance of PI3K signaling in TGF-β induced EMT, suggesting other TGF-β regulated signaling pathways are necessary for the full and irreversible promotion of EMT in cancer cells. In addition, our study implicates the great promise of optogenetics in cancer research for mapping input-output relationships in oncogenic pathways.
