Institute of Experimental Biomedicine-Chair I, University Hospital and Rudolf Virchow Center, 97080 Würzburg, Germany.
Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Baden-Württemberg, Germany.
Open Biol. 2022 Aug;12(8):220058. doi: 10.1098/rsob.220058. Epub 2022 Aug 17.
Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Cyclase opsin (Cyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. Cyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.
环磷酸鸟苷 (cGMP) 信号转导在许多细胞类型中发挥着基本作用，包括血小板。cGMP 已被牵涉到血小板的形成，但关于其在巨核细胞 (MKs) 中的时空调节的机制细节尚不清楚。光遗传学是一种允许在活细胞或生物体中对分子事件进行时空操作的技术。我们利用这种方法，在骨髓 (BM) 衍生的 MKs 中转基因表达了一种光激活的鸟苷酸环化酶，Cyclase opsin (Cyclop)，以精确地光调节 cGMP 水平。Cyclop-MKs 在光照后表现出明显增加的 cGMP 浓度，这强烈依赖于磷酸二酯酶 (PDE)5 的活性。这一发现通过实时成像 cGMP 信号得到了证实，揭示了药理学 PDE5 抑制也增强了 BM MKs 中一氧化氮触发的 cGMP 生成。总之，我们首次在原代 MKs 中建立了光遗传学，并表明 PDE5 是调节 MKs 中 cGMP 水平的主要 PDE。这些发现还表明，光遗传学允许对 MK 生物学进行精确操作。
