Optogenetic manipulation of cGMP highlights PDE5 as the predominant cGMP-hydrolyzing PDE in megakaryocytes

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 spatiotemporal regulation in megakaryocytes (MKs) is lacking. We expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin ( Be Cyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. Be 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 (NO) triggered cGMP generation in BM MKs. In summary, we established for the first time optogenetics in primary MKs and identified PDE5 as the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.