Optogenetic Induction of Subcellular Ca2+ Events in Megakaryocytes and Platelets Using a Highly Ca2+-conductive Channelrhodopsin

Calcium signaling is crucial across various cell types, but its spatiotemporal dynamics remain difficult to study due to limited methods. Optogenetics, with its high precision, can address this challenge. In this study, we introduced the channelrhodopsin variant ChR2 XXM2.0, which exhibits high light sensitivity and enhanced Ca ²⁺ conductance in Xenopus oocytes, into bone marrow-derived megakaryocytes through viral transduction, aiming to clarify the poorly understood role of Ca ²⁺ dynamics in these cells. ChR2 XXM2.0 expression was confirmed in megakaryocyte membranes, and its functionality validated through whole-cell patch-clamp and calcium imaging. Localized activation of ChR2 XXM2.0 at the cell periphery induced cell polarization, dependent on localized calcium influx, myosin IIA, and integrin αIIbβ3-fibrinogen interaction. Furthermore, we generated a transgenic mouse line with Pf4-Cre -dependent expression of ChR2 XXM2.0, enabling optogenetic manipulation of anucleate blood platelets via light-triggered calcium signaling. Illumination induced phosphatidylserine and P-selectin exposure in spread platelets. Our results highlight the importance of asymmetric subcellular calcium events in megakaryocyte polarity and demonstrate the feasibility of manipulating platelet function using optogenetics. Taken together, our study introduces the ChR2 XXM2.0 construct and its corresponding Cre-dependent transgenic mouse line as powerful tools for manipulating subcellular Ca ²⁺ signaling, with potential applications for different cell types.