Light-induced ion influx triggers megakaryocyte polarization

Objective Bone marrow megakaryocytes (MKs) extend proplatelets into sinusoidal blood vessels where these proplatelets undergo fission to release platelets. The molecular mechanisms that regulate megakaryocyte differentiation, polarization and proplatelet formation are only poorly understood. Here, we expressed an optogenetic construct in primary MKs, which allows us to study these complex processes by spatiotemporally controlling cellular activity using light. A widely used optogenetic construct is Channelrhodopsin2 (ChR2), which is a blue light-activatable, non-selective cation channel from the green alga Chlamydomonas reinhardtii. We modified ChR2 to obtain higher calcium conductance and named the protein ChR2 XXM2.0. We expressed ChR2 XXM2.0 in MKs to manipulate calcium signaling by light in a high spatiotemporal manner in order to better understand the influence of changes in intracellular Ca2+ levels on MK function.

Material and Methods ChR2 XXM2.0 was expressed in bone marrow-derived MKs after virus transduction. Whole cell patch-clamp was used to test the functionality of the channel. MKs were globally or locally illuminated and subsequent MK behavior was analyzed by light and fluorescence microscopy.

Results ChR2 XXM2.0 localized in the plasma membrane and the demarcation membrane system in MKs. Application of blue light induced a significant photocurrent in ChR2 XXM2.0 expressing MKs, which indicates cation influx into MKs. Increased intracellular calcium levels were observed with a Cal 590 sensor in ChR2 XXM2.0 positive MKs after illumination. Global illumination of ChR2 XXM2.0 expressing MKs resulted in a blebbing behavior and exposure of phosphatidylserine on the cell surface. Incubation with the apoptotic inhibitor, Q-VD-OPh, decreased the percentage of MK blebbing from 61 % to 9 %, indicating an apoptotic process. Local illumination of ChR2 XXM2.0 positive MKs spread on fibrinogen triggered increased stress fiber formation, cell polarization and motility towards the direction of light.

Conclusion Here, we established for the first time optogenetics in bone marrow-derived MKs. We observed cell polarization after local illumination of ChR2 XXM2.0 positive MKs due to a local increase of ion influx. Currently, we are expressing ChR2 XXM2.0 in genetically modified MKs to identify key proteins and mechanisms involved in calcium regulated MK polarization.

Publication History

Article published online:
18 June 2021