Optogenetic approaches to study platelet production and function

• Applicants: Professor Dr. Markus Bender; Dr. Shiqiang Gao, Ph.D.
• Subject Area: Hematology, Oncology ; Anatomy and Physiology ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525167920

The molecular mechanisms that regulate megakaryocyte (MK) differentiation, polarization and platelet formation are only poorly understood. The second messengers Ca2+, cAMP and cGMP were reported to play important roles in MKs but mechanistic details are lacking. We hypothesize that their modulatory effects are varying dynamically in spatial and temporal terms, which cannot be easily dissected by traditional methods, other than optogenetics. Optogenetics is a technique that uses light to precisely modulate molecular events in living cells or organisms. In previous studies, we expressed optogenetic constructs in primary MKs, which allowed us to study in vitro the role of ion influx and cyclic nucleotide second messengers in MK biology, using activating light. A widely used optogenetic construct is Channelrhodopsin-2 (ChR2), a blue light-activatable, non-selective cation channel from the green alga Chlamydomonas reinhardtii. In order to better understand the influence of changes in intracellular Ca2+ levels on MK function, we modified ChR2 to the variant ChR2 XXM2.0 with increased Ca2+ permeability. We expressed ChR2 XXM2.0 in MKs after viral infection and manipulated Ca2+ signaling by light in a high spatiotemporal manner. Local illumination of spread ChR2 XXM2.0 expressing MKs triggered increased stress fiber formation, cell polarization, and motility towards the direction of light. Using inhibitors and genetically modified mouse models, we could show that MK polarization was dependent on Cdc42 and myosin IIA activity. In another approach, MKs expressing photoactivated adenylyl cyclase (bPAC) or photoactivated guanylyl cyclase (BeCyclop) significantly increased cAMP or cGMP levels, respectively, after illumination. The intracellular increase of these second messengers was strongly dependent on phosphodiesterase (PDE) activity. We identified PDE5 as the predominant PDE in MKs, regulating cGMP levels. Based on the MK results obtained in vitro in our previous study, we have generated optogenetic ChR2 XXM2.0 and BeCyclop transgenic mice and now propose to manipulate MK function in vivo as well as platelet function in vitro and in vivo by light. To further explore the subcellular role of the small GTPase Cdc42, which participates in the light-induced Ca2+ signalling-mediated MK polarization, we propose to directly control the local activity of Cdc42 by light, which can be achieved by establishing a light-inducible translocation tool to control the localization of target proteins within MKs. We also aim to expand this to other proteins in order to precisely control and manipulate MK differentiation and platelet production with the help of light and thereby identify key proteins and mechanisms. Taken together, we have established for the first time optogenetics in bone marrow-derived MKs, and now intend to look deeper into the dynamic roles of Ca2+, cAMP/cGMP and proteins, such as small GTPases, in both MKs and platelets.

• DFG Programme: Research Grants