OC 35.1 - Light-induced Ca2+ signaling triggers megakaryocyte polarization

Monday, July 11, 2022

10:45 AM – 11:00 AM

Location: ExCel Center, ICC Capital Suite Room 8&9

Presentation Format: In-Person

Presenting Author(s)

Yujing Zhang, PhD student

PhD student
University Hospital Wuerzburg and Rudolf Virchow Center, University of Würzburg
wuerzburg, Bayern, Germany

Co-Author(s)

SG

Shiqiang Gao

Group leader
Department of Neurophysiology, Institute of Physiology, Biocenter, University of Würzburg
Wuerzburg, Bayern, Germany
JY

Jing Yu-Strzelczyk

PhD student
Department of Neurophysiology, Institute of Physiology, Biocenter, University of Würzburg
Wuerzburg, Bayern, Germany
HK

Hendrikje Kurz

Dr
Institute of Experimental Biomedicine - Chair I, University Hospital Wuerzburg and Rudolf Virchow Center, University of Würzburg
Wuerzburg, Bayern, Germany
GN

Georg Nagel

Professor
Department of Neurophysiology, Institute of Physiology, Biocenter, University of Würzburg
Wuerzbrug, Bayern, Germany
MB

Markus Bender

Principal Investigator
Institute of Experimental Biomedicine, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg
Würzburg, Bayern, Germany

Background: 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. 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-activated cation channel from the green alga Chlamydomonas reinhardtii. We modified ChR2 by molecular engineering to obtain higher Ca2+ conductance (ChR2-XXM2.0), which allows us to investigate the role of Ca2+ in MK function.

Aims: We expressed ChR2-XXM2.0 in MKs to manipulate Ca2+ signaling by light in a high spatiotemporal manner to better understand the influence of intracellular Ca2+ changes on MK function.

Methods: ChR2-XXM2.0 was expressed in bone marrow-derived MKs after viral 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 with confocal microscopy.

Results: ChR2-XXM2.0 localized in the plasma membrane and the demarcation membrane system (DMS) in MKs. Blue light induced a significant photocurrent in ChR2-XXM2.0 expressing MKs, which indicates cation influx. Global illumination of ChR2-XXM2.0 expressing MKs spread on fibrinogen resulted in intracellular Ca2+ increase, stress fiber formation and more pronounced MK spreading. Local illumination triggered MK polarization and motility towards the direction of light. Using inhibitors and genetically-modified mouse models, we show that MK polarization was dependent on Ca2+ influx, Cdc42 expression and myosin IIA activity. Impaired light-induced polarization of Cdc42-deficient MKs was rescued upon co-expression of the Cdc42/ChR2-XXM2.0.

Conclusion(s): We established optogenetics in bone marrow-derived MKs. Light-induced local Ca2+ influx triggers Cdc42- and myosin IIA-dependent MK polarization. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.