PB0353 - RhoB drives megakaryocyte transendothelial migration in RhoA-deficient mice

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Maximilian Englert, MSc

PhD Student
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany

Co-Author(s)

KA

Katja Aurbach

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

Tobias Heib

PhD
Institute of Experimental Biomedicine, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg
Ulm, Bayern, Germany
Isabelle C. Becker, PhD

Postdoctoral Fellow
Boston Children's Hospital
Boston, Massachusetts, United States
LW

Lou M. Wackerbarth

PhD Student
Institute of Experimental Biomedicine, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg
München, Bayern, Germany
Charly Kusch

PhD Student
University Hospital Würzburg, Germany
Würzburg, Bayern, Germany
UK

Ulla G. Knaus

Professor
Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland
Belfield, Dublin, Ireland
CS

Christian Stigloher

Professor
Imaging Core Facility, Theodor-Boveri Institute of Biosciences, Biocenter, Julius-Maximilians-Universität Würzburg
Würzburg, Bayern, Germany
IP

Irina Pleines

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

Bernhard Nieswandt

Professor
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany
ZN

Zoltan Nagy

Project Leader
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany

Background: Platelet-producing megakaryocytes (MKs) are large cells in the bone marrow (BM), localized in close vicinity to vascular sinusoids. The penetration of the endothelial barrier and subsequent intravascular protrusion formation by MKs are key steps in platelet biogenesis, however the molecular mechanisms regulating these processes are unclear. It was previously reported that entire MKs transmigrate into the BM sinusoids in mice lacking RhoA conditionally in the MK lineage (RhoAfl/fl;Pf4-Cre mice; here referred to as RhoA-deficient for simplicity). RhoB is closely related to RhoA and regulates cell migration and invasion in other cell types, although its role in MKs remains unknown.

Aims: To dissect the mechanisms underlying transendothelial migration of RhoA-deficient MKs in the BM.

Methods: RhoB-deficient (constitutive), RhoA-deficient (conditional), RhoA/RhoB double-deficient and RhoA-deficient/RhoB-heterozygous mice were analysed for platelet production. MK number, localization and transmigration were assessed in stained cryosections using immunofluorescence microscopy. RhoB protein levels in MKs and platelets were determined by Western blotting.

Results: We found that RhoB is expressed in wild-type (WT) MKs and platelets and becomes highly overexpressed in RhoA-deficient MKs. We hypothesized that increased RhoB levels might induce the observed MK transmigration phenotype in RhoA-deficient mice. While RhoB-deficient mice displayed microthrombocytopenia, but no evidence of increased MK transmigration, double-deficient mice exhibited severe macrothrombocytopenia, but reconstituted MK distribution at BM sinusoids to normal, compared to RhoA-deficient mice. Interestingly, we found that RhoA-deficient/RhoB-heterozygous mice, in which MKs express WT levels of RhoB, also showed a reverted phenotype with normal MK localization.

Conclusion(s): We conclude that the increased RhoB levels contribute to MK transmigration in RhoA-deficient MKs and that RhoA and RhoB have opposing roles in this lineage. These findings suggest that MK localization and proplatelet formation are critically regulated by a balanced expression of RhoA and RhoB.