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Red Cell Biology & its Disorders
Non-muscle myosin II drives vesicle loss during human reticulocyte maturation
Pedro L. Moura,1 Bethan R. Hawley,1 Tosti J. Mankelow,2,3
Rebecca E. Griffiths,2,3,4 Johannes G.G. Dobbe,5 Geert J. Streekstra,5 David J. Anstee,2,3 Timothy J. Satchwell1,2,3* and Ashley M. Toye1,2,3*
*These authors contributed equally to this work
ABSTRACT
The process of maturation of reticulocytes into fully mature ery- throcytes that occurs in the circulation is known to be character- ized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differ- ences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demon- strated that mechanical stimulus is necessary, but insufficient for reticu- locyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously unde- scribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.
Introduction
Reticulocytes are anucleate erythroid cells which undergo maturation to form the biconcave erythrocyte.1 The mechanism leading to maturation is relatively unde- fined, but it is known to occur in the peripheral circulation within 1-2 days after cells have egressed from the bone marrow.2 As the current endpoint of existing in vitro erythroid culture systems,3,4 interest in this cell type and the mechanisms and factors that may underlie and drive their maturation to erythrocytes has received renewed interest in recent years.
The phenotypic differences between reticulocytes and erythrocytes have been studied in detail. Reticulocyte maturation involves extensive membrane and cytoskeletal remodeling, with loss of approximately 20% of cell surface area during this process5-9 which allows the initially amorphous reticulocyte to acquire the char- acteristic erythrocyte biconcave morphology and accompanying increased resistance to shear stress. This remodeling is a highly selective process, characterized by protea- somal degradation and exocytosis of specific components (e.g. actin, myosin, talin) with preferential retention of the remainder (e.g. α or β-spectrin).7 Other notable hall- marks of maturation include a progressive loss of RNA content, an increase in cell deformability, and a decrease in both cytoplasmic and surface protein content through exocytosis, membrane shedding and autophagy-mediated pathways.6,10-12
Ferrata Storti Foundation
1School of Biochemistry, University of Bristol, UK; 2Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), UK; 3NIHR Blood and Transplant Research Unit, University of Bristol, UK; 4UQ-StemCARE, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Australia and 5Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, the Netherlands
Haematologica 2018 Volume 103(12):1997-2007
The most well-described pathway relating to loss of protein during reticulocyte
Correspondence:
t.satchwell@bristol.ac.uk or ash.m.toye@bristol.ac.uk
Received: June 1, 2018. Accepted: July 26, 2018. Pre-published: August 3, 2018.
doi:10.3324/haematol.2018.199083
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/12/1997
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