Reticulocyte and red blood cell deformation triggers specific phosphorylation events

Pedro Luís Moura, Maria A Lizarralde Iragorri, Olivier Francais, Bruno Le Pioufle, Johannes G. G. Dobbe, Geert J. Streekstra, Wassim El Nemer, Ash M Toye, Timothy Satchwell

Research output: Contribution to journalArticle (Academic Journal)peer-review

2 Citations (Scopus)
191 Downloads (Pure)

Abstract

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC’s response to mechanical stimuli and for the study of reticulocyte maturation.
Original languageEnglish
Pages (from-to)2653-2663
Number of pages11
JournalBlood Advances
Volume3
Issue number17
DOIs
Publication statusPublished - 10 Sep 2019

Keywords

  • erythrocytes
  • glycogen synthase kinase 3
  • phosphorylation
  • phosphotransferases
  • reticulocytes
  • shear stress
  • cytoskeleton
  • datasets
  • precipitating factors
  • retail clinics

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