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NMII participates in reticulocyte maturation
were left untreated or were treated with blebbistatin(-). In all cases, a reduction in the percentage of cells with mito- chondria upon circulation was identified, which was sig- nificantly affected by treatment with blebbistatin(-), as shown in Figure 5C. Thus, we have demonstrated a link between mitochondrial loss and NMII activity. In concert with the observation of mitochondrial co-localization with vesicle markers, proximity of NMIIA to those same vesicle markers and the impact of NMIIA activity in the loss of cross-sectional area in the circulating reticulocyte, we have demonstrated a functional link between NMIIA activity and vesicle clearance in reticulocyte maturation.
Discussion
The study of the process of human reticulocyte matura- tion has been massively enhanced by the ability to gener- ate large numbers of late reticulocytes by laboratory cul- ture methods.3 To date, much of the published literature has focused on comparison of in vitro-derived reticulocytes to circulating mature erythrocytes.20,21,39 Here we report the first quantitative protein abundance and qualitative phos- phoproteomic datasets comparing human donor-matched adult native reticulocytes, erythrocytes and in vitro culture- derived reticulocytes. These data highlight the broad pro- teomic equivalence of cultured reticulocytes to their natively derived counterparts and provide a basis for more detailed exploration of maturation processes and mecha- nisms that occur in vivo and in vitro.
In order to explore the contribution of circulatory shear stress to the maturation of reticulocytes and dissect this mechanical element from that of cell-cell interactions and other features of the in vivo circulatory system, a simple circulation system was developed that is easily adaptable and was shown to have a significant impact on reducing cross-sectional area, vesicle/mitochondrial content and general protein content of the circulated in vitro-derived reticulocytes in a way that recapitulates their natural pro- gression through maturation.
These ex vivo data highlight the influence that shear stress is able to exert on biological processes fundamental to reticulocyte maturation. We demonstrate that while the mechanical process of circulation results in a consistent reduction of cell volume and protein abundance, circulat- ed reticulocytes maintain partial expression of the trans- ferrin receptor, CD71. Loss of CD71 has been described in the literature as occurring via exosome release, a process which is independent from autophagosome release.8 Thus, our data provide further evidence for these path- ways being uncoupled in maturation.
Importantly, these data demonstrate that shear stress is necessary but not sufficient to generate a fully mature ery- throcyte and suggest that cell-cell interactions or other influencing aspects present during in vivo circulation are required to facilitate the complete maturation process.
In addition to loss of cell volume, loss of organellar con- tent is one of the distinguishing hallmarks of reticulocyte maturation in vivo, and here also shown to be stimulated by ex vivo circulation. Organelles such as mitochondria are cleared from the reticulocyte in a process partially depend- ent on autophagy.40 The presence of autophagic vacuoles in human reticulocytes was described by Kent et al. back in 1966.41 Subsequent studies demonstrated that the mat- uration of late circulating R2 reticulocytes involves the
generation of endocytic vesicles which fuse with autophagosomes to create large autophagic vesicles,4 cor- responding to the vacuoles described by Kent et al.41 However, the process leading to transport and extrusion of these vesicles is currently undefined.
Proteomic profiling of native and in vitro-derived reticu- locytes identified NMIIA as being among the most differ- entially expressed proteins in these cells compared to mature erythrocytes. NMIIA interacts with actin to con- tribute to various cellular processes, such as cell migration,42 adhesion43 and cytokinesis.44 NMIIA has also been implicated in autophagosome maturation and lyso- some fusion through association with autophagy-related receptors.45 In erythroid cells, the NMIIB isoform has been described as having an essential role in enucleation of the differentiating erythroblast.46 However, there is currently no defined role for NMIIA in erythroid cells other than as a component of the cytoskeleton.37 We show that reticulo- cytes exhibit a NMIIA phosphorylation that has been pre- viously associated with vesicle transport29 and is unde- tectable in the mature red blood cell, both by mass spec- trometric analysis and by immunofluorescence.
Phosphorylation of the S1943 site has been associated with filament destabilization,47 which could be necessary for the regulation of NMIIA assembly dynamics.48 Phosphorylated NMIIA localizes proximally to LC3B, a known autophagic vesicle marker which was observed to colocalize with other known erythroid autophagic vesicle markers. Moreover, the phosphorylated myosin light chain was found in a similar localization. The observed concurrent localization of the phosphorylated NMIIA heavy chain and the active light chain in proximity to reticulocyte vesicles led us to speculate that NMIIA is responsible for autophagic vesicle movement in the maturing reticulocyte. We explored this hypothesis through pharmacological inhibition of NMII activity by blebbistatin, a selective and potent inhibitor, and show that while inhibition of NMII activity does not affect retic- ulocyte viability or capacity to deform, it leads to a signif- icant decrease in the cell’s ability to respond to shear stress by loss of cell volume and autophagosome-mediated mitochondrial clearance.
Finally, it is notable that shear stress-induced calcium ion (Ca2+) influx is a well-described phenomenon in ery- throcytes49,50 as well as other cell types.51,52 NMIIA activity is regulated by phosphorylation of its light chain, which in turn is regulated by Ca2+ influx and interaction with calmodulin.32 It is therefore attractive to speculate that shear stress-mediated induction of Ca2+ influx may indi- rectly modulate NMIIA activity and thereby influence vesicle transport in the reticulocyte. However, further work is required to confirm this hypothesis.
In conclusion, our results have uncovered a previously undescribed mechanism of shear stress response in the human reticulocyte which is dependent on NMII activity for vesicle clearance and cell volume reduction.
Acknowledgments
We would like to thank Dr. Kate Heesom and Dr. Marieangela Wilson, as well as the proteomics facility of the University of Bristol, for proteomics sample processing and data acquisition. We also thank Dr. Emile van den Akker and Sanquin (Amsterdam, the Netherlands) for providing the Sanquin reticulocyte stabilizing reagent, Matthias Rust (arivis, Rostock, Germany) for the training provided with Vision4D, Dr.
haematologica | 2018; 103(12)
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