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P.L. Moura et al.
maturation concerns the removal of the transferrin recep- tor CD71.10 This mechanism of plasma membrane protein loss, which involves the internalization of receptors with- in endocytic vesicles, multivesicular body formation and the release of exosomes has been extensively studied and shown to also mediate the loss of other proteins.12 Other major mechanisms involved in the removal of proteins during reticulocyte maturation are encompassed by the process of autophagy. Autophagy occurs through the sort- ing of redundant, damaged or leftover organelles and other cytoplasmic content into autophagosomes,13 which in reticulocytes have been reported to fuse with lyso- somes and form autophagolysosomes that are later expelled from the cell.4,14 Parts of this process are highly specific: for instance, NIX-/- mice undergo autophagy and maturation, but not mitophagy, causing retention of mito- chondria in reticulocytes.15
Using reticulocytes cultured in vitro, the autophagosome was recently shown to be involved in the process of remodeling of the reticulocyte membrane which involves the release of an inside-out vesicle.4 It is also known that, in other mammalian cells, mechanical stress upregulates autophagy, and the removal of autophagic vesicles in the reticulocyte is triggered by passage through the sinusoidal walls of the spleen.16,17 However, the process of autophagic vesicle transport and the eventual release of these vesicles from reticulocytes is poorly understood.
In vitro-derived reticulocytes expanded and differentiated under conditions compatible with clinical use do not cur- rently emulate the final stages of maturation, which occur in vivo after egress from the bone marrow and within the cir- culation to generate definitive erythrocytes. However, trans- fusion of in vitro-derived reticulocytes into mouse models induced these final stages of maturation,3,18 indicating the involvement of as of yet undefined factors or stimuli not recapitulated in the in vitro culture process. During their time in the peripheral circulation, reticulocytes are exposed to a variety of new stimuli, including shear stress, dynamic pres- sure changes, contact with other cell types (endothelial cells, residing spleen and liver macrophages) and a varying pH, pO2 and pCO2. We hypothesized that shear stress may be a driver for maturation and demonstrate here that it is possi- ble to simulate the shear stress component of in vivo circula- tion using a simple ex vivo circulation mechanism, leading to loss of cell surface area and selective loss of protein content and of mitochondrial content in cultured reticulocytes. Finally, we delineate a novel role for non-muscle myosin IIA (NMIIA) in shear-responsive reticulocyte vesicle transport and maturation. We demonstrate its specific phosphoryla- tion and localization in the proximity of autophagic vesicle markers in reticulocytes and show that chemical inhibition of NMIIA leads to an inability to lose cell volume, as well as a reduction in mitochondrial clearance.
Methods
Antibodies
A list of antibodies used in this study is provided in the Online Supplementary Methods.
Native reticulocyte isolation and in vitro erythroid culture
Native CD71+ reticulocytes and CD71- erythrocytes were isolat- ed from the red blood cell fraction obtained by Histopaque sepa-
ration of healthy donor platelet apheresis waste blood using CD71 MicroBead (Miltenyi Biotec) isolation according to the manufac- turer’s instructions. In vitro-cultured reticulocytes were differenti- ated from CD34+ cells isolated from the mononuclear cell fraction according to previously published protocols.4 All source material was provided with written informed consent for research use given in accordance with the Declaration of Helsinki (NHSBT, Filton, Bristol). The research into the mechanisms of erythro- poiesis was reviewed and approved by the Bristol Research Ethics Committee (REC Number 12/SW/0199).
Proteomics experimental design, data acquisition and analysis
Two experiments were performed: (i) a comparison of erythro- cytes, endogenous reticulocytes and in vitro-derived reticulocytes with three biological repeats per cell type, for a total of nine indi- vidual samples which were processed through both quantitative tandem mass tag (TMT) proteomics and qualitative TiO2-enriched phosphoproteomics; and (ii) a comparison of circulated and non- circulated in vitro-derived reticulocytes with three biological repeats per condition, for a total of six individual samples, which were processed through quantitative TMT proteomics. Cells were washed three times with phosphate-buffered saline containing 1 mg/mL bovine serum albumin and 2 mg/mL glucose; 2x106 cells were counted and used per sample for quantitative TMT pro- teomics, and 10x106 cells for qualitative phosphoproteomics. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD009015, PXD009023 and PXD009024. Extended details on the methods for sample prepara- tion, data acquisition and analysis are provided in the Online Supplementary Methods.
Immunofluorescence and automated image processing
Details on the methods for fixed and live-cell immunofluores- cence are provided in the Online Supplementary Methods. For quan- titative analysis, tile scans composed of 10x10 images taken at 1024x1024 resolution of cells labeled with Calcein Blue and Mitotracker Deep Red were generated using confocal imaging and analyzed with arivis Vision4D (arivis, Germany). Further details on the analysis are provided in the Online Supplementary Methods.
Sample preparation for rheoscopy and cell analysis
Two million cells were diluted in 200 mL of a polyvinylpyrroli- done solution (polyvinylpyrrolidone viscosity 28.1; Mechatronics Instruments). Samples were assessed in an Automated Rheoscope and Cell Analyzer (ARCA)19 consisting of a plate–plate optical shearing stage (model CSS450) mounted on a Linkam imaging sta- tion assembly and temperature controlled using Linksys32 soft- ware (Linkam Scientific Instruments). The microscope was equipped with an LMPlanFl 50x with a 10.6 mm working distance objective (Olympus) illuminated by an X-1500 stroboscope (Vision Light Tech) through a band-pass interference filter (CWL 420 nm, FWHM 10 nm; Edmund Optics). Images were acquired using a uEye camera (UI-2140SE-M-GL; IDS GmbH). At least 1500 valid cells per sample were analyzed using bespoke ARCA analy- sis software.
Ex vivo cell circulation
Details on the construction of the circulation system are provid-
ed in the Online Supplementary Methods. Cells were packed and resuspended in a mixture of culture medium and Sanquin reticu- locyte stabilizer solution (Sanquin Blood Supply, the Netherlands) in a 4:1 ratio. The suspension was then circulated overnight at 37oC in 5% CO2, with the 5 rpm setting. A matched control sam-
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haematologica | 2018; 103(12)