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NMII participates in reticulocyte maturation
were selected for further investigation. Coincidentally, both NMIIA and IIB were consistently phosphorylated in native and/or cultured reticulocyte samples, but not in erythrocytes. Interestingly, the occurrence of phosphory- lation at the S1943 site on NMIIA has previously been implicated in cargo binding in natural killer cells.29 Since reticulocyte maturation involves vesicle extrusion,4 we examined whether phospho-NMIIA could be the driving force behind this activity.
Using a well-characterized commercially available phospho-residue specific antibody we co-labeled fixed and permeabilized reticulocytes for phospho-NMIIA together with its non-phosphorylated form. Non-phos- phorylated NMIIA localize at the plasma membrane (Figure 2A, Online Supplementary Figure S5), as anticipated based upon its known integration with the spectrin-actin cytoskeleton.30 However, phosphorylated NMIIA local- ized to punctae that were observed to be exclusive to reticulocytes. The distinct localization of phospho-NMIIA in reticulocytes was particularly striking, and we therefore decided to validate the signal of the phospho-antibody through a dephosphorylation assay with lambda phos- phatase. After lambda phosphatase treatment, the phos- pho-NMIIA signal was largely abrogated in the reticulo- cyte (Figure 2B), and this result was confirmed by western blotting (Figure 2C). Unexpectedly, not all phospho- NMIIA-positive structures were found to co-label with non-phosphorylated NMIIA. We speculate that this may reflect differing binding accessibility of the antibodies used against the phosphorylated and non-phosphorylated forms of the protein.
To further investigate the localization of phospho- NMIIA and to identify the adjacent vesicular compart- ment, reticulocytes were co-labeled with an antibody spe- cific to the lipidated form of LC3B, an established marker of autophagic membranes.31 Figure 2D shows fluorescent labeling of phospho-NMIIA immediately proximal to the autophagic vesicles. Phosphorylated myosin light chain (S20, MLC), a marker for increased myosin activity,32 was then detected with additional vesicle markers as shown in Figure 2E. Using protease treatment methodology to detect membrane proteins that have been internalized from the plasma membrane,33 phosphorylated MLC was found adjacent to glycophorin A-positive vesicles inside the cell, emerging from the cell and in trypsin-treated reticulocytes. These combined data suggest that NMIIA may have a role in the movement of vesicles in the retic- ulocyte.
In vitro circulation of cultured reticulocytes simulates the maturation process
The most striking difference between in vitro cell cul- turing systems and the conditions to which maturing reticulocytes are exposed in the body is the absence of shear stress caused by circulation in the bloodstream. Therefore, to investigate the effects of this influencing factor, we attempted to construct a system that could induce shear stress conditions similar to those of in vivo circulation. Microcirculation systems have previously been used to study the effects of circulation in other cell types,34,35 however, our objective was to build a relatively inexpensive and scalable system. Thus, rather than use lithography for the creation of microcapillary-scale tub- ing (as this would limit the volumes used), we decided to use wider and more flexible gas-permeable tubing. The
final system has a very simple design and can be con- structed with readily available parts, as shown in the dia- gram (Figure 3A).
Deformability and cross-sectional area of unstimulated reticulocytes and erythrocytes were measured using an Automated Rheoscope and Cell Analyzer (ARCA)19 and the measurements were used as a basis to assess alter- ations in these parameters in response to circulation. Figure 3B demonstrates a decrease in cross-sectional area to a range more akin to that observed for erythrocytes following overnight circulation of reticulocytes, provid- ing important evidence that circulatory shear stress ex vivo is able to induce alterations in the morphology of reticulocytes associated with maturation. Figure 3C shows that the erythrocyte and reticulocyte populations are visually distinct when the cross-sectional area and deformability measurements are combined, with the dis- tribution of circulated reticulocytes approaching the dis- tribution of erythrocytes. Figure 3D shows no reduction in the deformability index profile, confirming that the cells maintain their functional viability and do not form microspherocytes.
Matched control and circulated reticulocytes were sub- mitted for TMT-based quantitative proteomics (Online Supplementary Table S4), which showed that the global outlook is of reduced protein content after circulation (Figure 3E). However, not all proteins decreased in the same manner. As expected, the amount of CD71 decreased following circulation, albeit incompletely and to a variable degree between replicates. Figure 3F shows that the most greatly decreased proteins after circulation include a significant number of mitochondrial and riboso- mal proteins. Erythroid-specific proteins of the dataset are illustrated in Figure 3G, showing that the decrease is also consistent with previous literature on the protein changes inherent to reticulocyte maturation in mice7 (e.g. lower decreases of glycophorin A, band 4.1, 4.2 and spec- trins compared to adducin). Of note, a large number of serum-derived proteins were shown to exhibit increased abundance within the samples prepared from circulated compared to uncirculated reticulocytes. Since the cells were incubated in identical media, it is likely that circula- tion results in increased adhesion of these proteins to the extracellular membrane of the reticulocytes. Although this is likely inconsequential, we cannot exclude a role for serum protein binding in facilitating maturation, there- fore, a detailed list of these proteins is provided in Online Supplementary Figure S6.
Non-muscle myosin IIA inhibition significantly affects the response of reticulocytes to shear stress
Having established a system that can be used to charac- terize the response of cultured reticulocytes to shear stress, we next tested the impact of blocking NMIIA- mediated activity with the use of blebbistatin, a selective and high-affinity inhibitor of NMIIA and NMIIB which preferentially binds to the ATPase intermediate and slows down phosphate release.36 Blebbistatin is particularly use- ful for experiments of this nature because of the existence of both an active and an inactive enantiomer, blebbis- tatin(-) and blebbistatin(+), respectively, which allows for the use of a matched control. It has been reported that NMIIB levels are undetectable through western blotting of red blood cells, with NMIIA being the predominant iso- form in human erythrocytes.37 Therefore, although bleb-
haematologica | 2018; 103(12)
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