The final stages of mammalian terminal erythropoiesis involve cell cycle exit of orthochromatic erythroblast, enucleation of the condensed nucleus, and organelle clearance of the nascent reticulocytes. Although many critical factors in these processes have been discovered over the past years, several key questions remain unanswered. For example, what are the factors that regulate the exit of the last mitosis of erythroblast for enucleation? How does the nascent reticulocyte separate from the extruded nucleus? What are the signals involved in regulating the clearance of organelles in reticulocyte? Answers to these questions with mechanistic insights are important not only for our understanding of the basic biology of terminal erythropoiesis and pathophysiology of many red cell-related diseases, but also to provide clues for efficient strategies for in vitro or ex vivo generation of red blood cells in transfusion medicine.
Our work on formin family proteins, enzymes involved in linear actin filament polymerization, in erythropoiesis may shed light on the clues to these questions. We show in our published work that mDia2, one of the diaphanous-related formins, plays critical roles in enucleation and cytokinesis of erythroblasts. However, the mechanism of how mDia2 regulates these processes is unclear. In this study, we used mDia2 hematopoietic-specific knockout mouse model and revealed that mDia2 controls the motility of the nascent reticulocyte that is required for the detachment of the pyknotic nucleus. Reticulocytes in mDia2 deficient mice are rigid with extended spectrin chains, possibly due to disrupted actin protofilaments. Indeed, a stochastic optical reconstruction (STORM) high resolution microscopy analysis revealed that actin protofilaments were completely disrupted with loss of mDia2 in the reticulocytes. Using immuno-gold stain and electron microscopy, we further found that mDia2 localized at the junctional complex, confirming its critical role in the polymerization of actin protofilaments and maintenance of erythroid cytoskeleton. In addition to the cytoskeleton defects, reticulocytes from mDia2 deficient mice also showed enlarged volume with many organelles failed to be eliminated. Flow cytometry analyses showed that several membrane proteins destined to be downregulated, as well as mitochondria and lysosome markers, remained high in mDia2 deficient reticulocytes. We also performed a tandem mass tagging (TMT) mass spectrometry, which revealed numerous chromatin-associated proteins that failed to be downregulated. Together, these results demonstrated an important role of mDia2 in the reticulocyte maturation.
The erythroid phenotypes in mDia2 deficient mice, including failure of cytokinesis and organelle clearance, prompted us to investigate whether there are any defects in ESCRT complexes that are cellular components essential for these processes. Indeed, several ESCRT III complex and associated proteins, including Chmp5, Vta1, and Usp8, were significantly downregulated. mDia2 is known to function though actin polymerization to influence the transcriptional activity of SRF. We found that Chmp5 was a novel target of SRF through ChIP assay. Transplantation of mDia2 knockout c-kit positive progenitors transduced with Chmp5 into the lethally irradiated WT recipient mice dramatically reduced the percentages of bi-nuclear erythroblasts and reverted anemia. Consistent with phenotypes of Chmp5 knockout cells where increased late endosome and lysosome are commonly found, markers for late stage endosome and lysosome were significantly increased in mDia2 deficient erythroblasts and reticulocytes. These data support that mDia2 regulates endosome/MVB and lysosome discharge through Chmp5 during reticulocyte maturation. More importantly, overexpression of Chmp5 largely rescued the defects in lysosome and mitochondria clearance in mDia2 deficient reticulocytes.
Our study reveals mDia2 formin as a master regulator of the late stage terminal erythropoiesis in the maintenance of erythroid cytoskeleton and organelle clearance. The novel mDia2-SRF-ESCRT III complex pathway provides the first signaling axis that connects erythroid cytoskeleton to reticulocyte maturation, which may open a new field in signaling networks that modulate enucleation to reticulocyte formation.
Disclosures
Ji: Longbiopharma: Consultancy.
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