Red or green? Enucleation traffic light

In this issue of Blood, Keerthivasan and colleagues provide compelling support for the novel concept that the formation, movement, and fusion of endocytic vesicles in the region between the extruding nucleus and nascent reticulocyte are critical steps in erythroblast enucleation.¹ 

Erythroblasts complete terminal differentiation and enucleation in erythroid niches composed of erythroblastic islands nested in extracellular matrix proteins.²  Although approximately 2 million reticulocytes are generated per second, the mechanism(s) regulating mammalian erythroblast enucleation have remained largely mysterious. One popular postulate is that enucleation is a type of asymmetric cytokinesis: this is supported by the observation of concentrated actin in the region between the extruding nucleus and nascent reticulocyte, reminiscent of the acto-myosin ring present during cytokinesis.^3,4  Furthermore, it has been reported that enucleation requires normally functional actin³  and that Rac GTPases participate in enucleation by affecting the formation of the actin ring.⁵  One controversial theory is that microtubules may also participate in some phase of enucleation because in vitro and in vivo studies in rats show that a microtubule-depolymerizing agent inhibits nuclear extrusion.⁶  Hence investigations, to date, mainly offer hints as to players active in enucleation but present no well-delineated molecular mechanisms for the entire sequence of events leading to successful enucleation.

To address this important issue, Keerthivasan and colleagues more comprehensively explored the model of asymmetric cytokinesis. Treatment of postmitotic primary murine erythroblasts with well-characterized inhibitors of cytokinesis, such as blebbistatin, hesperadin, and nocodazole, did not block enucleation. The authors then turned to a fascinating and novel hypothesis based upon earlier electron micrographs that revealed an accumulation of vesicles in the region between the extruding nucleus and nascent reticulocyte. Could these vesicles participate in the enucleation process? After confirming the prior ultrastructural findings, Keerthivasan and colleagues elegantly examined different components of the vesicle trafficking pathway, using a diverse set of molecules to determine their impact on enucleation of adult spleen and fetal liver mouse erythroblasts. Inhibiting endocytosis with either dynamin inhibitors or sucrose, which blocks the formation of clathrin-coated pits, prevented enucleation. Monensin, which disrupts trafficking between endosomes and lysosomes, also inhibited enucleation. Importantly, these small molecules had little effect on differentiation or cell viability. Moreover, treatment with brefeldin A (which blocks endoplasmic reticulum and Golgi transport) or by A5 (an inhibitor of trafficking between the trans-Golgi network and endosomes) did not interfere with enucleation. These results clearly show that intact endocytic vesicle trafficking and the endosome/lysosome secretory pathway are essential for nuclear extrusion. Further evidence of the importance of vesicle trafficking included inhibition of enucleation in cultured human CD34⁺ cells by knockdown of clathrin and increased enucleation after inducing vacuole formation with vacuolin-1 treatment. Using this multifaceted experimental strategy, the authors conclude that endocytosis and coalescence of vesicles play a key role in nuclear extrusion (see figure).

This study raises several intriguing questions of importance to both clinical medicine and erythroid biology. In particular, could mutations affecting the vesicle trafficking pathway result in reticulocytopenia and hypoproductive anemia? This is a completely unexplored mechanism for ineffective erythropoiesis not easily detectable on microscopic examination of bone marrow samples. All stages of erythroid differentiation would be present, and the absence of extruded nuclei would not be readily apparent, as extruded nuclei are not frequently visualized, even in normal bone marrow, because of efficient phagocytosis.⁷  Another question is whether the presence of circulating nucleated red cells observed in disorders with extramedullary hematopoiesis results from an inhibition of enucleation produced by abnormal vesicle trafficking. One could theorize that extracellular matrix proteins, such as fibronectin, might be different in sites of extramedullary hematopoiesis compared with normal bone marrow, resulting in abnormal erythroblast/matrix adhesion, changes in erythroblast plasma membrane organization, and disruption of endocytosis. A third issue to consider is whether vesicles containing erythroblast membrane proteins are dispersed at the completion of enucleation and not incorporated into either expelled nuclei or young reticulocytes. This would be a novel and as yet unexplored mechanism for plasma membrane remodeling at the conclusion of terminal erythroid differentiation.