Vesicle trafficking plays a novel role in erythroblast enucleation

Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.

Endocytosis and Erythroblast Enucleation Are Linked by the Action of a Survivin-EPS15-Clathrin Complex

The mechanisms that drive the final step in the development of reticulocytes from nucleated erythroblasts remain largely undefined. We recently reported that the chromosome passenger complex (CPC) protein survivin is required for the maintenance of hematopoietic stem cells and for the proliferation and survival of erythroid progenitors. Several lines of evidence suggest that survivin also plays an additional role late in erythropoiesis. First, 20% of survivin heterozygous mice showed a decrease in the proportion of enucleated cells relative to nucleated erythroblasts. Second, survivin is highly expressed in orthochromatic erythroblasts undergoing enucleation, where it partially co-localizes with actin at the junction between the extruding nucleus and cytoplasm. Third, overexpression of survivin promoted enucleation during DMSO-induced murine erythroleukemia (MEL) cell differentiation. Since survivin plays an essential role during normal mitosis and because enucleation has been postulated to occur by asymmetric cytokinesis, we investigated the link between cytokinesis and enucleation. Using primary erythroblast cultures, we found that chemical inhibitors of cytokinesis failed to prevent enucleation. Furthermore, we could not detect co-localization of survivin with aurora-B or INCENP, two other members of the CPC, in enucleating primary human erythroblasts. To determine the mechanism by which survivin contributes to enucleation, we utilized an in vivo biotinylation strategy to isolate survivin protein complexes from extracts of MEL cells. Using mass spectrometry, we discovered that survivin complexes include clathrin and its partner EPS15 (EGFR pathway substrate clone 15). We confirmed the endogenous interaction between these three proteins by co-immunoprecipitation. Treatment of primary erythroblast cultures with an inhibitor of endocytosis led to a decrease in enucleation efficiency. In addition, siRNAs targeting survivin, EPS15, or clathrin each reduced the degree of enucleation of primary human erythroblasts, confirming a requirement for this protein complex in enucleation. Together, our data demonstrate that survivin promotes enucleation by a novel clathrin-mediated process. Furthermore, our data help explain electron microscopy (EM) observations that enucleation is accompanied by an accumulation of vesicles that coalesce to form U-shaped channels at the junction between the nucleus and plasmalemma of the erythroblast (Simpson and Kling, JCB35:237, 1967). These EM studies along with our new data suggest that vesicular trafficking may be one of the major driving forces in enucleating erythroblasts.