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.
Keerthivasan
Ganesan
G
Division of Hematology/Oncology, Northwestern University, 303 East Superior Street, Chicago, IL 60611, USA.
Small
Sara
S
Liu
Hui
H
Wickrema
Amittha
A
Crispino
John D
JD
D000818:Animals; D002478:Cells, Cultured; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D048749:Cytokinesis / Q000187:drug effects; D004705:Endocytosis; D004900:Erythroblasts / Q000166:cytology* / Q000187:drug effects; D004920:Erythropoiesis; D055785:Gene Knockdown Techniques; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000166:cytology; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D013154:Spleen / Q000166:cytology; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
D002966:Clathrin; D006576:Heterocyclic Compounds, 4 or More Rings; C503870:vacuolin-1; C472645:blebbistatin; D008985:Monensin; D015739:Nocodazole