Calcium Signaling Is Required for Erythroid Enucleation

Abstract

Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.

Fig 1. Orthochromatic erythroblasts display transient calcium bursts prior to enucleation.

Orthochromatic erythroblasts were isolated by FACS and incubated in the presence of Fluo-3 for 30min. Orthochromatic erythroblasts were then washed and allowed to settle on a microgrid for live cell imaging using the Leica Confocal Sp5 microscope. Images were taken every minute for 2h. (A) Images of the same orthochromatic erythroblast at various time points prior (-), during (0) and after (+) nuclear extrusion. For the cell demonstrated here, Fluo-3 fluorescence is most intense between 12 and 8 min prior to nuclear extrusion. (B) Analysis of Fluo-3 fluorescence intensity in different enucleating cells compared to non-enucleating ones.

Fig 2. Erythroid enucleation requires intracellular calcium signaling.

(A-B) Orthochromatic erythroblasts were FACS sorted and incubated in the presence of the compounds for 5h. Graphs showing percentages of enucleation in the presence of the indicated inhibitors at the indicated concentrations. Data are means (+/- SD) of 3 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). (C-E) Orthochromatic erythroblasts were incubated in media containing DMSO (vehicle control) or the indicated compounds at the indicated concentration for 5h and subsequently cytospun. For quantitative analysis cells were manually examined (Olympus BX-51 microscope; 100x/1.40 NA oil objective) using the Spot Advanced software (version 4.7)) and assigned a morphological class as per illustration. Data are means (+/- SD) of 3 independent experiments. Scalebar = 10μm.

Fig 3. Erythroid enucleation requires uptake of extracellular calcium.

(A-C) Left panels: Orthochromatic erythroblasts were incubated in the presence of the indicated compounds for 5h. Graphs showing percentages of enucleation in the presence of the indicated compounds at the indicated concentrations. Data are means (+/- SD) of 3–4 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). Middle and right panels: Cytospins and quantitative analysis of orthochromatic erythroblasts treated with the indicated compounds. Data are means (+/- SD) of 3 independent experiments. Scale bar = 10μm.

Fig 4. Erythroid enucleation requires calmodulin signaling.

(A) Immunofluorescence staining for calmodulin (CaM) in orthochromatic erythroblasts at different stages of the enucleation process. Scalebar = 5μm. (B-C) Left panels: Orthochromatic erythroblasts were incubated in the presence of the indicated compounds for 5h. Graphs showing percentages of enucleation in the presence of the indicated compounds at the indicated concentrations. Data are means (+/- SD) of 3–4 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). Middle and right panels: Cytospins and quantitative analysis of orthochromatic erythroblasts treated with the indicated compounds. Data are means (+/- SD) of 3 independent experiments. Scale bar = 10μm.

Fig 5. Model of potential roles for calcium in erythroid enucleation.

Model of potential actions of calcium during nuclear extrusion. Inhibitors that target calcium-dependent signaling pathways and resulted in arrest of enucleation are shown in red.