Hereditary spherocytosis and hereditary elliptocytosis: aberrant protein sorting during erythroblast enucleation

Abstract

During erythroblast enucleation, membrane proteins distribute between extruded nuclei and reticulocytes. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), deficiencies of membrane proteins, in addition to those encoded by the mutant gene, occur. Elliptocytes, resulting from protein 4.1R gene mutations, lack not only 4.1R but also glycophorin C, which links the cytoskeleton and bilayer. In HS resulting from ankyrin-1 mutations, band 3, Rh-associated antigen, and glycophorin A are deficient. The current study was undertaken to explore whether aberrant protein sorting, during enucleation, creates these membrane-spanning protein deficiencies. We found that although glycophorin C sorts to reticulocytes normally, it distributes to nuclei in 4.1R-deficient HE cells. Further, glycophorin A and Rh-associated antigen, which normally partition predominantly to reticulocytes, distribute to both nuclei and reticulocytes in an ankyrin-1-deficient murine model of HS. We conclude that aberrant protein sorting is one mechanistic basis for protein deficiencies in HE and HS.

Figure 1

Analysis of GPC sorting during enucleation of WT and 4.1R-null erythroblasts. Differential interference contrast (DIC) and immunofluorescent micrographs of wild-type (WT) and 4.1R-null enucleating erythroblasts, including nascent reticulocyte (R) and extruding nucleus (N), probed with fluorescein isothiocyanate–conjugated TER 119, specific for GPA (green), or Alexa Fluor 555–labeled rabbit anti–mouse GPC antibody (red). Nuclei were identified by Syto-17 staining (blue). The number of enucleating erythroblasts examined under each staining condition was 6 or more. The images were observed by the use of a Zeiss LSM META 510 Confocal microscope (Carl Zeiss Microimaging Inc) with an APOCHROMAT 63×/1.4 oil DIC objective and acquired by the use of Zeiss Laser Scanning Microscope LSM 510, Version 3.2 SP2 software with a Zeiss AxioCam HRm Rev. 2/3.3V camera. The images were processed with the use of Adobe Photoshop (Adobe Systems Inc).

Figure 2

Analysis of band 3, RhAG, and GPA sorting during enucleation of WT and nb/nb erythroblasts. DIC and immunofluorescent micrographs of WT and nb/nb-enucleating erythroblasts, including nascent reticulocyte (R) and extruding nucleus (N); probed with rabbit anti–mouse GPC and Alexa Fluor 594–labeled goat anti–rabbit; IgG (red; A); probed with rat anti–mouse TER 119 and Alexa Fluor 488–conjugated donkey anti–rat IgG (green) or rabbit anti–mouse band 3 and Alexa Fluor 594–labeled goat anti–rabbit IgG (red; B); or probed with rat anti–mouse TER 119 and Alexa 488–conjugated donkey anti–rat IgG (green) or rabbit anti–mouse RhAG and Alexa Fluor 594–labeled goat anti–rabbit IgG (red; C). Nuclei were identified by 4′,6-diamidino-2-phenylindole staining (DAPI; blue). Dashed lines outline the spherical portion of extruding nuclei in the red and green images in which there is no fluorescent labeling of the nucleus. The number of enucleating erythroblasts examined under each staining condition was 6 or more. Of note, during extrusion the nucleus transiently deforms, and a portion of it is visualized within the nascent reticulocyte, as evidenced in these images.