Immunofluorescent localization of various nuclear pore and spliceosome proteins in erythroid progenitors. (A) Diagram illustrating localization within nuclear pore structure of proteins probed with specific fluorescent antibodies: Nup153 and Tpr (nucleoplasmic basket), Nup62 (central transporter), and Nup358 (cytoplasmic fibrils). Fluorescence micrographs show early and late mouse erythroblasts from freshly harvested bone marrow labeled with antibody to Nup153, Tpr, Nup62, or Nup358 (green). Micrographs of enucleating erythroblasts show cells stained for erythroid-specific marker Glycophorin A (GPA) (Ter119, red), nuclear pore (green), and DNA (Hoechst, blue). Nuclear pore proteins exhibit a more uniform rim fluorescence in early erythroblasts, but then they redistribute to form an irregular punctuate pattern in late and enucleating erythroblasts. Bar is 2.5 μm. (B) Bright-field micrograph of a live enucleating erythroblast, freshly harvested from mouse bone marrow, that was identified by positive staining for erythroid-specific marker GPA and DNA. In the paraformaldehyde-fixed antibody and Hoechst-stained enucleating cells shown in panel A, reticulocytes are smaller than extruding nuclei because of a decrease in reticulocyte volume secondary to fixation. In the live-cell image, the nascent multilobulated reticulocyte and extruding nucleus appear similar in size. Because the reticulocyte is multilobulated, a visual comparison in size to the spherical nucleus can be misleading. In an earlier report we determined the surface area of multilobular young reticulocytes and extruding nuclei to be 83 μm² and 60 μm², respectively.⁴⁵  (C) Micrographs of early and late erythroblasts probed with anti-Sm (top panels; green) and anti-SC35 (bottom panels; green) show that these spliceosome components maintain their irregular nucleoplasmic cluster patterns in late-stage erythroblasts. Bar is 2.5 μm.