Localization of spectrin isoforms within proplatelets. Micrographs of immunofluorescence studies performed with spectrin antibodies reveal differential localizations for spectrin isoforms within proplatelets. The top panels (A-D) show proplatelet-producing MKs that were fixed before staining. The bottom panels (E-H) denoted “cytoskeleton,” show proplatelets permeabilized in 0.1% Triton-X 100 before fixation to remove soluble and membrane-associated structures. All micrographs are merged images of cells that were double-labeled with spectrin isoform antibodies (green) and F-actin staining by phalloidin (red). The isotype probed and the fluorophore of the secondary antibody are indicated in each panel. (A-D) α1 and β1 spectrins decorate punctate spots distributed throughout the proplatelets. α2 and β2 spectrins localized strongly along proplatelet shafts, proplatelet tips, and swellings. Both α2 and β2 spectrin isoforms colocalized with F-actin. All 4 spectrin isoforms tested were retained in the cytoskeleton of permeabilized cells (E-H) and displayed a similar localization pattern to nonextracted cells. Spectrin 1 isoforms stained in a punctate pattern throughout the proplatelet skeleton, whereas spectrin 2 isoforms displayed a more cytoskeletal localization in permeabilized cells. Scale bar indicates 5 μm.