Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets, then preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy reveals that proplatelets, like circulating platelets, have a dense membrane skeleton whose main fibrous component is spectrin; unlike other cells, megakaryocytes and their progeny express both erythroid and non-erythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, as expression of a spectrin tetramer-disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the "barbell-shapes" of the penultimate stage in platelet production, since addition of the tetramer-disrupting construct converts these barbell-shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. Overall, this study provides evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure.

  • Submitted January 14, 2011.
  • Accepted April 27, 2011.