The megakaryocyte DMS accumulates PI-4,5-P₂ late in differentiation. MKs were retrovirally transduced with EGFP-tagged pleckstrin homology (PH) domain derived from phospholipase C (PLC)δ1, a specific probe for membrane-associated PI-4,5-P₂, and examined by fluorescence deconvolution microscopy. (A-D) In young MKs, only the plasma membrane shows PI-4,5-P₂ accumulation, as described for other cells. (A) EYFP merged with nuclear DAPI stain, (B) EYFP alone, (C) CD61 stain alone, (D) merger of EYFP and CD61 signals, showing overlap. (E) In MKs of advanced differentiation, internal membranes stain strongly with PH-PLCδ1. The cell-surface signal is weak, and the predominant staining is internal. (F) Continued cytologic maturation, judged by increased cell size and nuclear complexity, results in elaborate staining of internal membranes, which resemble the DMS as seen by electron microscopy and in EYFP^ki mice, with virtual loss of cell-surface signal (see also Video S1). (G-I) Costaining of PH(PLCδ1)–EGFP–expressing MKs (G) with CD41 antibody (H), a surface and internal marker of MKs. Double staining (I) highlights the cortical absence of PI-4,5-P₂. (J-K) Proplatelets demonstrate PI-4,5-P₂ staining in contiguity (arrows) with internal membranes. Scale bars: A-D, 10 μm; E, F, J, K, 15 μm; G-I, 20 μm.