DMS onset and expansion in cultured MKs. (A-D) Confocal images from MK cultures fixed after 4 hours. Cells were stained with anti-GPIbβ (green) and DAPI (blue); full Z-stacks of the representative DMS developmental stages are assembled as maximum intensity projections. (A-B) Distinct GPIbβ-positive territories at and just beneath the cell surface (arrowheads) were observed in MKs. Note the connection with the cell surface (supplemental Movies 1 and 2). (C-D) In larger MKs, GPIbβ-positive areas (pre-DMS) were centrally located between the nuclear lobes and displayed numerous tubular connections with the cell surface (arrowheads in D). Bars, 10 µm. (E-H) Pulse-chase kinetics of anti-GPIbβ in cultured MKs. Lin⁻ BM cells were pulse-labeled for 15 minutes with Alexa488-conjugated anti-GPIb (green), washed, and subsequently fixed after 5 minutes and 1, 2, and 4 hours, respectively. Representative maximal projections of whole cells are shown. Bars, 10 µm (I-L) Ultrastructural characterization of the pre-DMS in cultured MKs. TEM images showing pre-DMS in MKs cultured for 4 hours. (I) At this stage, the pre-DMS is a well-defined circular membrane network located between the lobes of the nucleus. (J) Higher magnification of the network. (K) Immunogold labeling with anti-GPIbβ (10 nm protein A gold particles) showing that the pre-DMS contains GPIbβ. (L) Tannic acid staining reveals that the pre-DMS is connected o the cell surface. Bars, 1 µm. er, endoplasmic reticulum; g, golgi; n, nucleus.