Actin fibers assemble near MK internal membranes and are required for proplatelet formation. (A-F) Wild-type MKs infected with PH(PLCδ1)–EGFP retrovirus were fixed, permeabilized, and labeled with Texas Red–phalloidin and DAPI nuclear stain on culture day 4. A central z-section in panel A reveals internal, PI-4,5-P₂–containing membranes. Phalloidin-stained actin filaments appear in a reticular network that colocalizes partially with PH(PLCδ1)–EGFP at one cell pole (B), as judged by merger of fluorescent signals (C). PI-4,5-P₂–positive proplatelet membranes also associate closely with actin filaments, suggesting continuity of the spatial relationship established in the cell body (D-F). (G-I) MKs were infected with retroviruses to express EGFP-tagged N-WASp CA domain, and infected cells were identified by fluorescence microscopy. The tagged CA domain colocalizes extensively with Texas Red–labeled Arp3, as shown by the merger of fluorescent signals. (J) Phalloidin staining of WASp-CA–expressing cells confirms disruption of actin filaments, which also failed to colocalize with the EGFP-CA fragment (data not shown). (K) EGFP-CA and control retroviruses were used to infect MKs on culture day 2, and proplatelet formation was scored on day 4. Expression of the isolated CA domain substantially reduced proplatelet formation. Results are averaged from 3 independent experiments, and are presented with standard deviations (error bars). (L) WASP-CA–infected MKs are large, have multilobed nuclei, and extend rare proplatelets (inset), findings that argue against general toxicity. (M) Proplatelet formation by cytochalasin D–treated MKs on culture day 4. Results show the effects of introducing cytoD into MK cultures between days 2.5 and 3.5 and are averaged over 3 independent experiments. Ctrl indicates DMSO treatment for 2.5 days. (N) Representative EYFP^ki MK treated with cytoD on culture day 2, showing the characteristically elaborate internal membrane structure. (O) Cells treated with DMSO (control) or with 0.2 μM cytoD on culture days 2.5 or 3 were assessed for cell size on day 4. The fraction of FSC^high MKs (CD61⁺ cells) is shown from 2 independent experiments. MKs were reduced slightly in size but showed little difference over the same interval in which proplatelet formation (M) was notably inhibited. Scale bars: A-I, N, 15 μm; J, L, 10 μm.