Myosin light chain regulates PPF negatively in a myosin-IIA–dependent manner. (A) Constitutively activated (D₁₈D₁₉), dominant-negative (A₁₈A₁₉), or wild-type GFP-tagged MLC constructs were introduced into fetal liver–derived blood progenitors by retroviral transduction. Wild-type MLC significantly delayed the onset and peak of PPF, whereas D₁₈D₁₉-MLC blocked PPF and A₁₈A₁₉-MLC enhanced PPF less than 2-fold. (B) PPF morphology after exogenous MLC expression. Abnormal grape-like clusters of proplatelets appeared with expression of wild-type MLC, compared with fully extended PPF in control GFP-expressing MKs; D₁₈D₁₉-MLC showed restricted localization within MKs (bright green spots) and blocked PPF. (C) GFP-D₁₈D₁₉-MLC incorporates into actomyosin structures formed within fibroblasts in the same primary cultures. (D) D₁₈D₁₉-MLC also blocked PPF in MKs derived from normal ES cells (data not shown) but not in those from Myh9^−/− ES cells. RFP-fused D₁₈D₁₉-MLC was introduced by retroviral transduction into blood progenitors derived from GFP⁺Myh9^−/− ES cells on differentiation day 5 and again localized in a few spots within cells. A neighboring MK not expressing RFP-D₁₈D₁₉-MLC is also shown (red arrowhead). Both cells show fully extended proplatelets that appear normal. (E) A₁₈A₁₉-MLC enhanced PPF in wild-type MKs. MKs derived from blood progenitors doubly transduced with GFP and RFP-A₁₈A₁₉-MLC generated considerably more proplatelets than MKs expressing only GFP. All scale bars represent 15 μm.