Figure 1. PIP₃-binding class I myosins are required for normal cell growth and development.

(A) The domain structure of seven class I myosins in the Dictyostelium genome. (B) Immunoblotting of whole cell lysates prepared from Dictyostelium cells expressing myosin I-GFP fusions using anti-GFP antibodies. (C) Lipid dot blot assays show interactions of myosins ID, IE, and IF with PIP₃. Images are representative of more than three independent experiments. (D) Localization of myosin IE-GFP and PHcrac-RFP in wild-type, pi3k-null, and pten-null cells (n ≧ 3, more than 10 cells analyzed in each experiment). Fluorescence intensity was quantified along the lines shown in Fig. S3. (E) Cells expressing myosin IE-GFP and PHcrac-RFP, a biomarker for PIP₃, were observed before and after cAMP stimulation, in the presence or absence of latrunculin A or LY294002 (n ≧ 2, more than 25 cells analyzed in each experiment). (F) Cellular localization of myosin IE-GFP and LimEΔcoil-RFP, a biomarker for F-actin, in a cAMP gradient (n ≧ 6, more than 5 cells analyzed in each experiment). Arrow indicates the direction of cell migration. (G) Cell growth in wild-type and myosin I-null cells. Cells were counted daily. Values represent the mean ± SEM from more than four independent experiments. (H) Cells were plated on non-nutrient (DB) agar and examined for development. Images are representative of more than three independent experiments.