Myosin I links PIP3 signaling to remodeling of the actin cytoskeleton in chemotaxis

Abstract

Class I myosins participate in various interactions between the cell membrane and the cytoskeleton. Several class I myosins preferentially bind to acidic phospholipids, such as phosphatidylserine and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], through a tail homology 1 (TH1) domain. Here, we show that the second messenger lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) binds to the TH1 domain of a subset of Dictyostelium class I myosins (ID, IE, and IF) and recruits them to the plasma membrane. The PIP3-regulated membrane recruitment of myosin I promoted chemotaxis and induced chemoattractant-stimulated actin polymerization. Similarly, PIP3 recruited human myosin IF to the plasma membrane upon chemotactic stimulation in a neutrophil cell line. These data suggest a mechanism through which the PIP3 signal is transmitted through myosin I to the actin cytoskeleton.

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.

Figure 2. The IQ and TH1 domains are required for PIP3-myosin IE interactions

(A) Truncated and mutated forms of myosin IE used in the present study are shown. (B) Immunoblotting of whole cell lysates confirms expression of myosin I constructs in Dictyostelium cells. (C) Results of lipid dot blot assays show that the IQ and TH1 domains are necessary and sufficient for PIP₃ interactions. Images are representative of more than two independent experiments. (D) Mutations in the TH1 domain (A4), but not in the motor domain (E391A), block myosin IE localization in undifferentiated triple knockout cells (n = 3, more than 50 cells analyzed in each experiment). (E) Wild-type and triple knockout cells expressing the indicated myosin IE constructs were examined for cell growth. Cells were counted daily with a hemocytometer. Values represent the mean ± SEM from more than four independent experiments. (F) cAMP-stimulated membrane recruitment in differentiated triple knockout cells expressing different myosin IE-GFP constructs (n ≧ 2 experiments, more than 50 cells analyzed in each experiment).

Figure 3. Myosins ID, IE, and IF function in chemotaxis and phagocytosis

(A) The movement of wild-type cells and triple knockout cells expressing the indicated myosin IE constructs toward a micropipette containing cAMP was tracked. (B) Chemotaxis speed was calculated as the distance travelled towards the micropipette divided by the elapsed time (20 min) (n ≥ 3 experiments). (C) Chemotaxis index was defined as the distance traveled in the direction of the gradient divided by the total distance traveled in 20 min (n ≥ 3 experiments). At least 15 cells were analyzed in each experiment in (B) and (C). (D) cAMP-stimulated actin polymerization was determined in wild-type and triple knockout cells expressing the indicated myosin IE constructs. At the indicated time points after stimulation, 5×10⁶ cells were harvested and lysed, and amounts of F-actin were determined (36). Values represent the mean ± SEM from more than six independent experiments. (E) Localization of myosin IE-GFP and PHcrac-RFP was examined during phagocytosis. Circles indicate yeast cells in the first three time points. (F) Quantification of yeast uptake during phagocytosis. At the indicated time points, samples were collected and phagocytosed yeast cells were quantified. Values represent the mean ± SEM from more than four independent experiments. (G) Wild-type and triple knockout cells expressing LimEΔcoil-RFP were observed during phagocytosis of yeast cells. Arrows indicate yeast cells released from phagocytic cups.

Figure 4. PIP₃ regulates the intracellular localization of human myosin IF

(A) The domain structure of human myosin IF. An asterisk indicates the location of the mutations introduced in the TH1 domain (K770A, R780A). (B) Immunoblotting of whole cell lysates prepared from COS-7 cells expressing YFP-human myosin IF using anti-GFP antibodies. (C) Lipid dot blot assays show that interactions between YFP-human myosin IF and PIP₃ depend on the TH1 domain. YFP was used as a negative control and PH_AKT-YFP was used as a positive control. Images are representative of >3 independent experiments. (D) Localization of YFP-human myosin IF, YFP-human myosin IF (K770A, R780A), and PH_AKT-YFP was examined in COS-7 cells before and after EGF treatment (n = 3 experiments, more than 10 cells analyzed in each experiment). In the presence of LY294002, EGF-stimulated membrane recruitment of YFP-myosin IF and PH_AKT-YFP was blocked (+LY). (E) Localization of YFP-human myosin IF, YFP-human myosin IF (K770A, R780A), PH_AKT-YFP, and YFP was evaluated in HL-60 cells stimulated by fMLP (n = 3 experiments). Arrows indicate the direction of cell migration.