Figure 5.
Figure 5. MTM-1 and PIKI-1 coordinate to regulate PtdIns3P levels during phagosome formation. (A–G) DIC and fluorescent images of the gonads in the indicated strains expressing YFP::2xFYVE. White arrowheads and boxes indicate germ cell corpses labeled by YFP::2xFYVE, and yellow ones designate unlabeled corpses. Quantifications are shown in G. At least 15 animals were scored in each strain. (H and I) Time-lapse images of germ cell corpses in mtm-1(qx322);controlRNAi (H) or mtm-1(qx322);piki-1(ok2346);vps-34RNAi (I) animals coexpressing mCHERRY::MTM-1(C378S) and YFP::2xFYVE. White and yellow arrowheads point to labeled and nonlabeled extending pseudopods, respectively. (J) The percentage of worms containing 2xFYVE-positive pseudopods in the gonads was quantified in the indicated strains. At least 40 animals were examined in each strain. (K–K2’) A cross section of a pair of gonadal sheath cells is shown in K. The red line indicates the plasma membrane (PM) of the sheath cell, and the arrow points to the sheath cell cytosol separated by germ cell nuclei. DIC and fluorescent images of a gonad (K1 and K1’) and an embryo (K2 and K2’) expressing GFP::PLCδ1-PH. The red box in K1’ indicates the surface of a gonadal sheath cell, designated by the red line in the cross section (K). (L–T) Fluorescent images of gonadal sheath cells in the indicated strains expressing YFP::2xFYVE were captured at the top focal plane to show labeling on the cell surface. The curves indicate outlines of the gonads, and arrowheads point to 2xFYVE-positive endosomes or phagosomes beneath the cell surface. (U) Quantification of the relative intensity of 2xFYVE labeling on the cell surface in the strains shown in L–T. Three different regions were chosen in each animal, and ≥10 animals were quantified in each strain. (V) Time-lapse images of a cell corpse in a wild-type embryo coexpressing GFP::PIKI-1 and mCHERRY::2xFYVE. “0 min” represents the time point when PIKI-1 was first detected around the cell corpse. (G and U) Data are shown as mean ± SD. A one-way ANOVA with Tukey’s post-test was performed. (H, I, and V) Images in 20–25 z series were captured. Representative images are shown. **, P < 0.0001. Bars, 5 µm.

MTM-1 and PIKI-1 coordinate to regulate PtdIns3P levels during phagosome formation. (A–G) DIC and fluorescent images of the gonads in the indicated strains expressing YFP::2xFYVE. White arrowheads and boxes indicate germ cell corpses labeled by YFP::2xFYVE, and yellow ones designate unlabeled corpses. Quantifications are shown in G. At least 15 animals were scored in each strain. (H and I) Time-lapse images of germ cell corpses in mtm-1(qx322);controlRNAi (H) or mtm-1(qx322);piki-1(ok2346);vps-34RNAi (I) animals coexpressing mCHERRY::MTM-1(C378S) and YFP::2xFYVE. White and yellow arrowheads point to labeled and nonlabeled extending pseudopods, respectively. (J) The percentage of worms containing 2xFYVE-positive pseudopods in the gonads was quantified in the indicated strains. At least 40 animals were examined in each strain. (K–K2’) A cross section of a pair of gonadal sheath cells is shown in K. The red line indicates the plasma membrane (PM) of the sheath cell, and the arrow points to the sheath cell cytosol separated by germ cell nuclei. DIC and fluorescent images of a gonad (K1 and K1’) and an embryo (K2 and K2’) expressing GFP::PLCδ1-PH. The red box in K1’ indicates the surface of a gonadal sheath cell, designated by the red line in the cross section (K). (L–T) Fluorescent images of gonadal sheath cells in the indicated strains expressing YFP::2xFYVE were captured at the top focal plane to show labeling on the cell surface. The curves indicate outlines of the gonads, and arrowheads point to 2xFYVE-positive endosomes or phagosomes beneath the cell surface. (U) Quantification of the relative intensity of 2xFYVE labeling on the cell surface in the strains shown in L–T. Three different regions were chosen in each animal, and ≥10 animals were quantified in each strain. (V) Time-lapse images of a cell corpse in a wild-type embryo coexpressing GFP::PIKI-1 and mCHERRY::2xFYVE. “0 min” represents the time point when PIKI-1 was first detected around the cell corpse. (G and U) Data are shown as mean ± SD. A one-way ANOVA with Tukey’s post-test was performed. (H, I, and V) Images in 20–25 z series were captured. Representative images are shown. **, P < 0.0001. Bars, 5 µm.

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