Figure 3.
Figure 3. SPDL-1 localizes to and targets MDF-1 to kinetochores that face away from the mono centrosome. (A) Immunofluorescence images of AB or P1 cells of ZYG-1–depleted embryos in the second mitosis. Indicated proteins (red) were stained with each specific antibody. DNA (white) and tubulin (green) were also stained with DAPI and anti-tubulin antibody, respectively. The numbers before and after the slash in parentheses are, respectively, the numbers of cells in which the indicated protein localized at both sides of kinetochores and those of cells in which the indicated protein localized only at the outer side of the chromosome arc. KNL-1, NDC-80, BUB-1, and CZW-1 localize at both sides of the chromosomes along the longitudinal axis, showing two parallel bars (white arrows). In contrast, SPDL-1 and MDF-1 localize along the side of the chromosome that faces away from the centrosome (white arrows). KNL-1 and BUB-1 also localize on the plus-end terminus region of microtubules that extends beyond chromosomes. Bar, 10 μm. (B) Embryos expressing GFP–MDF-1 and mCherry–histone H2B (RFP–H2B) were depleted of the indicated genes and subjected to time-lapse fluorescence microscopy. Images of embryos at metaphase of the second mitosis in AB cells are shown. The anterior of the embryo is at the right. Kinetochore localization of MDF-1 depends on both SPDL-1 and BUB-1. Bar, 20 μm. (C) Immunoprecipitations were performed on extracts from wild-type (WT), zyg-1(b1) homozygotes (zyg-1ts), and mdf-1(gk2); fzy-1(h1983) homozygotes (mdf-1Δ) using an anti–SPDL-1 antibody, an anti–MDF-1 antibody, or control IgG. Immunoprecipitants were separated on a gel, transferred onto a nitrocellulose membrane, and probed with antibodies to MDF-1, SPDL-1, and MDF-2. SPDL-1 was detected in MDF-1 immunoprecipitants and MDF-1 and MDF-2 in SPDL-1 immunoprecipitants. (D) Schematic model of the position of SPDL-1 in the hierarchical dependency of kinetochore assembly. SPDL-1 is downstream of CZW-1 and required for unattached kinetochore localization of MDF-1. BUB-1 is also required for unattached kinetochore localization of MDF-1 but not for that of SPDL-1. Together, MDF-1 targeting is regulated by two independent pathways, one includes SPDL-1 and the other includes BUB-1.

SPDL-1 localizes to and targets MDF-1 to kinetochores that face away from the mono centrosome. (A) Immunofluorescence images of AB or P1 cells of ZYG-1–depleted embryos in the second mitosis. Indicated proteins (red) were stained with each specific antibody. DNA (white) and tubulin (green) were also stained with DAPI and anti-tubulin antibody, respectively. The numbers before and after the slash in parentheses are, respectively, the numbers of cells in which the indicated protein localized at both sides of kinetochores and those of cells in which the indicated protein localized only at the outer side of the chromosome arc. KNL-1, NDC-80, BUB-1, and CZW-1 localize at both sides of the chromosomes along the longitudinal axis, showing two parallel bars (white arrows). In contrast, SPDL-1 and MDF-1 localize along the side of the chromosome that faces away from the centrosome (white arrows). KNL-1 and BUB-1 also localize on the plus-end terminus region of microtubules that extends beyond chromosomes. Bar, 10 μm. (B) Embryos expressing GFP–MDF-1 and mCherry–histone H2B (RFP–H2B) were depleted of the indicated genes and subjected to time-lapse fluorescence microscopy. Images of embryos at metaphase of the second mitosis in AB cells are shown. The anterior of the embryo is at the right. Kinetochore localization of MDF-1 depends on both SPDL-1 and BUB-1. Bar, 20 μm. (C) Immunoprecipitations were performed on extracts from wild-type (WT), zyg-1(b1) homozygotes (zyg-1ts), and mdf-1(gk2); fzy-1(h1983) homozygotes (mdf-1Δ) using an anti–SPDL-1 antibody, an anti–MDF-1 antibody, or control IgG. Immunoprecipitants were separated on a gel, transferred onto a nitrocellulose membrane, and probed with antibodies to MDF-1, SPDL-1, and MDF-2. SPDL-1 was detected in MDF-1 immunoprecipitants and MDF-1 and MDF-2 in SPDL-1 immunoprecipitants. (D) Schematic model of the position of SPDL-1 in the hierarchical dependency of kinetochore assembly. SPDL-1 is downstream of CZW-1 and required for unattached kinetochore localization of MDF-1. BUB-1 is also required for unattached kinetochore localization of MDF-1 but not for that of SPDL-1. Together, MDF-1 targeting is regulated by two independent pathways, one includes SPDL-1 and the other includes BUB-1.

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