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First published online 8 October 2003
doi: 10.1242/dev.00790

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LET-99 opposes G{alpha}/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling

Meng-Fu Bryan Tsou*, Adam Hayashi and Lesilee S. Rose{dagger}

Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA



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  		Fig. 4. GPR-1/2 asymmetry depends on PAR-3 and LET-99. (A-N) Confocal images of wild-type embryos (A-D), par-3 embryos (E-I) and let-99 embryos (J-N) stained with anti-GPR-1/2 antibodies (top panels) and DAPI (bottom panels). (A,E,J) One-cell prophase embryos. (K) One-cell metaphase embryos. (B,F,L,M) One-cell late anaphase embryos. (C,G,N) Two-cell interphase embryos. (D,H,I) Four-cell interphase embryos. (O) Confocal images of wild-type and gpr-1/2(RNAi) anaphase embryos stained with anti-LET-99 antibodies. Scale bar: 10 µm. (P) Quantification of relative intensity of GPR-1/2 staining in wild-type, gpb-1(RNAi) and let-99(or81) one-cell prophase embryos.

 


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  		Fig. 1. G{alpha} and Gß mutants display opposite phenotypes and the Gß phenotypes are due to gain of G{alpha} activity. (A) DIC images of live one-cell embryos recorded by time-lapse video microscopy in various genetic backgrounds (as indicated) undergoing the first division. Black arrowheads indicate the current position of centrosomes in each image. White arrowheads indicate the position of the centrosome in the previous image during anaphase spindle pole oscillations. White arrows indicate multiple nuclei. Relative time points are indicated (minutes:seconds). Note the rapid changes in centrosome position in gpb-1(RNAi) embryos (rocking), and the lack of anaphase spindle pole oscillations in G{alpha} mutant embryos. (B) Spherical G{alpha}(RNAi) embryos. Anterior is towards the left in this and all subsequent figures. Scale bar: 10 µm.

 


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  		Fig. 2. gpr-1/2(RNAi) embryos have similar phenotypes to those seen in G{alpha} mutants. DIC images of live gpr-1/2(RNAi) and gpr-1/2(RNAi); gpb-1(RNAi) one-cell embryos recorded by time-lapse video microscopy undergoing the first division. Black arrowheads mark the current position of centrosomes. Short arrows mark the current position of the spindle poles on the AP axis during spindle elongation, and long arrows indicate the original position of the spindle poles before spindle elongation onset. White arrowheads indicate multiple nuclei. Note the lack of hyperactive nuclear rocking and lack of anaphase spindle pole oscillations (compare with Fig. 1). Relative time points are indicated (minutes:seconds). Scale bar: 10 µm.

 


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  		Fig. 3. Localization of GPR-1/2. Confocal sections of wild-type (A-I), gpr-1/2(RNAi) (J), G{alpha}(RNAi) (K-O) and gpb-1(RNAi) (P-T) embryos stained with anti GPR-1/2 antibodies (top panels for each series) and DAPI to visualize DNA (bottom). (A,P) One-cell prophase embryos. (B,K,Q) One-cell metaphase embryos. (C,D,L) One-cell anaphase embryos. (E) One-cell telophase embryo. (F,J,M,R) Two-cell interphase embryos. (G) Two-cell prophase embryo. (H) Two-cell embryo where P1 is at metaphase. (I,N,O,S,T) Four-cell interphase embryos. White arrowheads indicate the boundaries of the domains enriched GPR-1/2. Scale bar: 10 µm.

 


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  		Fig. 5. Hyperactive nuclear and spindle movements of let-99 embryos are suppressed in G{alpha}; let-99 and gpr-1/2; let-99 double mutants. DIC images of live let-99, G{alpha}(RNAi); let-99 and gpr-1/2(RNAi); let-99 one-cell embryos recorded by time-lapse video microscopy. Centrosomes are marked as in Fig. 2. Note the lack of hyperactive nuclear rocking, and lack of asymmetric anaphase movements. Relative time points are indicated. Scale bar: 10 µm.

 


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  		Fig. 6. The asymmetric distribution of GPR-1/2 and LET-99 in EMS cells is dependent on MES-1/SRC-1 signaling. Confocal images of wild-type (A-I), mom-5 and mes-1 (J), and let-99(or204ts) (K) four-cell embryos stained with anti-GPR-1/2 or anti-LET-99 antibodies (as indicated) and DAPI (lower panels). (A,D) EMS in interphase. (B,C,E,F,G-K) EMS in prophase. (G-I) EMS in prophase double stained with both GPR-1/2 and LET-99 antibodies. Merged image (I) shows LET-99 in green and GPR-1/2 in red. Scale bar: 10 µm.

 


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  		Fig. 7. G{alpha} and let-99 are required for nuclear rotation in EMS cells. DIC images of live wild-type (A-E), gpa-16(it143ts) (F-J) and let-99(or204ts) (K-O) embryos recorded by time-lapse video microscopy after shifting to 25°C as described in text. Black arrowheads mark the current position of centrosomes. Note the normal division of P1 (F,G,K,L), but the absence of EMS nuclear rotation in the mutants. Scale bar: 10 µm.

 


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  		Fig. 8. Models for the roles of G{alpha}/GPR-1/2 and LET-99 in transmitting polarized signals during nuclear rotation in P lineage and EMS cells. LIN-5 is required for cortical localization of GPR-1/2 in P cells and EMS cells, and the enrichment of LIN-5 and GPR-1/2 at the EMS/P2 boundary is MES-1 dependent (Srinivasan et al., 2003Go). LET-99 asymmetry (blue band) and its downstream effects are shown in blue. G{alpha}/GPR-1/2 (red outline) and its downstream effects are shown in red. Notice that the G{alpha}/GPR-1/2 levels at the EMS/P2 boundary are particularly high (thick red line), where LET-99 is absent. Orange arrows indicate the types of nuclear rotation (free versus directed). See text for details.

 

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© The Company of Biologists Ltd 2003