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, 315 (1), 42-54

Dynamic Localization of LIN-5 and GPR-1/2 to Cortical Force Generation Domains During Spindle Positioning

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Dynamic Localization of LIN-5 and GPR-1/2 to Cortical Force Generation Domains During Spindle Positioning

Dae Hwi Park et al. Dev Biol.

Abstract

G protein signaling pathways regulate mitotic spindle positioning during cell division in many systems. In Caenorhabditis elegans embryos, G alpha subunits act with the positive regulators GPR-1/2 and LIN-5 to generate cortical pulling forces for posterior spindle displacement during the first asymmetric division. GPR-1/2 are asymmetrically localized at the posterior cortex by PAR polarity cues at this time. Here we show that LIN-5 colocalizes with GPR-1/2 in one-cell embryos during spindle displacement. Significantly, we also find that LIN-5 and GPR-1/2 are localized to the opposite, anterior cortex in a polarity-dependent manner during the nuclear centration and rotation movements that orient the forming spindle onto the polarity axis. The depletion of LIN-5 or GPR-1/2 results in decreased centration and rotation rates, indicating a role in force generation at this stage. The localization of LIN-5 and GPR-1/2 is largely interdependent and requires G alpha. Further, LIN-5 immunoprecipitates with G alpha in vivo, and this association is GPR-1/2 dependent. These results suggest that a complex of G alpha/GPR-1/2/LIN-5 is asymmetrically localized in response to polarity cues, and this may be the active signaling complex that transmits asymmetries to the force generation machinery during both nuclear rotation and spindle displacement.

Figures

Figure 1
Figure 1
Asymmetric localization of LIN-5 and GPR-1/2. (A) Confocal images of wild-type one-cell embryos double-labeled with LIN-5 and GPR-1/2 antibodies at the stages indicated. Scale bars: 10µm. (B) Fluorescence intensity plots of the embryos shown in (A), from running averages of line scans. All embryos shown but early anaphase were stained with the LIN-5 PAb, which gives higher intensities. (C) Control staining for antibody specificity of LIN-5 PAb and GPR. (D) Average fluorescence intensity and relative cortical (cortical/cytoplasmic ratio) plots for wild-type embryos. n=10 cortices each, which differs from the text because only embryos stained with LIN-5 PAb were averaged.
Figure 2
Figure 2
Defective centration and nuclear rotation in lin-5 and gpr-1/2 embryos. A) Centration and nuclear rotation are slower in lin-5 and gpr-1/2 embryos. Speeds of centration in wild type (0.068± 0.014µm/s, n=10), lin-5ts (ev571ts) (0.042± 0.009µm /s, n=9), and gpr-1/2 (RNAi) (0.046± 0.007µm /s, n=9) embryos. Speed of a centrosome during nuclear rotation in wild type (0.134± 0.024µm/s), FM102 (lin-5ts) (0.080± 0.012µm /s, n=9), and TH32 gpr-1/2 (RNAi) (0.079± 0.017µm /s, n=8,) embryos. Note that TH32 (n=10) and AZ244 (n=9) data were combined to calculate the speed of a centrosome in wild type because the results are not statistically different (p=0.138). Error bars indicate SD. P<0.002 for wild type versus lin-5 and gpr-1/2 in centration and P≪0.001 in nuclear rotation. B) Quantification of the extent of nuclear-centrosome rotation at NEB in lin-5 and gpr-1/2 embryos. Each dot represents a single embryo, with 0° indicating complete rotation of the complex onto the A/P axis. The average rotation angle ± SD and p values for wild type versus lin-5ts and gpr-1/2 (RNAi) are also given.
Figure 3
Figure 3
Cortical asymmetry of LIN-5 requires polarity and GPR-1/2. (A, B) Confocal images of embryos stained as indicated. Scale bars: 10µm.
Figure 4
Figure 4
Average fluorescence intensity and relative cortical intensity plots for LIN-5 and GPR-1/2 in mutant embryos during prophase (A) and metaphase/anaphase (B). n= number of cortices examined. Wild-type plots are from Fig. 1.
Figure 5
Figure 5
LIN-5 cortical localization depends on Gα. Confocal images of wild-type embryos double-labeled with the antibodies indicated. (A) Anaphase embryos. (B) Metaphase wild-type and lin- 5(RNAi) embryos and an anaphase lin-5(ev571ts) embryo. Scale bars: 10µm. (D) Western blots of Gα immunoprecipitation products. Anti- Gα antibody was used for immunoprecipitations (IP) from wild type and gpr-1/2(RNAi) embryo extracts. 1/40 dilution of input is shown for comparison. Products were probed with LIN-5 MAb, Gα and GPR antibodies.

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