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First published online 14 September 2005
doi: 10.1242/dev.02039

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Cortical localization of the G protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

¹ Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology (EPFL), CH-1066 Lausanne, Switzerland
² Department of Pharmacology, Lineberger Comprehensive Cancer Center and Neuroscience Center, The University of North Carolina, Chapel Hill, NC 27599-7365, USA

View larger version (108K): [in a new window]   Fig. 1. GPA-16 distribution in early embryos. (A) Western blot analysis using GPA-16 antibodies on wild-type or gpa-16(RNAi) embryonic extracts. The blot was reprobed with -tubulin antibodies as a loading control (bottom). (B-H) Wild-type embryos (B, one-cell stage late telophase; C, four-cell stage), as well as four-cell stage embryos of the indicated genotypes stained with antibodies against GPA-16 (red) and -tubulin (green); DNA is shown in blue. Left panels show GPA-16 staining alone, right panels the merge of the three signals. Rectangles highlight a region of the cortex at the ABp/EMS boundary to ease comparison of GPA-16 levels; this region does not exhibit the variability in staining intensity sometimes observed on the cortex facing the outside. Insets represent 2.5x magnified view of the approximate region indicated by the rectangles. Analogous distributions are observed in one-cell stage embryos (data not shown). Arrows in C,D indicate signal around microtubule asters, which persists in gpa-16(RNAi) embryos. In this and other figures, anterior is leftwards, posterior is rightwards. Scale bar: 10 µm.

View larger version (59K): [in a new window]   Fig. 2. RIC-8 is required for interaction between GPA-16 and GPR-1/2, and for normal GPA-16 protein levels. (A) Embryonic extracts of the indicated genotypes were immunoprecipitated with GPA-16 antibodies and analyzed by western blot using RIC-8, GPR-1/2 or GPA-16 antibodies, as indicated on the left by arrowheads. The top panel shows inputs (1/50 of starting materials). Whereas GPA-16 antibodies co-immunoprecipitate both RIC-8 and GPR-1/2, RIC-8 or GPR-1/2 antibodies do not co-immunoprecipitate GPA-16 (data not shown). Lanes 1-5 and 7-11 are from the same experiment; lanes 6 and 12 from a different experiment performed with appropriate controls. GPA-16 levels are diminished in inputs from the gpb-1(RNAi) embryonic extract. Quantifications of the intensity of the GPA-16 band from four experiments, using the -tubulin or the GPR-1/2 band as loading control, indicate that the amount of GPA-16 in ric-8(md1909) and in gpb-1(RNAi) embryos is essentially identical [ratio of gpb-1(RNAi) versus ric-8(md1909):1.15; s.d.=0.4]. RIC-8 is truncated in ric-8(md1909) mutant embryos (Afshar et al., 2004) and is present at a lesser abundance than in the wild type. (B) Western blot analysis of embryonic extracts of the indicated genotypes using sequentially antibodies against GPA-16, GOA-1 and RIC-8, as well as -tubulin as a loading control, as indicated on the left with arrowheads. RIC-8 antibodies also detect a minor non-specific species, which co-migrate with the truncated protein in ric-8(md1909) mutant embryos (Afshar et al., 2004).

View larger version (18K): [in a new window]   Fig. 3. GPA-16 and GOA-1 become entirely dispensable for generation of pulling forces following GPB-1 depletion. Average peak velocities±s.e.m. of anterior (A) and posterior (P) spindle poles after spindle severing in one-cell stage embryos of the indicated genotypes. Actual values are given in Table S1 in the supplementary material. We found an analogous outcome when examining the movements of centrosomes during centration/rotation prior to mitosis: whereas centration/rotation is gradual, as in wild type, in embryos compromised for either goa-1 or gpa-16 function, it is abrupt and accompanied by back and forth movements in all genotypes in which gbp-1 function is compromised (see Movies 1-7 in the supplementary material). Values for wild type, goa-1(sa734) and gpb-1(RNAi) are from Afshar et al. (Afshar et al., 2004).

View larger version (116K): [in a new window]   Fig. 4. Cortical GPR-1/2 distribution. Early two-cell stage embryos of the indicated genotypes stained with antibodies against GPR-1/2 (red) and -tubulin (green); DNA is shown in blue. Left panels show GPR-1/2 staining alone, right panels the merge of the three signals. See also Figs S2 and S3 in the supplementary material for four-cell stage embryos and corresponding quantifications of GPR-1/2 cortical distribution. Scale bar: 10 µm.

View larger version (36K): [in a new window]   Fig. 5. GPR-1/2 is a GDI for GPA-16; RIC-8 is not a GEF for GPA-16. (A,B,E) Surface plasmon resonance was used to analyze the binding of GPA-16 to GST-GPR-1/2 (amino acids 374-476) (A) and to GST-RIC-8 (B), as well as of GOA-1 to GST-RIC-8 (E). GST fusion proteins were immobilized on a GST antibody biosensor surface. `Analyte' (30 µl of 2 µM GPA-16 or GOA-1), in each of the indicated nucleotide bound states, was injected over the biosensor surface. Non-specific binding to GST was subtracted from each curve. (C) Time-course of [35S]GTPS binding to 100 nM GPA-16 in the presence or absence of 10 µM GPR-1/2 peptide (amino acids 423-461). Results are the mean±s.e.m. of duplicate samples. Observed association rate constants (with 95% confidence intervals in parentheses) were: GPA-16, 0.73 (0.44-1.0) minutes-1; GPA-16+GPR-1/2, 0.069 (0.050-0.087) minutes-1. (D,F) Time-course of [35S]GTPS binding to 100 nM GPA-16 (D) or 100 nM GOA-1 (F) in the presence or absence of 2 µM RIC-8 (D) or 200 nM RIC-8 (F). Results are the mean±s.e.m. of duplicate samples. Observed association rate constants (with 95% confidence intervals in parentheses) were: GPA-16, 0.32 (0.24-0.40) minutes-1; GPA-16+RIC-8, 0.35 (0.26-0.43) minutes-1; GOA-1, 0.052 (0.044-0.061) minutes-1; GOA-1+RIC-8, 0.12 (0.093-0.14) minutes-1. (G,H) Co-immunoprecipitation of the same wild-type embryonic extracts with GPA-16 (G) or GOA-1 (H) antibodies either alone (lanes 1) or in the presence of 100 µM GDP (lanes 2) or 100 µM GTP-S (lanes 3). The co-immunoprecipitated material was detected using RIC-8, GPR-1/2, GPA-16 or GOA-1 antibodies, as indicated on the left with arrowheads. A previous study reported that spindle positioning in ric-8(md1909) goa-1(RNAi) embryos is similar to that of ric-8(md1909) embryos (Couwembergs et al., 2004); the difference with our results may reflect the use of distinct RNAi conditions.

View larger version (149K): [in a new window]   Fig. 6. Compromising the function of GOA-1 and RIC-8 is more detrimental than compromising that of GPA-16 and RIC-8. (A-G) Early two-cell stage embryos of the indicated genotypes (see also Movies 8-14 in the supplementary material). Arrowheads indicate cleavage furrow position. (H) Average cleavage furrow positions along the AP axis (0% egg-length, anterior-most; 100% egg-length, posterior-most), along with standard deviations, for embryos of the genotypes illustrated in A-G. Number of embryos examined for each genotype: A, 10; B, 7; C, 8; D, 7; E, 6; F, 8; G, 8. Scale bar: 10 µm.

View larger version (17K): [in a new window]   Fig. 7. Working model of GPA-16 activation during asymmetric cell division. RIC-8 binds to GPA-16·GDP, counteracting the formation of the GPA-16/Gß heterotrimer; this allows association of GPR-1/2 and LIN-5 with GPA-16. A complex of GPA-16·GDP-GPR1/2-LIN-5 promotes generation of pulling forces on spindle poles. As some GPR-1/2 co-immunoprecipitates with RIC-8 (Afshar et al., 2004), RIC-8 may still be present in this complex, as illustrated. As RIC-8 does not exhibit GEF activity towards GPA-16, RIC-8-independent nucleotide exchange may promote formation of GPA-16·GTP. Co-immunoprecipitation experiments (see Fig. 5G) suggest that RIC-8 can associate with GPA-16·GTP, although this is not illustrated. RGS-7 GAP activity promotes GTP hydrolysis and formation of GPA-16·GDP and is thus is required for the activation cycle (Hess et al., 2004).