Pulling forces on spindle poles at 16 °C are normal in gpa-16(it143) but not gpa-16(it143)/goa-1(RNAi) embryos. Average peak velocities of the anterior (A) and posterior (P) spindle poles (±S.E.) following spindle severing of C. elegans embryos of the indicated genotypes. Experiments were performed as indicated either at 16 °C (this study) or at 25 °C (as previously described in Refs. 7, 9, 10 or in this study for gpa-16(it143)/goa-1(RNAi) embryos). For values and statistical tests, see supplemental Table 2.

Protein-protein interactions of wild type and Gly-to-Asp mutant Gα subunits. Interactions between wild type (WT) or indicated Gly-to-Asp mutated Gα subunits and Gβ₁γ₁ (A and B), PCP-2 (C and D), Ric-8A (E and F), the RGS domain of RGS14 (G and H), and PDEγ(aa 63–87) (I and J) were measured using surface plasmon resonance. Proteins were immobilized using biotin-streptavidin coupling (A, B, I, and J) or anti-GST antibody capture (C–H). Indicated concentrations of Gα subunits in the GDP (blue), GTPγS(red), or (green) loaded forms were injected over biosensor surfaces at a flow rate of 20 μl/min as denoted by arrows. Binding curves were generated after subtracting nonspecific binding to mNOTCH peptide (A, B, I, and J) or GST (C–H) control surfaces.

Spindle oscillations occur normally in gpa-16(it143) but not gpa-16(it143)/goa-1(RNAi) embryos at 16 °C. Images from time-lapse differential interference contrast microscopy of wild type (16 °C) (A), gpa-16(RNAi) (16 °C) (B), gpa-16(it143) (25 °C) (C), gpa-16(it143) (16 °C) (D), gpa-16(it143)/goa-1(RNAi) (16 °C) (E), and gpa-16(it143)/goa-1(RNAi) (25 °C) (F) embryos during anaphase (see corresponding supplemental movies 1–6). Black lines depict the distance between the two centrosomes of the spindles, and the dashed circles indicate the position of the posterior spindle pole. Elapsed time is indicated in minutes and seconds; embryos are about 50 μm long, and anterior is to the left, posterior to the right. Note that spindle oscillations occur in both wild type and gpa-16(it143) embryos at 16 °C, but not in embryos of the other genotypes (B, C, E, and F).

The G202D mutation compromises proper switch II rearrangement upon GTPγS binding. A, time course of Gα_i1 intrinsic tryptophan fluorescence enhancement by GTPγS. The intrinsic fluorescence of buffer (No Protein), 100 nm wild type (WT) Gα_i1, or 100 nm Gα_i1(G202D) was measured at 30 °C. 100 μm GTPγS was added at 0 s as indicated by the arrow. RFU = relative fluorescence units. Data were fit to a single exponential association function to determine the activation rate constant (95% confidence intervals in parentheses): WT 0.0139 (0.0137–0.0142) min^-1. B, time course of Gα_i1(G202D) intrinsic tryptophan fluorescence enhancement by GTPγS. The intrinsic fluorescence of 100 nm Gα_i1(G202D) was measured at 30 °C. 100 μm GTPγS was added at 0 s, as denoted. Data were normalized to percentage change in fluorescence. Data were fit to a single exponential association curve (95% confidence interval in parentheses): G202D+GTPγS 0.149 (0.13–0.16) min^-1. Data from intrinsic tryptophan fluorescence experiments (as illustrated in A and B; n = 3 independent experiments) were also analyzed to determine mean percent fluorescence enhancement over GDP-bound basal state (with mean ± S.E. in parentheses as follows: wild type Gα_i1, 57% (±4%); Gα_i1(G202D), 4% (±1%).

Structural features of GDP-bound Gα_i1(G202D) compared with GDP-bound and GTPγS-bound wild type Gα_i1. A, superposition of GDP-bound Gα_i1(G202D) (green) with wild type Gα_i1·GDP/Gβ₁γ₂ heterotrimer (Gα_i1 (yellow), Gβ₁ (gray), Gγ₂ (wheat); Protein Data Bank code 1GP2). Aside from the N-terminal helix, Gα_i1(G202D) is largely unaltered compared with wild type. However, the β3/α2 loop containing the G202D mutation is displaced from the nucleotide-binding pocket relative to wild type, Gβγ-bound Gα_i1. The partially ordered switch II region that proceeds from the β3/α2 loop does not assume the helical nature typical of Gβγ-bound and activated conformations of Gα. B, superposition of Gα_i1(G202D)·GDP (green) and Gα_i1·GTPγS (yellow; Protein Data Bank code 1AS0). Switch I and III regions are in a similar orientation; however, the orientations of switch II differ dramatically, most notably in the N-terminal portion (i.e. the β3/α2 loop). In wild type Gα, binding of GTPγS induces a rigid helical conformation in switch II (α2) that results in its movement toward the nucleotide-binding pocket. However, in the G202D mutant, switch II is deflected away from the nucleotide. Importantly, the Asp²⁰² side chain demonstrates a significant steric and electrostatic clash with the γ-phosphate of the modeled GTPγS molecule. C, depiction of the GDP-binding pocket illustrating the orientation of the Asp²⁰² side chain relative to GDP. Notably the acidic side chain of Asp²⁰² is oriented directly toward the β-phosphate of GDP. Residues of switch I critical for GTP hydrolysis (Arg¹⁷⁸ and Thr¹⁸¹) are shown along with Glu⁴³ in the phosphate-binding loop region. The confidence of the structural model is highlighted by a 2F_o - F_c simulated annealing omit electron density map contoured at 1.0σ (gray mesh).

Temperature-dependent nucleotide binding, protein stability, and protein interactions of wild type and G202D Gα subunits. A and B, time course of GTPγS binding by wild type and G202D Gα_i1 measured at 30 °C (A) or 15 °C (B). 100 nm Gα was incubated with 1 μm [³⁵S]GTPγS, and bound nucleotide was measured at indicated times. Data were fit to single exponential association curves (95% confidence intervals in brackets) as follows: 30 °C wild type, 0.017 (0.015–0.019) min^-1; 30 °C G202D, data could not be fit; 15 °C wild type, 0.0025 (0.0021–0.0028) min^-1; 15 °C G202D, 0.027 (0.021–0.032) min^-1. C and D, CD in millidegrees (mdeg) of 4.4 μm wild type Gα_i1 (C) or G202D Gα_i1 (D) was measured at 208 nm in both GDP- and GTPγS-bound conformations. Thermal melting curves were generated by measuring CD values at 1 °C intervals. Data are graphed as mean ± S.E. The mean melting temperatures (S.E. in parenthesis; n = 3) for wild type Gα_i1 (C) were GDP 50.2 °C (0.4) and GTPγS 77.2 °C (0.4) and for G202D Gα_i1 (D) were GDP 49.5 °C (0.8) and GTPγS 50.1 °C (0.01). E, co-immunoprecipitation conducted at 16 °C using wild type or gpa-16 (it143) embryonic extracts and GPA-16 antibody, either without (w/o) exogenous nucleotides or in the presence of 100 μm GDP or GTPγS. Immunoprecipitated material was analyzed by Western blot using antibodies against RIC-8, GPR-1/-2, or GPA-16. Input corresponds to 1/70 of starting material.

A model for heterotrimeric G-protein function during C. elegans asymmetric cell division and functions of the GPA-16(it143) temperature-sensitive mutant at both the restrictive and permissive temperatures. A, this model, based on published data (7, 9, 10), assumes that Gα·GDP bound to GPR-1/2 is crucial for pulling force generation. In the wild type embryo, GOA-1·GDP and GPA-16·GDP interact with the GoLoco motif (“GL”) proteins GPR-1/2 at the cell cortex to mediate pulling forces. Gα·GDP/GPR-1/2 concentration is determined by an equilibrium between the levels of free Gα·GDP, the amount of free Gβγ, and the amount of Gα·GDP/Gβγ. For simplicity, we have omitted from this model other regulatory components that likely participate such as RIC-8 (10), RGS-7 (11), and LIN-5 (55). B, GPA-16(it143) is unstable at 25 °C and likely misfolds or has defects in tertiary structure. Loss of functional GPA-16(it143) prevents the formation of GPA-16·GDP/GPR-1/2, thereby decreasing pulling forces. Although not formally tested, it is possible that the loss of functional GPA-16(it143) protein may also lead to an increase in free Gβγ subunits, as illustrated here. This would increase the amount of GOA-1·GDP/Gβγ and consequently reduce the amount of GOA-1·GDP/GPR-1/2, thus decreasing pulling forces. C, GPA-16(it143) is stable at 16 °C and has lost the normal nucleotide-state dependence in its Gβγ and GoLoco motif interactions (denoted “GxP”; see Figs. 1E and 2 and Table 1). This leads to an increased amount of GPA-16/Gβγ, thereby reducing the amount of free Gβγ available for formation of GOA-1·GDP/Gβγ. The consequence of this is an increase in free GOA-1·GDP, a resultant increase in the amount of GOA-1·GDP/GPR-1/2, and thus increased pulling forces. (Although a strong nucleotide-independent interaction of GPA-16(it143) with Gβγ likely explains the observed phenotype at the permissive temperature, we cannot formally rule out that GPA-16(it143) alone (freed of Gβγ sequestration) may be competent for generating pulling forces. This notion is particularly interesting as GPA-16(it143) has appreciable, although nucleotide-state independent, binding to regulatory proteins such as RIC-8 and GPR-1/2. Thus, it is also possible that increased levels of GPA-16/GPR-1/2 at the permissive temperature may cause increased pulling forces.) D, loss of all GOA-1 protein by RNA interference prevents accumulation of GOA-1·GDP/GPR-1/2, thus decreasing pulling forces. Furthermore, increased amounts of free Gβγ sequesters the stable GPA-16(it143) mutant and prevents formation of GPA-16/GPR-1/2 complexes. Thus pulling forces are potentially reduced by two mechanisms.