Structural Determinants Underlying the Temperature-sensitive Nature of a Gα Mutant in Asymmetric Cell Division of Caenorhabditis elegans*

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Gαo) and GPA-16 (Gαi) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Gαi1(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Gα. At the permissive temperature, the mutant Gα was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Gα bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Gα subunit function in ACD.

G-protein-coupled receptors (GPCRs) 5 mediate the actions of a variety of sensory and metabolic stimuli (1). Heterotrimeric G-proteins are molecular switches that transduce GPCR activation into intracellular changes and are composed of a G␣ subunit and a G␤␥ dimer (1,2). GPCR-promoted activation of G␣␤␥ causes G␣ to exchange GDP for GTP, which in turn causes G␣⅐GTP and G␤␥ to dissociate. G␣⅐GTP and liberated G␤␥ then regulate effector systems to alter cell physiology (1,2). This canonical, GPCR-driven "G-protein cycle" is reset by the intrinsic GTP hydrolysis activity of G␣ (1,2).
A noncanonical, yet evolutionarily conserved, heterotrimeric G-protein regulatory module has been identified in Caenorhabditis elegans, Drosophila, and mammalian asymmetric cell division (ACD) that is important for generating diversity during development (1,(3)(4)(5). An initial step in ACD is generation of cell polarity. Cell fate determinants then segregate to different sides of the cell (3), and the mitotic spindle is positioned to permit proper distribution of determinants to daughter cells. Integral to accurate spindle positioning is a heterotrimeric G-protein module thought to be independent of GPCRs and involving GDP dissociation inhibitors (GoLoco motif proteins), guanine nucleotide exchange factors (i.e. RIC-8), and GTPase-accelerating proteins (RGS proteins) (1,3,4).
In the Caenorhabditis elegans zygote, G-protein involvement in ACD has been extensively studied using genetic and cell biological approaches (6). Two G␣ subunits, GPA-16 and GOA-1, are important for generating pulling forces on astral microtubules critical for spindle positioning (7). These two G␣ subunits are required for force generation in concert with the GoLoco motif proteins GPR-1/-2 and the coiled-coil protein LIN-5 (7). GPA-16 and GOA-1 exert partially redundant functions in ACD (8) but differ in some respects, e.g. RIC-8 is required for cortical localization of GPA-16 but not GOA-1 (9). The mechanisms by which GOA-1, GPA-16, and their binding partners mediate force generation during ACD are gradually being revealed (10 -14) but remain incompletely understood.
Whereas the contribution of GOA-1 to ACD has been ana-lyzed solely using null alleles and RNAi-mediated inactivation, the role of GPA-16 in ACD was discovered using both RNAi and a unique temperature-sensitive allele gpa- 16(it143). At 25°C, 70% of gpa- 16(it143) worms die during embryogenesis, with a significant proportion of adult escapers showing reversal of left-right body axis asymmetry (15). At 16°C, only 2% of gpa- 16(it143) worms die during embryogenesis (15), suggesting that GPA-16(it143) somehow supports function at the permissive temperature; however, an analysis of ACD in one-cell stage embryos at this particular temperature has not been conducted previously. The gpa-16(it143) allele encodes a point mutation causing a glycine 202 to aspartate (G202D) change in switch II of GPA-16 (15). We have used enzymology, crystallography, genetics, and cell biology to delineate the molecular mechanism underlying the critical contribution of this residue to GPA-16 function during ACD. Purifying GPA-16 from baculovirus-infected insect cells yields only micrograms of purified protein (9), preventing detailed biochemical and, especially, x-ray diffraction crystallographic structural studies. We have thus also examined the G202D mutation in the context of the most rigorously characterized and experimentally tractable G␣ subunit that is most closely related to GPA-16, namely mammalian G␣ i1 (9).

EXPERIMENTAL PROCEDURES
Materials-Unless otherwise specified, all reagents were of the highest purity obtainable from Sigma or Fisher (Pittsburgh, PA). Site-directed mutagenesis was conducted using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA).
Protein Purification-Full-length His 6 -tagged human G␣ i1 (wild type and G202D) was purified as described (16,17). Fulllength His 6 -tagged (wild type and G198D) chimeric G␣ t /G␣ i1 subunits (18) were purified using standard chromatography methods (16,19). Care was taken to keep all G␣ subunits GDPbound and at 4°C throughout their respective purification processes. A GST fusion of rat Ric-8A was purified as described (20). Recombinant G␤ 1 ␥ 1 dimer incorporating the biotinylation sequence was produced and purified as described (21), with DNA coding for the biotin ligase substrate motif (GLN-DIFEAQKIEWHE) inserted upstream of the G␥ 1 coding sequence within the baculoviral shuttle vector pFASTBacHT (Invitrogen). Biotin protein ligase (BirA) was incubated with 50 M G␤ 1 ␥ 1 dimer for 24 h at 25°C under the conditions described by the manufacturer (Avidity, Denver, CO). Resulting protein was buffer-exchanged into phosphate-buffered saline and stored at Ϫ80°C. Full-length GST-tagged human PCP-2 was prepared as described (17). The RGS domain of rat RGS14 was prepared as a GST fusion protein as described (16). Wild type and scrambled RGS12 GoLoco motif synthetic peptides are described in Ref. 22. An N-terminal biotinylated peptide comprising amino acids 63-87 of bovine rod PDE␥ is described in Ref. 23.
GTP␥S Binding-[ 35 S]GTP␥S (PerkinElmer Life Sciences) binding was measured using a filter binding assay as described previously (10). Rate constants were obtained by subtracting nonspecific binding (counts/min obtained in the presence of 100 M unlabeled GTP␥S) and fitting binding curves to a single exponential function in GraphPad Prism version 4.0 (San Diego, CA).
C. elegans Strains and RNAi Conditions-The N2 strain was used as wild type and maintained according to established procedures (24). The gpa-16(it143) strain (15) was maintained at 16°C and shifted for 20 -24 h to 25°C before analysis at the restrictive temperature. For inactivation of gpa-16 or goa-1 by RNAi, bacterial feeding strains (10) were used to feed L3/L4 worms for 36 h at 25°C or for 48 h at 16°C.
Coimmunoprecipitation and Western Blot Analysis-Generation of worm embryonic extracts and coimmunoprecipitation experiments were performed as described (10) with the following modifications. For each experiment, ϳ1.5 mg of protein extract and 3 g of GPA-16 antibodies (9) were utilized. As specified, GDP or GTP␥S were included at final concentrations of 100 M. For testing the interactions at 16°C, lysates were incubated with antibodies and nucleotides at 16°C for 40 min; 15 l of protein G-Sepharose were added, and the incubation was continued at 4°C overnight. Following immunoprecipitation, SDS-PAGE and Western blot analysis were performed according to standard procedures. RIC-8, GPR-1/2, and GPA-16 primary antibodies (9, 10) were diluted 1:1000 and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (GE Healthcare) 1:2000; the signals were revealed by standard chemiluminescence (GE Healthcare).
Circular Dichroism-All CD experiments were performed in Buffer C (10 mM phosphate buffer (KH 2 PO 4 /K 2 HPO 4 ), pH 7.5, 50 mM NaCl, 5 mM MgCl 2 ). For CD experiments, G␣ subunits were purified as described (16,19), but the final purification step was by Sephacryl S200 gel filtration (GE Healthcare) using Buffer C. For CD measurements, 50 M of G␣ subunits were loaded with 100 M GDP or GTP␥S at 15°C for the times determined to give Ϸ100% binding, based on rate constants (see Fig.  1B). Proteins were then diluted to 4.4 M in Buffer C and kept at 4°C. CD was measured using a PiStar-180 spectrophotometer (Applied Photophysics, Surrey, UK). CD was measured at 208 nm (slits 4.0 nm) for 30 s at each temperature. The temperature ramp was conducted using 1°C steps with a tolerance of Ϯ0.2°C. Apparent melting temperatures were calculated as the minima of first derivatives with respect to the reciprocal of temperature (25). First differential minima were calculated using Rt-Plot (version 2.7, Horst Reichert, Eppstein, Germany) using the Akima interpolation with a weighting of 1 (26).
Crystallization, Structure Determination, and Refinement-To aid crystallization of the labile G202D mutant of G␣ i1 , we were careful to ensure strict temperature control throughout protein induction (14°C), purification (4°C), and crystallization (18°C). Moreover, crystallization trials were conducted immediately following concentration of purified protein, as freeze-thawed protein was incapable of reproducing crystal growth. Crystals of G␣ i1 (G202D) were obtained by vapor diffusion from hanging drops containing a 1:1 (v/v) ratio of protein solution (10 -20 mg ml Ϫ1 in 50 mM HEPES buffer, pH 8.0, 1 mM EDTA, 100 M GDP, and 5 mM dithiothreitol) to well solution (1.9 M ammonium sulfite and 100 mM sodium acetate, pH 6.0). Crystals (ϳ0.6 ϫ 0.3 ϫ 0.2 mm) were formed in 3-5 days in the  For data collection at 100 K, crystals were transferred to a solution containing well solution supplemented with 20% (w/v) glycerol for 90 s followed by immersion in liquid nitrogen. A native data set was collected on a single crystal using an R-Axis IVϩϩ detector with Rigaku (The Woodlands, TX) rotating anode generator and osmic confocal "blue" optics at the University of North Carolina, Chapel Hill, x-ray facility. Diffraction data were scaled and indexed using HKL2000 (30). The structure of G␣ i1 ⅐GDP (Protein Data Bank code 1AS3), excluding residues 177-184 and 195-220, GDP, waters, and other heterogeneous molecules, was used as a molecular replacement model for G␣ i1 (G202D) using Phaser in CCP4 (31). Model building was achieved using the programs O and Coot (32,33). Model refinement was conducted using real space refinement protocols in Coot as well as a combination of rigid body, simulated annealing, energy minimization, and b-factor protocols in CNS (34). All structural images were made with PyMol (DeLano Scientific, San Carlos, CA).
Fluorescence Spectroscopy-Intrinsic tryptophan fluorescence was measured using an LS55 spectrometer (PerkinElmer Life Sciences). Excitation and emission wavelengths were 292 and 342 nm respectively, with slit widths of 2.5 FIGURE 3. Structural features of GDP-bound G␣ i1 (G202D) compared with GDP-bound and GTP␥Sbound wild type G␣ i1 . A, superposition of GDP-bound G␣ i1 (G202D) (green) with wild type G␣ i1 ⅐GDP/ G␤ 1 ␥ 2 heterotrimer (G␣ i1 (yellow), G␤ 1 (gray), G␥ 2 (wheat); Protein Data Bank code 1GP2). Aside from the N-terminal helix, G␣ i1 (G202D) is largely unaltered compared with wild type. However, the ␤3/␣2 loop containing the G202D mutation is displaced from the nucleotide-binding pocket relative to wild type, G␤␥-bound G␣ i1 . The partially ordered switch II region that proceeds from the ␤3/␣2 loop does not assume the helical nature typical of G␤␥-bound and activated conformations of G␣. B, superposition of G␣ i1 (G202D)⅐GDP (green) and G␣ i1 ⅐GTP␥S (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 ␤3/␣2 loop). In wild type G␣, binding of GTP␥S induces a rigid helical conformation in switch II (␣2) 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 Asp 202 side chain demonstrates a significant steric and electrostatic clash with the ␥-phosphate of the modeled GTP␥S molecule. C, depiction of the GDP-binding pocket illustrating the orientation of the Asp 202 side chain relative to GDP. Notably the acidic side chain of Asp 202 is oriented directly toward the ␤-phosphate of GDP. Residues of switch I critical for GTP hydrolysis (Arg 178 and Thr 181 ) are shown along with Glu 43 in the phosphate-binding loop region. The confidence of the structural model is highlighted by a 2F o Ϫ F c simulated annealing omit electron density map contoured at 1.0 (gray mesh). a Dissociation constants (K D in micromolar) were determined using surface plasmon resonance spectroscopy (see representative SPR sensorgrams in Fig. 2). For experiments with G␣ i1 , equilibrium resonance units of specific binding were graphed versus G␣ concentration and fit to the equation Y ϭ (B max ⅐ X)/(K D ϩ X). Data are presented with 95% confidence intervals in parentheses. For G␣ t/i1 /PDE␥ experiments, affinity was calculated by kinetic analysis (S.E. in parentheses): k a ϭ 1.6 ϫ 10 2 (0.9) M Ϫ1 s Ϫ1 , k d ϭ 8.33 ϫ 10 Ϫ4 (0.1) s Ϫ1 . b Note that the dissociation constant (K D ) established for the binding of His 6 -G␣ i1 ⅐ GDP to immobilized G␤ 1 ␥ 1 is higher than expected from comparable studies given our use of non-lipid-modified G␣ and G␤␥ subunits as well as a high concentration of free magnesium (Sarvazyan et al. 1998  nm. Fluorescence was measured in temperature-controlled cuvettes containing 1 ml of 10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 5 mM MgCl 2 , 150 mM NaCl. Controls were performed to account for any nonspecific effects of GTP␥S addition on fluorescence. Microscopy and Spindle Severing-Preparation of embryos, time-lapse differential interference contrast microscopy, and spindle severing were performed as described (10,35,36). Experiments were conducted using a homemade device that consists of a thermostat and a cooling/heating element coupled to a fan blowing air at the appropriate temperature onto the objective and the microscope stage. The temperature of the embryo during the experiment was monitored using a thermometer inserted in the agarose pad. Measurements of peak velocities of spindle poles following spindle severing were performed essentially as described (36).

RESULTS AND DISCUSSION
G202D Mutation Gives Temperature-dependent Lability to the Activated G␣ State-We purified wild type and G202D G␣ i1 to homogeneity as assessed by SDS-PAGE (data not shown). G␣ i1 (G202D)⅐GDP migrated in size exclusion chromatography as a monomer and had a circular dichroism spectrum consistent with properly folded G␣ (data not shown) (37). We first tested the ability of purified G␣ i1 to bind nucleotide at 30°C to approximate conditions found in vivo at the restrictive temperature. Wild type G␣ i1 bound GTP␥S in a saturable manner with an association rate comparable with published values (38) (Fig.  1A). By contrast, G␣ i1 (G202D) rapidly bound GTP␥S but in a biphasic manner, peaking at 30 min then rapidly decaying over time. We also examined GTP␥S binding at the permissive temperature (Fig. 1B). GTP␥S binding by G␣ i1 (G202D) at 15°C was rapid, monophasic, and stable for up to 15 h. These data suggest that G␣ i1 (G202D), and by extrapolation GPA-16(it143), undergoes nucleotide state-dependent inactivation at 30°C but not 15°C. The rate of GTP␥S binding by G␣ i1 (G202D) at 15°C was over an order of magnitude faster than the wild type protein (Fig. 1B), suggesting that the G202D mutation also enhances spontaneous GDP release.
High spontaneous GDP release engendered by the G202D mutation may contribute to G␣ inactivation at the restrictive temperature by promoting the GTP-bound form of the protein.
Alternatively, it is well described that nucleotide-free G␣ subunits are inherently unstable (20,39,40); thus, increased residence of G␣(G202D) in the nucleotide-free state could also contribute to inactivation. This is suggested to be the mechanism of temperature sensitivity of the human G␣ s (A366S) mutant, which bears an Ala-to-Ser point mutation in the GDPbinding pocket that increases spontaneous GDP release causing constitutive activity (40,41). Uniquely, this mutation is permissive at the temperature of the human testis (32-33°C) causing testotoxicosis because of the overproduction of testosterone. In other tissues, the protein is nonfunctional at 37°C causing pseudohypoparathyroidism, typical of G␣ s reduction of function (42).
Thermal melting of the G␣ i family proteins under examination in this study gave a single cooperative transition from predominantly ␣-helical structure to random coil, as measured by circular dichroism. Normally, activated G␣ is significantly more thermostable than inactive G␣; wild type G␣ i1 ⅐GDP had a melting temperature of 50°C, whereas wild type G␣ i1 ⅐GTP␥S had a melting temperature of 77°C (Fig. 1C) consistent with structural data that the three switch regions of GDP-bound G␣ are conformationally flexible, and GTP␥S binding induces a distinct, stable switch region conformation (43). In contrast to wild type, GTP␥S binding to G␣ i1 (G202D) did not induce a thermostable protein-nucleotide complex; both GDP-and GTP␥S-bound G␣ i1 (G202D) had melting temperatures of 50°C (Fig. 1D). These data suggest a mechanism for the temperature-sensitive loss-of-function in GPA-16(it143); at the permissive temperature, GPA-16(it143) is relatively stable in both the GDP-and GTP-bound forms, whereas at the restrictive temperature GPA-16(it143) is unstable in the GTP-bound state, becoming rapidly inactivated. Compatible with this view, the phenotype of gpa-16(it143) embryos at 25°C is indistinguishable from that of embryos depleted of gpa-16 by RNAi (9,10).

GPA-16(it143) Is Also Altered at Permissive Temperature
Wild type G␣ i1 also exhibited GDP-specific binding to the GoLoco motif protein PCP-2 and the guanine nucleotide exchange factor Ric-8A (Fig. 2, C and E; Table 1), consistent with previous studies (9,17,20). In contrast, G␣ i1 (G202D) interacted in a nucleotide-independent manner with PCP-2 and Ric-8A (Fig. 2, D and F), and with markedly reduced affinities versus wild type G␣ i1 ⅐GDP (K D for PCP-2 of 7-8 M versus 800 nM, K D for Ric-8A of 4 -5 M versus 400 nM; Table 1). We and others have previously shown RGS14 and G␣ i subunits to be involved in mammalian spindle formation and orientation (reviewed in Refs. 1,5). Whereas wild type G␣ i1 exhibited a high affinity, AlF 4 Ϫ -dependent interaction with the RGS14 RGS domain (Fig. 2G) as shown previously (22), no binding was observed between any form of G␣ i1 (G202D) and RGS14 (Fig. 2H). As RGS domains and G␣ effectors bind distinct (44), but at times overlapping (50), regions of switch II, we examined if the G202D mutation also altered effector binding in the context of a chimeric transducin/ G␣ i1 protein (G␣ t/i1 ) (18) known to exhibit activation-dependent binding to a fragment of the ␥ subunit of cGMP PDE␥ (23). Wild type G␣ t/i1 bound, in an activation-dependent manner, to immobilized PDE␥ peptide as expected ( Fig. 2I; Table 1); however, no binding was seen between ground state (nor activated) G␣ t/i1 (G198D) and PDE␥ ( Fig. 2J; Table 1), suggesting that the Gly-to-Asp substitution abrogated the effector binding properties of G␣ t/i1 .

GPA-16(it143) Interacts with GPR-1/-2 and RIC-8 in a Nucleotide-independent Manner-
The binding studies detailed above establish that the G202D mutation within switch II renders G␣ unable to interact properly in vitro with many G␣ regulators, including those that bind activated states (i.e. RGS domains, effectors). We confirmed that GPA-16(it143) exhibits this lack of proper nucleotide stateselective interactions by co-immunoprecipitation using C. elegans embryo extracts. Using this approach, we previously showed that wild type GPA-16 interacts robustly with the GoLoco motif proteins GPR-1/-2 in the presence of GDP but much less so in the presence of GTP␥S, whereas wild type GPA-16 interacts with RIC-8 equally well in the presence of either nucleotide (9). Here we used embryonic extracts from worms grown at 16°C and conducted co-immunoprecipitation at 16°C to investigate the behavior of native complexes at the permissive temperature. The interaction between GPA-16(it143) and GPR-1/-2 was decreased compared with wild  AUGUST 1, 2008 • VOLUME 283 • NUMBER 31 type GPA-16, whereas that between GPA-16(it143) and RIC-8 was not diminished (Fig. 1E). The interaction between GPA-16(it143) and GPR-1/-2 occurred equally well in the presence of GDP or GTP␥S, in contrast to wild type GPA-16 (Fig. 1E). This finding is compatible with the in vitro results of Table 1 and supports the view that GPA-16(it143)⅐GDP and GPA-16(it143)⅐GTP can both associate with GPR-1/-2 at the permissive temperature in vivo. Overall, these findings suggest that the G202D substitution renders G␣ proteins unable to interact properly in vivo with their regulatory proteins.

GPA-16(it143) Is Also Altered at Permissive Temperature
Structural Changes Caused by the G202D Mutation-We determined the crystal structure of G␣ i1 (G202D)⅐GDP to 2.2 Å (Fig. 3; supplemental Table 1). The overall structure of G␣ i1 (G202D) is largely unaltered from wild type G␣ i1 (51) with an overall root mean square deviation of 0.38 Å (Fig. 3A). Surprisingly, switch I and III of GDP-bound G␣ i1 (G202D) adopt conformations similar to those of activated conformations of G␣ (Fig. 3B). Arg 178 and Thr 181 , important residues to GTP hydrolysis found in switch I (51), do not undergo significant alterations in side-chain conformation compared with activated G␣ structures, despite a slight alteration in the switch I backbone conformation ( Fig. 3B and data not shown). The conformation of switch II is unique and likely a direct result of the G202D mutation. Although residues Lys 208 -Trp 211 of switch II are disordered, the ␤3/␣2 loop (within which the G202D mutation resides) folds back toward the ␣2/␣3 cleft and away from the nucleotide pocket (Fig. 3A). This altered conformation may underlie enhanced spontaneous GDP release seen with the G202D mutant (Fig. 1B), as the ␤3/␣2 loop is thought to be an occlusive barrier to the release of GDP (2). Additionally, the orientation of the Asp 202 side chain toward GDP (Fig. 3C) may introduce an electrostatic repulsion causing enhanced GDP release. Although the Asp 202 side chain orients directly at the GDP ␤-phosphate, the distance separating its electronegative carboxylate from the ␤-phosphate (ϳ5 Å) is great enough to prevent an outright electrostatic or steric clash. However, GTP binding would present significant electrostatic and steric clashes with the Asp 202 side chain (Fig. 3B). Such a clash may lead to protein instability, an effect likely exacerbated at elevated temperatures.
We also measured G␣ conformational change upon activation by GTP␥S. G␣ subunits contain a tryptophan in switch II (Trp 211 in G␣ i1 and GPA-16) that shifts from being solventexposed when GDP is bound to a hydrophobic pocket when GTP␥S is bound (reviewed in Ref. 52). Wild type G␣ i1 gave a substantial increase in Trp fluorescence upon exposure to GTP␥S (Fig. 4A); in contrast, G␣ i1 (G202D) gave a minimal increase in fluorescence upon incubation with GTP␥S (Fig. 4A). This weak fluorescence enhancement was reproducible and specific (Fig. 4B), albeit severely diminished in magnitude (4% increase over GDP-bound basal fluorescence versus 57% increase of wild type).
Taken together, these findings indicate that G␣ i1 (G202D) alters the conformation of switch II in response to GTP␥S binding, most likely by preventing complete rotation and translation toward the GTP-binding pocket (53,54). Combined with the observation that switch I and III conformations in G␣ i1 (G202D)⅐GDP are similar to activated G␣ i1 , the G202D-induced changes in switch II conformation help resolve not only the reduced affinity of G␣ i1 (G202D) seen for certain binding partners (e.g. PCP-2, Ric-8A, and RGS14; Table 1) but also the loss of nucleotide selectivity for such interactions (Fig. 2).
Normal Pulling Forces Seen at the Permissive Temperature in gpa-16(it143) Embryos Are GOA-1-dependent-We also evaluated the consequences of the G202D mutation on GPA-16 function in vivo by analyzing pulling forces on astral microtubules in gpa-16(it143) embryos at 16°C. In wild type embryos, the posterior aster undergoes characteristic oscillations transverse to the longitudinal axis, reflecting the extent of pulling forces acting on the spindle poles ( Fig. 5A and supplemental movie 1) (7). Although oscillations are largely abolished in gpa-16(it143) embryos at 25°C (Fig. 5C and supplemental movie 3) (9), they are indistinguishable from wild type oscillations in gpa-16(it143) embryos at 16°C (Fig. 5D and supplemental movie 4), suggesting that pulling forces are intact at the permissive temperature. We conducted in vivo laser microbeam-mediated spindle severing to reveal the extent of net pulling forces acting on each spindle pole (36). Pulling forces in wild type embryos were reduced at 16°C compared with 25°C ( Fig. 6 and supplemental Table 2), presumably a reflection of a global slowing at the lower temperature of biochemical reactions, such as microtubule dynamics known to be important for pulling force generation (14). However, net pulling forces were not decreased in gpa-16(it143) embryos at 16°C compared with 25°C and, more surprisingly, were even slightly increased on the anterior spindle pole in comparison with wild type at 16°C (Fig. 6). Intact pulling forces in gpa-16(it143) embryos at 16°C cannot be ascribed to GPA-16 being dispensable for pulling forces at this temperature, because gpa-16(RNAi) embryos at 16°C did not exhibit oscillations (supplemental Movie 2, Fig.  5B, and supplemental movie 2) and had decreased pulling forces (Fig. 6).
To test whether normal pulling forces at 16°C in gpa-16(it143) embryos may be sustained by GOA-1 function, given that GPA-16 and GOA-1 are partially redundant for force generation (8), we inactivated goa-1 using RNAi in gpa- 16(it143) embryos. Oscillations were absent and pulling forces substan- 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. exhibit a clear reduction in pulling forces much like gpa-16(RNAi) embryos (9,10). Future studies will help to further clarify the differential biochemistry and spatiotemporal dynamics of GPA-16 and GOA-1 in C. elegans embryos and thus better illuminate the conserved actions of G␣ subunits in governing ACD across metazoan evolution.