s2fieldsofstudy: Biology Chemistry

text:
Regulatory Interactions between the Amino Terminus of G-protein βγ Subunits and the Catalytic Domain of Phospholipase Cβ2*

We previously identified a 10-amino acid region from the Y domain of phospholipase Cβ2 (PLCβ2) that associates with G-protein βγ subunits (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148–7154). We mapped the site for cross-linking of a synthetic peptide (N20K) corresponding to this Y domain region to Cys25 within the amino-terminal coiled-coil domain of Gβγ (Yoshikawa, D. M., Bresciano, K., Hatwar, M., and Smrcka, A. V. (2001) J. Biol. Chem. 276, 11246–11251). Here, further experiments with a series of variable length cross-linking agents refined the site of N20K binding to within 4.4–6.7 Å of Cys25. A mutant within the amino terminus of the Gβ subunit, Gβ1(23–27)γ2, activated PLCβ2 more effectively than wild type, with no significant change in the EC50, indicating that this region is directly involved in the catalytic regulation of PLCβ2. This mutant was deficient in cross-linking to N20K, suggesting that a binding site for the peptide had been eliminated. Surprisingly, N20K could still inhibit Gβ1(23–27)γ2-dependent activation of PLC, suggesting a second N20K binding site. Competition analysis with a peptide that binds to the Gα subunit switch II binding surface of Gβγ indicates a second N20K binding site at this surface. Furthermore, mutations to the N20K region within the Y-domain of full-length PLCβ2 inhibited Gβγ-dependent regulation of the enzyme, providing further evidence for aGβγ binding site within the catalytic domain of PLCβ2. The data support a model with two modes of PLC binding to Gβγ through the catalytic domain, where interactions with the amino-terminal coiled-coil domain are inhibitory, and interactions with the Gα subunit switch II binding surface are stimulatory.

Activation of G-protein-coupled receptors releases the G␤␥ subunit from G␣-GTP, leaving G␤␥ free to interact with effector molecules such as enzymes or ion channels (1)(2)(3). A particular group of enzymes regulated by G␤␥ belong to the phosphoinositide-specific phospholipase C␤ (PLC␤) 1 class. Once activated, PLC␤ cleaves phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into the two second-messengers, diacylglycerol and inositol 1,4,5-trisphosphate. All PLC␤ isoforms have an N-terminal pleckstrin homology (PH) domain, four sets of EF-hand domains, X and Y domains comprising the catalytic center, and a C2 domain followed by a C-terminal extension (4). There are four isoforms of PLC␤, ␤1-4; however, only PLC␤2 and PLC␤3 are regulated by ␤␥ subunits (5).
G-protein ␤ subunits have two distinct domains, an N-terminal ␣ helix, which forms a coiled-coil interaction with the ␥ subunit, and a seven-blade ␤-propeller structure composed of seven WD repeating motifs (6,7). Amino acids within blades one, two, and three make extensive contacts with ␣-GDP (7,8).
Since ␣ subunits block G␤␥-dependent regulation of all effectors, effector-binding sites on G␤␥ were predicted to overlap with the ␣ subunit binding site. Indeed, mutagenesis of particular amino acids on G␤␥ important for ␣ subunit binding blocked activation of PLC␤2 (9,10). Furthermore, mutations to the outer strands of blades 2, 6, and 7, which do not make direct contact with G␣, rendered G␤␥ unable to effectively activate PLC␤2 (11).
The mechanism for regulation of PLC␤2 by G␤␥ subunits is not fully understood. Approaches for elucidating the activation mechanism have been to characterize the effects of a variety of mutants, peptides, and fusion proteins on regulation of PLC␤2 by G␤␥. Two major regions on PLC␤2 have been implicated as G␤␥-binding sites, the N-terminal PH domain and a portion of the catalytic Y domain. A large body of evidence supports a role for interactions between the catalytic domain of PLC␤2 and G␤␥ subunits. Overexpression of the Y domain in COS-7 cells blocked receptor-mediated G␤␥-dependent, but not G␣ qdependent activation of PLC. A purified glutathione S-transferase fusion protein comprising amino acids 526 -641 of the Y-domain bound directly to purified G␤␥ subunits (12). Overlapping peptides representing a portion of the Y-domain directly cross-linked to both G␤ and G␥ subunits with the heterobifunctional cross-linker SMCC, and this was blocked by purified PLC␤2 but not ␣ i . The peptides also inhibited G␤␥ regulation of PLC␤2 with an EC 50 of 30 -50 M (13). The crystal structure of PLC␦ reveals that the region implicated by the peptide studies corresponds to the ␣ 5 helix on the surface of the catalytic domain and therefore is accessible to interact with G␤␥ subunits (14). We hypothesized that direct binding of this helix within the catalytic domain to G␤␥ is involved in G␤␥-dependent PLC␤2 activation.
To define the site cross-linking of one of the catalytic domain peptides, N20K (amino acids 565-574 in PLC␤2), to the G␤ subunit and identify a potential Y-domain interaction site on G␤, we used a systematic cysteine mutagenesis approach where each cysteine on G␤ 1 was individually mutated to alanine. The cysteine residue critical for the majority of peptide cross-linking was cysteine 25 within the amino-terminal coiledcoil region of G␤ (15). This region is outside the G␣ subunit-binding site, complementing data demonstrating that G␣ i1 could not block cross-linking of N20K to G␤␥ (13,15). Previous studies have not implicated the amino terminus of G␤␥ as an important effector signaling site in mammals, although it has been implicated to be important in the yeast pheromone pathway (16,17).
These data suggest that the amino-terminal coiled-coil of G␤␥ directly binds to the ␣ 5 helix of the Y-domain to regulate PLC activity. The goals of this study were 3-fold: 1) to further delineate the specific region within the amino terminus of G␤␥ subunits that interact with PLC␤; 2) to demonstrate a functional role for the amino terminus of G␤ in mammalian effector regulation; and 3) to demonstrate the functional importance of the N20K region within full-length PLC for G␤␥ regulation. These results would be the first demonstrating a specific region on PLC interacting with a specific region on G␤␥, leading to a better understanding of how G␤␥ subunits regulate PLC.

EXPERIMENTAL PROCEDURES
Materials-Peptides (N20K, Ac-NRSYVISSFTELKAYDLLSK; SIGK, Ac-SIGKAFKILGYPDYD; L9A, Ac-SIRKALNIAGYPDYD) were purchased from Alpha Diagnostic International (San Antonio, TX) and had purity greater than 90% based on HPLC chromatography analysis. The masses of the peptides were confirmed by mass spectroscopy. All crosslinking reagents used in this study were from Pierce. Ni 2ϩ -nitrilotriacetic acid resin was from Qiagen. [ 3 H]PIP 2 and [ 32 P]NAD were from PerkinElmer Life Sciences. Pertussis toxin was purchased from List Biologicals. All molecular biology reagents were from Invitrogen, unless otherwise indicated. Baculoviruses encoding wild type G␤ 1 , His 6 -G␣ i , and G␥ 2 were obtained from the laboratory of Dr. Alfred Gilman.
Construction of G␤ Subunit and PLC␤2 Mutants-Mutants in rat G␤ 1 and in human PLC␤2 were constructed by overlap-extension PCR with Pfu polymerase (Stratagene) using standard protocols. Final constructs were sequenced to confirm the presence of the mutation. Bacoluviruses were generated as per the manufacturer's instructions (Invitrogen).
Purification of ␤␥ Subunits-G␤ 1 ␥ 2 subunits were purified from 2 liters of Sf-9 cells triply infected with His 6 -G␣ i1 , wild type or mutant G␤ 1 subunits, and G␥ 2 subunits essentially as described (18) with the following changes. Sf-9 cell membranes were prepared as described (18) and then extracted in buffer A containing 50 mM HEPES, pH 8.0, 3 mM MgCl 2 , 10 mM ␤-mercaptoethanol, 10 M GDP, 50 mM NaCl, 1% polyoxyethylene 10 lauryl ether (C 12 E 10 ), and a protease inhibitor mixture for 2 h at 4°C. Detergent-extracted proteins were diluted 5-fold with buffer B, 20 mM HEPES, pH 8.0, 1 mM MgCl 2 , 10 mM ␤-mercaptoethanol, 10 M GDP, 100 mM NaCl, 0.5% C 12 E 10 , and a proteaseinhibitor mixture and loaded onto a 4-ml Ni 2ϩ -nitrilotriacetic acidagarose column at 4°C overnight. The column was washed with 100 ml of buffer B containing 300 mM NaCl and 5 mM imidazole, warmed at room temperature for 15 min, and washed with 12 ml of the same buffer followed by 5 ml of buffer B containing 300 mM NaCl, 5 mM imidazole, and 1% n-octyl-␤-D-glucopyranoside (OG). G␤␥ subunits were eluted with 3 column volumes of buffer C, 20 mM HEPES, pH 8.0, 10 M GDP, 50 mM NaCl, 1% OG, 3 mM imidazole, 50 mM MgCl 2 , 30 M AlCl 3 , and 10 mM NaF followed by 4 volumes of buffer C with 1% cholate instead of OG. With this modified procedure, endogenous Sf-9 G␤␥ elutes in the first fraction, and the expressed G␤␥ elutes in fractions 2-5. Peak elutions, devoid of Sf-9 endogenous G␤␥, were combined, concentrated on a 0.5-ml hydroxyapatite column, and eluted with 20 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM DTT, 1% OG, and 200 mM KP i , pH 8.0. Protein concentrations were determined by an Amido Black assay. To confirm purity, 500 ng of purified wild type or mutated G␤␥ subunits were loaded onto a 12% SDS-polyacrylamide gel and stained with Coomassie Blue G250 stain. To confirm proper assembly of the mutant G␤ subunits with G␥ subunits, equal amounts of wild type and mutant G␤␥ subunits were run on a 17% SDS-PAGE gel, transferred to nitrocellulose, and blotted with G␥ subunit antibody, X263.
Purification of Wild Type and Mutant PLC-Wild type His 6 -PLC␤2 and His 6 -PLC␤3 were purified from 1 liter of Sf-9 cells as described (19). These wild type PLC␤s were used for measuring activation by mutated G␤␥ subunits. Mutant PLCs were purified using a protocol similar to the wild type 1-liter protocol from 50-ml cultures of Sf-9 cells, except buffer volumes were scaled down, and 1 ml of nickel resin was used (the heparin column was omitted). For experiments comparing the activities of PLC␤2 mutants, wild type and mutant enzymes were purified simul-taneously and in parallel, snap-frozen in liquid N 2 in single use aliquots, and stored at Ϫ70°C.
Phospholipase C Assays-PLC assays were performed as described (19). The concentrations of PLC, G␤␥, and detergent are indicated in the figure legends. Assays measuring peptide or G␣ subunit inhibition of G␤␥-dependent PLC activation were performed exactly as described (13). Assays measuring G␣ q activation of PLC were performed as described (5). In all experiments, a maximum of 20% of the PIP 2 substrate was hydrolyzed.
Measurement of ADP-ribosylation of G␣ i1 -Pertussis toxin-mediated ADP-ribosylation of 15 pmol of G␣ i1 in the presence of G␤␥ was measured as described (20). Myr-G␣ i was purified from Escherichia coli as described (21).
Chemical Cross-linking-All cross-linkers used in this study, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC), N-[⑀-maleimidocaproyloxy]succinimide ester, N-[␥-maleimidobutyryloxy]succinimide ester, N-[␣-maleimidoacetoxy]succinimide ester, and Nsuccinimidyl iodoacetate, were heterobifunctional, containing both sulfhydryl and amine reactive groups. The functional groups for all crosslinkers except N-succinimidyl iodoacetate had N-hydroxysuccinimideester and maleimide reactive moieties; N-succinimidyl iodoacetate had an iodoacetate group instead of a maleimide. All of the peptides used contained no cysteine residues and were N-terminally acetylated, so cross-linking was through the ⑀-amine of lysine residues. To prevent cross-linking of G␤ to G␥, peptides were preincubated with cross-linker prior to the addition to G␤␥ by use of the following protocol. Peptides at 2 times their final concentration (see figure legends for concentrations) were incubated with 400 M cross-linker in cross-linking buffer containing 50 mM HEPES, pH 7.4, 75 mM NaCl, 1 mM MgCl 2 , and 0.1% C 12 E 10 at room temperature for 10 min. Ethanolamine, pH 8, was then added to 100 mM to quench all unreacted N-hydroxysuccinimide-ester groups. 50 l of the peptide/cross-linker mix was added to 50 l of 60 nM G␤␥ diluted in cross-linking buffer and allowed to incubate at room temperature before quenching with 33 l of 4ϫ sample buffer (4ϫ sample buffer: 125 mM Tris, pH 6.8, 20% glycerol, 4% SDS, 1.4 M ␤-mercaptoethanol, and bromphenol blue) (times for each reaction are listed in the figure legends). The final concentrations were 30 nM G␤␥ and 200 M cross-linker; the peptide concentrations are listed in the figure legends. 10 l of the reactions were loaded onto a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with a G␤ subunit-specific antibody B-600 followed by an anti-rabbit horseradish peroxidase-conjugated antibody. Proteins were visualized using enhanced chemiluminescence detection reagents (PerkinElmer Life Sciences).

Delineation of the N20K Binding Site on G␤␥-Previous
studies in our laboratory demonstrated two overlapping peptides from the catalytic domain of PLC␤2, N20K, and E20K (amino acids 564 -584 and 574 -594, respectively) could be chemically cross-linked to G␤ subunits (13,15). Based on this, and other studies, we hypothesized the 10-amino acid region of overlap between these peptides, ELKAYDLLSK (amino acids 574 -584), representing an ␣ helix on the surface of PLC␤2, directly bound to G␤␥ subunits. To define this potential PLC␤2 interaction site on G␤␥, we mapped a cross-linking site for the N20K peptide using a cysteine to alanine mutagenesis approach (15). From this data, it was determined the sulfhydryl group of cysteine 25 is within reach of the peptide-binding site. However, since the cross-linker, SMCC, had a spacer arm length of 12 Å, we could only conclude the peptide was binding within a 15-Å radius of cysteine 25 (the length of the spacer arm in the SMCC cross-linker plus the length of a lysine side chain). To further refine the nature of the N20K interaction within G␤␥, we determined which residue(s) in N20K was involved in cross-linking. Since N20K has no cysteine residues and is acetylated at the amino terminus, the cross-linking must be through a one or both of the two ⑀-amine moieties on the lysine residues within the last 10 amino acids of N20K. Two N-terminally acetylated peptides were constructed, R1 and R2, where each lysine residue was individually replaced with arginine (Fig. 1A), abolishing a cross-linkable primary amine but retaining the peptide's charge. Both R1 and R2 cross-linked to ␤␥ with SMCC ( Fig. 1B), indicated by the appearance of a higher molecular weight immunoreactive G␤ subunit band only in the presence of peptide and cross-linker, although neither was as effective as N20K. Cross-linking of both of these peptides to G␤ 1 subunits where Cys 25 was mutated to alanine (G␤ 1 (C25A)) was substantially inhibited.
Purification of Amino-terminal Site-directed Mutants of ␤␥ Subunits near Cys 25 -The cross-linking data suggest a PLC binding site in the amino terminus of G␤␥ within 7 Å of cysteine 25. Therefore, surface amino acids in the coiled-coil region of G␤␥ within 10 Å of cysteine 25 were chosen for site-directed mutagenesis to confirm a functional role for the G␤␥ N terminus in PLC regulation. Six amino acids on G␤ and six amino acids on G␥ neighboring cysteine 25 were mutated three amino acids at a time. The mutations were named G␤ 1 (17)(18)(19)(20)(21) and G␤ 1 (23-27) based on the stretches of amino acids where the mutations were located ( Fig. 2A). Similar mutations made in G␥ 2 failed to dimerize with G␤ 1 and were not analyzed further. Wild type and mutant G␤␥ dimers were purified simultaneously and in parallel to near homogeneity in one step based on binding to His 6 -G␣ i and elution by activation of the G␣ subunit with AlF 4 Ϫ (Fig. 2B). This purification strategy insured that the mutants were properly assembled, folded, and functional. Proper assembly with the G␥ subunit was confirmed by immunoblotting for the presence of G␥ subunits in the purified mutant preparations (Fig. 2C). The proteins were Ͼ90% pure based on Coomassie Blue staining. No detectable endogenous Sf-9 G␤ (which runs at a higher molecular weight than overexpressed mammalian G␤ 1 ) was detected on either a Coomas-sie Blue-stained gel or immunoblots with an antibody that recognizes both endogenous Sf-9 and overexpressed G␤ 1 .
Activation of PLC␤ by Wild Type and Mutated G␤␥ Subunits-To demonstrate a functional role for the amino-terminal amino acids of G␤␥ in mammalian PLC␤2 regulation, the purified mutated dimers were tested for activation of PLC␤2. Surprisingly, G␤ 1 (23-27)␥ 2 activated PLC␤2 2-4-fold more effectively than wild type with no significant change in the EC 50 ( Fig. 3A and Table I). There was no significant difference in either the -fold activation or the EC 50 for activation by G␤ 1 (17-21) (Fig. 3A and Table I). Activation of PLC␤2 was abolished by boiling the mutant prior to performing the assay, indicating that the enhancement was not due to a nonprotein contaminant within the buffer (data not shown). The addition of GDP-loaded G␣ i blocked activation of PLC␤2 by G␤ 1 (23-27)␥ 2 , ensuring that the enhancement was due to the addition of G␤␥ and not a contaminating protein (Fig. 3B). The addition of purified PLC was required to observe accumulation of inositol 1,4,5-trisphosphate, indicating that no endogenous Sf-9 phospholipases were co-purified with the mutant G␤␥ (data not shown). These results were repeated with three different preparations of G␤ 1 (23-27)␥ 2 and two different preparations of wild type G␤ 1 ␥ 2 , demonstrating the reproducibility of the results with different batches of protein. As a final confirmation that both proteins were properly folded and functional postpurification, the ability of both G␤ 1 ␥ 2 and G␤ 1 (23-27)␥ 2 to support pertussis toxin-mediated ADP-ribosylation of G␣ i was tested. Both proteins supported ADP-ribosylation of G␣ i with no significant difference in either potency or efficacy (Fig. 3C). These data confirm that Cross-linking reactions were performed as described under "Experimental Procedures." Reactions were performed with SMCC and were carried out for 1.5 min at room temperature before quenching with sample buffer. The final concentration of the peptide was 30 M. C, cross-linking of R1 and R2 to wild type G␤ 1 ␥ 2 using cross-linkers with various lengths between functional groups. Reactions were performed as in B. The length of the spacer arm is given below the cross-linker.

FIG. 2. Construction and purification of N-terminal ␤ subunit mutants.
A, locations of the mutations are highlighted on the crystal structure of G␤␥ as determined by Sondek et al. (6). The sulfhydryl moiety of cysteine 25 is shown in dark blue, G␤ 1 (17)(18)(19)(20)(21) is shown in red, and G␤ 1 (23)(24)(25)(26)(27) is shown in purple. The changes in the amino acid sequences are shown on the right. Dashes indicate no substitution at that position. B, to ensure purity and confirm concentrations, 500 ng of purified wild type or mutated G␤ 1 ␥ 2 dimers were analyzed on a 12% SDS-polyacrylamide gel and stained with Coomassie Blue G250. C, to confirm assembly with ␥ subunits, 10 ng of the G␤ 1 ␥ 2 subunit preparations were resolved on a 17% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with the ␥ subunit antibody X-263.
both proteins are properly folded and functional and that there are no errors in the protein concentrations.
To determine whether this enhancement of activity was specific for PLC␤2, activation of PLC␤3 by mutant and wild type G␤␥ was tested. In this case, both WT and G␤ 1 (23-27)␥ 2 activated PLC␤3 to a similar extent, but ␤ 1 (23-27)␥ 2 was nearly 2-fold more potent than wild type ( Fig. 3D and Table I). These data strongly support a function of the amino terminus in the regulation of both PLC␤2 and PLC␤3. In both cases, the role of the amino terminus appears to be inhibitory, although the mechanisms are different. Others have reported differential activation of PLC␤2 and PLC␤3 by G␤␥ subunits (10,11,22).
Interaction between N20K and Mutated ␤␥ Dimers-The amino acids chosen for mutation were amino acids that, due to their close proximity to Cys 25 , were likely candidates to directly interact with the N20K peptide. To determine whether the G␤ subunit mutations eliminated N20K peptide binding, we tested whether N20K could still cross-link to the amino-terminal mutants. G␤ 1 (17-21)␥ 2 did cross-link to N20K, although higher concentrations of peptide were required for cross-linking compared with wild type, whereas G␤ 1 (23-27)␥ 2 substantially lost its ability to cross-link to N20K (Fig. 4A). Normally, N20K cross-linking to G␤␥ generates two higher molecular weight bands. N20K cross-linking to G␤ 1 (23-27)␥ 2 produces only one faint higher molecular weight band. We predicted that if the functional effects of N20K on activation of PLC␤ by G␤␥ subunits were through binding at the amino terminus, then elimination of this binding site would eliminate N20K inhibition of G␤␥-dependent activation of PLC␤2. Unexpectedly, N20K still inhibited G␤ 1 (23-27)␥ 2 activation of PLC␤2 with a similar potency as wild type. G␤ 1 (23-27)␥ 2 -dependent activation of PLC was inhibited by 50%, whereas wild type G␤␥-dependent activation was inhibited 85%. The inability of N20K to fully inhibit PLC activation by G␤ 1 (23-27)␥ 2 may be due to the increased efficacy of G␤ 1 (23-27)␥ 2 . Taken together, the crosslinking data and the peptide inhibition data suggest that there may be two binding sites for N20K on G␤␥, one within the amino terminus of G␤␥ eliminated by the G␤ 1 (23-27)␥ 2 mutation and one remaining intact. The ability of N20K to inhibit G␤ 1 (23-27)␥ 2 activation of PLC would be through binding to this second site. From these experiments, we hypothesize that Y-domain binding to the amino terminus of G␤␥ inhibits PLC activity, whereas Y-domain binding to this second site increases PLC activity.  3. Activation of PLC by wild type and mutated ␤␥ subunits. A, wild type and mutated G␤ 1 ␥ 2 subunits were incubated with 2 ng of PLC␤2 and sonicated lipid vesicles containing 25 M PIP 2 and 100 M liver phosphatidylethanolamine. All reactions contained 0.06% octyl glucoside. Reactions were initiated by the addition of 100 nM free Ca 2ϩ and transfer to a 30°C water bath for 7 min before quenching by the addition of ice-cold 10% trichloroacetic acid. Data shown are mean Ϯ S.E. of duplicate determinations from one of three (G␤ 1 (17)(18)(19)(20)(21)) or seven (G␤ 1 (23)(24)(25)(26)(27)) independent experiments. B, 100 nM G␣ i1 -GDP blocked the activation of PLC␤2 by 100 nM G␤ 1 (23)(24)(25)(26)(27). Data are from one of two independent experiments. C, G␤ 1 (23-27)␥ 2 supported ADP-ribosylation of G␣ i1 . 15 pmol of purified G␣ i1 was incubated with various amounts of wild type or mutated G␤ 1 ␥ 2 subunits in the presence of pertussis toxin and [ 32 P]NAD. Data shown are mean Ϯ S.E. of duplicate determinations. This experiment was repeated three times with similar results. D, wild type and mutated G␤ 1 ␥ 2 subunits were incubated with 5 ng of PLC␤3 as in A, except reactions were at 30°C for 10 min. The data are mean Ϯ S.E. of duplicate determinations from a representative experiment. This experiment was repeated three times with similar results. All curves were fit with a single site binding hyperbolic function using Graph Pad Prism Software. IP 3 , inositol 1,4,5-trisphosphate.
Defining a Second Site for N20K Interaction-Cross-linking data presented here and in our previous publications support the notion of two N20K binding sites. Cross-linking of N20K to G␤␥ produces two higher molecular weight bands (Figs. 1B and 4A). The first higher molecular weight band is G␤ cross-linked to one N20K peptide. The nature of the second band had been unclear, but its appearance was prevented by preincubation with purified PLC␤ or mutation of Tyr 15 to Gln in the peptide (13), indicating that it was not nonspecific. Moreover, replacement of Cys 25 with alanine still produced one faint higher molecular weight band (15). We propose that this second higher molecular weight band is G␤ cross-linked to two N20K peptides, one at the amino terminus and one elsewhere. We believe the residual cross-linking of peptide to the G␤ 1 (23-27) mutant and G␤ 1 (C25A) is through binding at the second site.
In order to define the second N20K binding site, we utilized the G␤ 1 (C25A) mutant. Our previous data showed that N20K cross-linked weakly to G␤ 1 (C25A)␥ 2 . Since there are no cysteine residues near Cys 25 capable of interacting with the crosslinker, G␤1(C25A) should have abolished all cross-linking if the N terminus was the sole site of binding. This finding indicates that there is a second N20K binding site outside of the amino terminus, and binding to this site results in the residual crosslinking to G␤ 1 (C25A). N20K has sequence similarity to G␤␥ binding peptides identified in a random peptide phage display screen conducted in our laboratory (23). This sequence similarity led us to hypothesize that these peptides were binding to a similar site on G␤␥ as the Y-domain of PLC (23). Recently, a binding site for one of these peptides was clearly defined in the x-ray crystal structure of one of these peptides, SIGKAF-KILGYPDYD (SIGK), bound to G␤ 1 ␥ 2 . 2 SIGK was identified in a doping mutagenesis screen to identify derivatives of one of the original peptides obtained in the phage display screen, SIRKALNILGYPDYD (SIRK). SIGK binds to G␤␥ with higher affinity than SIRK but retains most of the characteristics of SIRK. 3 The SIGK peptide bound to the top surface of G␤␥ overlapping with the switch II binding site of G␣. To determine whether this site was the second N20K binding site, we tested the ability of SIGK to compete away cross-linking of N20K to G␤␥ (SIGK itself does not cross-link to G␤␥ under these conditions). Fig. 5A shows that replacement of Cys 25 with alanine greatly reduces, but does not completely eliminate, cross-linking of N20K, confirming our previous studies (due to poor resolution of the second higher molecular weight band, we are focusing only on the lower band, which is a combination of G␤ cross-linked to N20K at either one of the two sites). The addition of SIGK at concentrations above the K d (ϳ1-5 M) partially reduces N20K cross-linking to wild type G␤␥, suggesting that binding to one of the two sites has been blocked by SIGK, but one remains intact. However, incubation with SIGK completely eliminated cross-linking of N20K to ␤ 1 (C25A)␥ 2 (Fig. 5,  A and B). The addition of a control peptide that does not bind ␤␥, L9A (23), has no effect on N20K cross-linking (Fig. 5A). These data demonstrate that N20K has two distinct binding sites on G␤, one within the amino terminus near Cys 25 and one at the G␣ interface, at the SIGK binding site.
We hypothesized that N20K blocked G␤ 1 (23-27)␥ 2 -dependent activation of PLC␤2 by interacting with the site outside of the amino-terminal binding site. Our cross-linking studies presented in Fig. 5, A and B, show that this second site overlaps with the SIGK peptide binding site; therefore, SIGK should also block PLC␤ activation by both wild type G␤␥ and G␤ 1 (23-27)␥ 2 subunits. Fig. 5C demonstrates that the SIGK peptide blocked G␤␥-dependent activation of PLC␤2 by both wild type and G␤ 1 (23-27)␥ 2 with no significant change in either the maximal inhibition or the IC 50 . From this finding, it is reasonable to conclude that the ability of N20K to block activation of PLC␤2 by G␤ 1 (23-27)␥ 2 was through binding to this site.
Regulation of Catalytic Domain Mutants of PLC␤2 by G␤␥ Subunits-This study and previous data suggesting that this peptide from the Y-domain of PLC binds to two sites are taken as evidence that the ␣ 5 helix on PLC represented by this peptide can bind to these two sites. If N20K is truly a model of the Y-domain ␣ 5 helix in full-length PLC, then mutations of the region corresponding to N20K within full-length PLC should have altered G␤␥-dependent activation. We constructed three triple alanine mutants (Fig. 6A) corresponding to the last 10 amino acids of N20K and the ␣ 5 helix in full-length PLC␤2. The basal Ca 2ϩ -dependent activities of all three PLC␤2 mutants were similar or slightly less than wild type (Fig. 6B), indicating that they were properly folded and active. The three PLC␤2 mutants were then tested for their ability to be activated by saturating concentrations of G␤ 1 ␥ 2 . PLC␤2(ELK/ AAA) had a significant decrease in its G␤␥-dependent activation as compared with wild type. PLC␤2(YDL/AAA) was also significantly deficient, whereas PLC␤2(LSK/AAA) was not significantly different from wild type. Full titration curves for G␤␥-dependent activation of PLC␤2(ELK/AAA) indicate that this mutant was activated very poorly at all concentrations tested (Fig. 6D). To confirm that the G␤␥-dependent activation resulted from specific alteration of G␤␥-dependent PLC activation, activation by G␣ q was tested. Previous studies have shown that the regions in PLC␤ necessary for G␣ q -dependent activation are different from those required for G␤␥ (25). The data in Fig. 6E show that PLC␤2(ELK/AAA) was activated to a greater extent than wild type PLC␤2, indicating that this mutant has no global deficiency in the catalytic machinery or in the ability to be activated in general. The nature of the enhanced activation by G␣ q is unknown and will be the subject of further investigation. Overall, these data demonstrate that there is a functional interaction between G␤␥ subunits and the ␣ 5 helix of PLC␤2.

DISCUSSION
In this work, we have reinforced and extended previous data suggesting that the catalytic domain of PLC␤2 can bind to the amino-terminal coiled-coil region of G␤␥ subunits near cysteine 25. Importantly, the mutagenic analysis demonstrates a functional role for interactions between the catalytic domain of PLC␤2 and both the N terminus and propeller regions of G␤␥. The amino-terminal region we identified in mammalian G␤␥ is the same region previously identified as an effector binding site in yeast G␤␥ subunits involved in the pheromone response pathway (17,26). Another study with single point mutations in the N terminus of mammalian ␤ failed to produce a mutant deficient in G␤␥-dependent effector activation when transfected into COS-7 cells, but interestingly, one mutant, K20A, showed a slight gain of function with respect to c-Jun N-termi- nal kinase activation but not activation of PLC␤2 (22). This study did not include mutations to Lys 23 or Ala 24 , which were mutated in our analysis. Other mutagenesis studies of G␤ subunits did not test for effects of mutations in the aminoterminal coiled-coil region, focusing on regions at the G␣ subunit interface and other amino acids within the propeller region (9 -11, 27).
Despite apparently eliminating a binding site for N20K in the G␤ 1 (23)(24)(25)(26)(27) mutant, N20K still inhibited the ability of this mutant to activate PLC␤2 with a similar potency as wild type (Table I, Fig. 4B), indicating that N20K must still be associating with the mutant. To explain this result, we hypothesize that there are two binding sites for N20K on the G␤ subunit, one eliminated by the G␤ 1 (23-27)␥ 2 mutation and one still intact. This hypothesis is supported by our cross-linking studies in which both G␤(C25A)␥ 2 and G␤ 1 (23-27)␥ 2 still displayed some cross-linking to N20K (Figs. 5A and 4A, respectively). Any cross-linking to G␤(C25A) must be through binding of the peptide to a second binding site on G␤␥. Since the cross-linking of G␤ 1 (23-27)␥ 2 is similar to G␤(C25A), we suspected that this residual cross-linking is due to N20K binding at the site outside of the amino terminus. Our previous work (13,15) and Fig. 1B support this idea, showing that cross-linking of wild type G␤␥ to N20K leads to two cross-linked species on a Western blot. The lowermost band is uncross-linked G␤. We believe that the first cross-linking band corresponds to G␤ cross-linked to one N20K peptide at one of two binding sites, and the upper band is G␤ cross-linked to two N20K peptides. This band is not G␤ cross-linked to G␥ due to the nature of the cross-linking protocol and confirmation by immunoblotting (data not shown; see Ref. 13).
We recently identified a family of peptides in a random peptide phage display screen that have homology to N20K. The binding site for a peptide derived from this screen, SIGK, has recently been defined at the G␣ subunit-switch II binding interface on the ␤-propeller. 2 Since SIGK competes for the residual cross-linking of N20K to G␤ 1 (C25A), the second N20K binding site must overlap with the SIGK binding site. This surface on ␤␥ subunits has been shown to be important for PLC regulation (9,10). There are two surface cysteine residues in proximity to this site, Cys 204 and Cys 271 , which could be participating in the cross-linking reaction. This interface is ϳ30 Å from Cys 25 . With SMCC as a cross-linker with a 12-Å spacer arm, it is not possible for the peptide to be binding at the amino terminus and use either of these two cysteine residues for cross-linking; nor is it possible for the peptide to be solely binding at this interface and use cysteine 25 for cross-linking. Binding of SIGK to this surface is able to block G␤␥-dependent activation of PLC, implying that it is a functional site for PLC binding.
Previous data suggesting that the Y-domain of PLC␤2 interacts with G␤␥ subunits has been inferred through indirect experiments from peptide and small domain binding and inhibition assays. Other data have suggested involvement of the PH domain in ␤␥-dependent regulation of PLC␤ (28). To conclusively demonstrate that the Y-domain is indeed important for G␤␥ regulation of PLC␤2, we made mutations in the ␣ 5 helix within the Y-domain on the surface of PLC␤ corresponding to the last 10 amino acids N20K. PLC␤2(ELK/AAA) was significantly impaired with respect to G␤␥-dependent activation relative to wild type PLC␤2 (Fig. 6, C and D). However, none of the three mutations tested completely blocked G␤␥-dependent activation. Previous work by Illenberger et al. (29) demonstrated that replacement of the PH domain of PLC␤2 with the PH domain of PLC␤1 (a phospholipase poorly regulated by G␤␥ subunits) blocked some but not all of the activa-tion by G␤␥ subunits, leading the authors to believe that interactions between G␤␥ subunits and the catalytic domain of PLC␤2 were both necessary and sufficient for G␤␥-dependent activation. Thus, data suggesting the PH domain is important for PLC regulation by G␤␥ subunits, taken together with the data presented here and previously, suggest that both the PH domain and the Y-domain of PLC␤2 play important roles in regulation of the enzyme by G␤␥ subunits. We also found that PLC(ELK/AAA) was activated to a greater extent by G␣ q relative to wild type PLC␤2. This is an interesting observation that needs to be investigated in further detail but clearly demonstrates that these mutants are selectively impaired with respect to G␤␥-dependent activation of PLC.
The results of this study suggest that the Y-domain of PLC␤2 can bind two distinct regions on the G␤␥ subunit, the N-terminal ␣ helix and the G␣ subunit switch II binding interface. Fig. 7 outlines a simple model for PLC regulation through binding at these two distinct sites. In the model, binding of the Y-domain of PLC␤2 (shown as a triangle, with the N20K region highlighted) to the amino terminus of G␤␥ is inhibitory. This complex is in equilibrium with a G␤␥-PLC complex where the Y-domain is bound to the propeller region. This contact is stimulatory and necessary for enzymatic activation of PLC. During interactions between wild type PLC and wild type G␤␥, these two processes are in equilibrium, with the balance resulting in a net activation of the enzyme. For the G␤ 1 (23)(24)(25)(26)(27) mutant, disruption of the interaction between the amino terminus of G␤␥ and PLC relieved this inhibitory constraint, resulting in the enhanced activation. The N20K peptide was still able to block this mutant's activation of PLC, because the stimulatory binding contact between the Y-domain and the blade region remained intact. Mutations to the N20K region within full-length PLC␤2 would be expected to reduce the ability of the enzyme to be activated by G␤␥, since binding to both the stimulatory and the inhibitory region would be equally affected. This was confirmed with the PLC␤2(ELK/ AAA) mutant.
There are two simple mechanisms by which binding to the amino terminus of G␤␥ could result in an inhibition in the activation of the enzyme. First, binding of the Y-domain to this region would sequester it from binding to the propeller region, which is necessary to stimulate PLC activation, as shown by our N20K and SIGK competition data. Second, the N20K region within the Y-domain of PLC␤ is within close proximity to the substrate binding site of the enzyme (14). Recent low resolution structures of G␤␥ bound to membrane tubules place the N-terminal region of G␤␥ far from the membrane surface (30). Therefore, if the Y-domain of PLC␤ is bound to the N terminus, it would be sequestered from its substrate. Some possible physiological implications for an inhibitory binding constraint within the amino terminus of G␤␥ for PLC␤2 can be imagined. First, inhibitory binding to the amino terminus could be a mechanism for regulating the activation of the enzyme. There are data to suggest that G-proteins can negatively regulate PLC activity. It had previously been demonstrated that the addition of a nonhydrolyzable version of GTP, Gpp(NH)p, to solubilized membranes from cortical neurons, decreased inositol phosphate production at low concentrations, whereas it stimulated production at high concentrations (31,32). Moreover, activation of G␣ i/ o-coupled receptors, such as the D2 dopamine receptor and the A1 adenosine receptors, could antagonize inositol phosphate production resulting from activation of the G␣ q -linked thyrotropin-releasing hormone receptor (24,33,34). These experiments suggest G␤␥ could negatively regulate PLC activation in vivo. Alternatively, the amino terminus could be functioning as a scaffold, anchoring the PLC near its activator and its substrate, while the G-protein is in its inactive heterotrimer and the propeller region is blocked by G␣. Upon activation of the G-protein signaling cascade and release of ␣-GTP, the Y-domain could dissociate from the amino terminus, and both the Y-domain and the PH domain of PLC could now make contact with the stimulatory areas within the propeller region, leading to enzyme activation.
Three novel conclusions have evolved from the course of this study. First, we clearly demonstrate that the amino-terminal coiled-coil region of G␤␥ is directly involved in effector regulation in mammalian isoforms of G␤␥ subunits, the first such data to map a mammalian effector binding site outside of the propeller region. Second, we show that mutations within the catalytic domain of PLC␤2 had reduced activation by G␤␥ subunits, supporting previous data with synthetic peptides and GST fusion proteins suggesting that the Y-domain of PLC was important for regulation by G␤␥ subunits. Finally, we show evidence suggesting that the N20K peptide may bind two distinct regions on the G␤␥ subunit, leading to the possibility the Y-domain of full-length PLC may also bind G␤␥ at two distinct sites as well. What is becoming clearly apparent with G␤␥ regulation of effectors is that no single binding interaction defines all of the contact interfaces. Thus, the mechanisms for G␤␥ binding and effector activation appear to be significantly more complex than had been previously imagined.


extfieldsofstudy: Biology Medicine

text:
Role of early experience in ant enslavement: a comparative analysis of a host and a non-host species

Background Ants use the odour of the colony to discriminate nestmates. In some species, this odour is learned during the first days following emergence, and thus early experience has a strong influence on nestmate discrimination. Slave-making ants are social parasites that capture brood of other ant species to increase the worker force of their colony. After emerging in the slave-maker nest, slave workers work as if they were in their own colony. We tested the hypothesis that early experience allows the deception of commonly enslaved species, while non-host species use a different mechanism, which does not involve learning. Results Pupae of a host species, Temnothorax unifasciatus, and a non-host species, T. parvulus, were allowed to emerge in the presence of workers of one of two slave-maker species, Chalepoxenus muellerianus or Myrmoxenus ravouxi. When T. unifasciatus was exposed to slave-makers for 10 days following emergence, they were more aggressive towards their own sisters and groomed the slave-maker more. T. parvulus gave a less clear result: while workers behaved more aggressively towards their sisters when exposed early to C. muellerianus workers, this was not the case when exposed early to M. ravouxi workers. Moreover, T. parvulus workers allogroomed conspecific nestmates less than T. unifasciatus. Allogrooming activity might be very important for the slave-makers because they are tended by their slaves. Conclusion Our findings show that early experience influences nestmate discrimination in the ant T. unifasciatus and can account for the successful enslavement of this species. However, the non-host species T. parvulus is less influenced by the early environment. This might help to explain why this species is never used by social parasites.

Background
The early stages of an animal's life constitute a critical period during which the environment can have dramatic and sometimes irreversible effects on adult behaviour. The best example is imprinting, a process by which the individual develops an irreversible reference pattern from a stimulus present during a short period of time during its development. Lorenz [1] was the first to conceptualize the imprinting process and gave one of the most striking examples: during the few minutes after hatching, young birds developed an attachment to their parents if present, or a parental surrogate, such as Lorenz himself. Imprinting is only a special case of the processes by which early experience affects subsequent adult behaviour. For instance, in monkeys and humans, Harlow & Harlow [2] and Bowlby [3] noted the importance of attachment during the early phase after birth for the development of adult behaviour.
Despite a much simpler brain structure, insects display similarly pronounced plastic responses to the early environment. Influences of the preimaginal environment on adult behaviour have been demonstrated as early as the late 1930's in Drosophila [4], and were later documented and shown to be involved in various contexts in many other species (see review in [5]). These contexts can have a social dimension in social insects. Some ants, for example, learn the colony odour during the early phase of imaginal life and later use this template for recognizing brood [6][7][8][9][10][11] and discriminating among adults [12][13][14][15]. This critical period determines who benefits from altruistic behaviours shown by worker ants, which rarely reproduce directly but rely on indirect fitness benefits via reproduction of related individuals [16].
Slave-making ants are social parasites that exploit the labour force of other ant species. Slave-maker workers are specialized for conducting raids, wherein they seize brood from nearby host ant colonies and bring them back to their own nest [17]. When they emerge, the slave ants behave as if they were in their own colony. Among other routine ant tasks, they rear the slave-maker brood, defend the nest, and sometimes feed and groom the slave-maker workers. Altruistic acts of slaves are thus directed toward unrelated individuals. One hypothesis suggests that slave deception is possible because slaves are captured as pupae and learn the slave-maker colony odour after emergence [18][19][20].
Myrmoxenus ravouxi and Chalepoxenus muellerianus are among the most common slave-makers in Western Europe. They use several related species as slaves but show a preference for Temnothorax unifasciatus [21]. In the Mediterranean area, M. ravouxi, C. muellerianus and T. unifasciatus can be found in sympatry with T. parvulus, though T. unifasciatus seems to prefer more arid environments. T. parvulus has never been found as a slave, but the reason for this is unknown. We postulated that slavery would be prevented if nestmate discrimination in T. parvulus was not achieved through early learning of colony odour, because slave workers emerging in the slave-maker colony would recognize the slave-maker brood and workers as aliens.
Our study had two aims. First, we tested the hypothesis that deception of the host T. unifasciatus ensues from early learning of colony odour as a mechanism for nestmate discrimination. Second, we tested the hypothesis that early experience (within the first 10 days following emergence) affects the discrimination capabilities of T. parvulus workers less than those of T. unifasciatus. After manipulat-ing the early experience of T. unifasciatus and T. parvulus, we tested and compared their discrimination capabilities.

Results
T. unifasciatus workers reared with M. ravouxi (batch A) or C. muellerianus (batch B) were significantly more aggressive towards their unfamiliar sisters than towards the familiar M. ravouxi or C. muellerianus workers (Table 1). Moreover they spent significantly more time allogrooming the familiar M. ravouxi or C. muellerianus workers than their unfamiliar sisters. Similarly, T. unifasciatus workers reared with conspecifics (batch C) were less aggressive towards their familiar sisters than towards the unfamiliar M. ravouxi and C. muellerianus workers. They also spent significantly less time allogrooming the unfamiliar M. ravouxi or C. muellerianus workers than the familiar sisters (Table 1).
T. parvulus workers reared with M. ravouxi (batch A) did not show a significant difference in aggressive behaviour or in the time spent allogrooming between their unfamiliar sisters and the familiar M. ravouxi workers (Table 1). Conversely, T. parvulus workers reared with C. muellerianus (batch B) were significantly more aggressive towards their unfamiliar sisters than towards the familiar C. muellerianus workers, and they spent significantly more time allogrooming the familiar C. muellerianus workers than the unfamiliar sisters. T. parvulus workers reared with conspecifics (batch C) were significantly less aggressive towards their familiar sisters than towards the unfamiliar M. ravouxi or C. muellerianus workers, but the time they spent allogrooming the unfamiliar M. ravouxi or C. muellerianus workers and their familiar sisters was much reduced and did not differ significantly (Table 1).

Discussion
Our results suggest that the environment experienced by T. unifasciatus during the first 10 days after emergence influences nestmate discrimination, at least in our artificial laboratory conditions. Indeed, workers that were exposed early to M. ravouxi and C. muellerianus displayed a very aggressive behaviour towards their own sisters and spent much time grooming the slave-makers. This suggests that the influence of the early environment could potentially account for the fact that T. unifasciatus slaves care for and defend the slave-makers. However, as host workers never emerge only with slave-makers present the mechanisms allowing enslavement could be different in nature. The same result was obtained for T. parvulus workers that were exposed to C. muellerianus. However, when exposed to M. ravouxi, they were not more aggressive toward their sisters than toward the slave-maker. These results partly confirm our hypothesis that the effect of early experience on the discrimination capabilities is reduced in T. parvulus when compared with T. unifasciatus workers.
The use of tethered, but live ants to record the reaction of test workers could have influenced their behaviour through the performance of antennation, biting, stridulation, etc., and the emission of pheromones. Indeed, several species of social parasites are known to use repellent, appeasement, and/or propaganda substances to usurp host nests [22]. The occurrence of such substances in our focal slave-maker species is likely and could have reduced aggressiveness of test workers. Moreover, several pupae of the test species were allowed to emerge in the glass tubes containing slave-makers. The early experience of test workers could then have been influenced by the other emerging test workers. Another potential source of bias in our results is the unequal representation of collected populations of Temnothorax in the experimental groups. The observed differences between our T. parvulus and T. unifasciatus might be due to differences between populations rather than species, if early experience had a smaller influence on nestmate discrimination in the Italian population. However, this possibility seems unlikely because species differences for fundamental mechanisms (such as the determinant of nestmate discrimination) are expected to be unaffected by population differences. The observed differences between the two species cannot be due to a mere difference in aggressiveness, because their levels of aggression towards slave-makers in the control experi- ment were not significantly different. However the difference between their levels of allogrooming was highly significant. A low basic level of allogrooming activity for T. parvulus workers was probably responsible for the fact that they did not groom their sisters more than the slavemakers in the control experiment. This makes it difficult to test the influence of early experience on allogrooming behaviour of T. parvulus.
Few other studies have demonstrated that early experience with slave-maker workers or brood elicits slave-maker care in the host species [23][24][25]. These studies dealt with species in the subfamily Formicinae; specifically, the slavemaker Formica sanguinea and its hosts F. fusca and F. cunicularia. Slave-making ants are known from only two subfamilies: the Formicinae and the Myrmicinae [26]. In the only study considering Myrmicinae, Alloway and Hare [27] showed that early learning was not necessary to explain the acceptance of the brood of the slave-maker Protomognathus americanus by enslaved T. longispinosus workers. Indeed, slave-maker larvae were preferentially accepted even when T. longispinosus workers were exposed early to conspecific larvae. Our results confirm that early experience can be important for successful ant enslavement in other myrmicine systems. Together, these studies across subfamilies seem to confirm the hypothesis that early behavioural plasticity of certain ant species has permitted or at least facilitated the evolution of slave-making habits [28], and show that other mechanisms are possible [27] but seem less common. This suggests that early behavioural plasticity could be a general prerequisite for the evolution of slave-making in ants [20]. The fact that early experience is important for the integration of the young slaves into the slave-maker colony might explain why M. ravouxi and C. muellerianus do not capture adult hosts during raids [29,30]. In fact, out of 10 genera displaying interspecific slavery, only one is known to result in the mixing of adult slaves into the slave-maker colony [26]: raids of the slave-making Strongylognathus commonly end in the fusion with the Tetramorium host nest [31]. However, the fusion occurs after a prolonged fight that often results in casualties on both sides. It is noteworthy that intraspecific colony fusion has been reported in the genus Tetramorium as a result of territorial competition [26].
For some slave-making species, host colony take-over by slave-maker queens requires acceptance by adult host workers that have had no previous exposure to slave-makers. The founding queen of many slave-making species has to enter a host nest, be accepted by the host workers, then kill and replace the resident queen [19]. Once the slave-making queen manages to kill the resident queen, the workers care for the new queen and rear her brood. The acceptance of the new queen does not require previ-ous exposure to slave-makers because specialised behavioural and chemical strategies are involved [22]. For example, in the genus Polyergus, the young slave-maker queen does not bear any odour, and thus prevents the host workers from detecting her. The slave-maker queen then acquires the cuticular hydrocarbons of the host queen by physical contact and the host workers accept her as though she were there natal queen [32,33]. The founding queen of M. ravouxi enters the Temnothorax host nest, reaches the queen, and slowly throttles her to death [34]. The chemical mechanism responsible for the acceptation of the M. ravouxi queen by the host workers remains to be elucidated.
Queens of other slave-making species including C. muellerianus, Harpagoxenus and Protomognathus evict all adult ants from the host nest and keep only the host brood. They thus rely completely on the manipulation of early experience even during colony foundation. The life history of slave-making ants suggests that the use of adult slaves without previous exposure to the slave-maker is costly or involves a highly specialised strategy. Manipulation of early experience by the slave-maker appears as the most parsimonious strategy, which reinforces the suggestion that it facilitated the evolution of slavery.
Early behavioural plasticity is a widespread mechanism in animals and is involved in a number of host-parasite systems. However, in most cases, this mechanism is used by the parasite to find its host. The European cuckoo is known to rely on habitat, probably in addition to other cues, which is learned when reared by the host [35], but it does not seem to imprint directly on the host [36]. In parasitoid wasps, preimaginal imprinting is involved in host selection [5]. In slave-making ants, imprinting was also shown to influence host selection during raids and colony foundation [37,38].
A striking result of this study was that manipulating the early experience of T. parvulus had different consequences depending on the slave-maker species to which it was exposed; nestmate discrimination seemed to be influenced by C. muellerianus, but not by M. ravouxi. If T. parvulus was insensitive to experience at emergence due to a strict genetic system of odour discrimination or an earlier sensitive period, we would expect the same outcome for both slave-maker species. A possible explanation is that there is a limited set of odours that T. parvulus can learn and/or perceive at emergence. M. ravouxi and C. muellerianus likely have different chemical profiles and T. parvulus might be chemically more similar to C. muellerianus than to M. ravouxi. Thus, the latter could be out of the range of potentially learned patterns. As we show in our experiments, phylogenetically closely related ant species can display various sensitivities to the early environment. Thus, perhaps the mechanisms of nestmate discrimination differ as well.
Nestmate discrimination has been shown to be less influenced by social environment at emergence in the ant genus Camponotus than in the genus Formica [7]. It is therefore interesting that no species of Camponotus is parasitized by slave-makers, while many Formica species are hosts to slave-makers. Similarly, our results on T. parvulus, the non-slave species, suggest that plasticity at emergence is not the only determinant of nestmate discrimination. A first alternative possibility is that nestmate discrimination is determined during an earlier sensitive period. Indeed, studies in Camponotus floridanus and Cataglyphis cursor demonstrated the importance of larval stages for nestmate discrimination [39,40]. As most slave-makers capture pupae preferentially, a nestmate discrimination mechanism based on larval experience might prevent these species from being enslaved. A second possibility is that nestmate discrimination in T. parvulus has a genetic component or is based on self-referent phenotype matching.
T. nylanderi, a species very closely related to T. parvulus, has nestmate discrimination cues based on colony environment, and especially nest site material [41]. Even if T. parvulus can be expected to rely in part on a similar mechanism in the field, it could not have influenced our results because colonies were reared in identical artificial nests. Whatever the determinant of nestmate discrimination in this species, it can be an obstacle to slavery because at least some elements of the template involved in discrimination are established before the slaves are captured. Moreover, T. parvulus workers do not groom their nestmates often, which could be problematic for the slavemakers because they are tended by the slaves. These characteristics might partly explain why T. parvulus has never been found as slave. However, other factors could explain why M. ravouxi and C. muellerianus does not enslave T. parvulus. Social parasites in the tribe Formicoxenini are phylogenetically closely-related to their hosts [42]. It might be impossible for the social parasites to exploit more distant species as hosts because they would not share the same ecological and microhabitat requirements, or the same communication system. Slave-maker species could also be under selection to match the colony odour of the host species. Coevolution occurs between slavemaking ants and their hosts [43,44] and it has been shown that in slave-maker colonies both species have similar cuticular hydrocarbon profiles [45,46]. It might therefore be easier for the host species to learn the more similar profile of the parasite, than for the non-host species.

Conclusion
Early experience can have important consequences on the behaviour of adult ant workers and consequently on their inclusive fitness. Manipulation of early experience in an experimental (this study) or natural (social parasitism) context influences nestmate discrimination. However, we showed that the effect varies across species: it was more pronounced in the host than in the non-host species. Social parasitism is not evenly distributed in ants: most of the 200 known species occur in only two subfamilies (the Formicinae and the Myrmicinae), and in these subfamilies it is concentrated in a few genera [47]. The reasons for this distribution are still unknown and elucidating the fine scale mechanisms involved in nestmate discrimination across taxa might help to explain the evolution of slavery in ants.

Methods
Colonies of the two slave-maker species and of both host species were collected in northern Italy (Lago di Garda) and southern France (Vaison-la-Romaine and Grasse, we found no slave-makers near Grasse) in the spring and summer 2003. Because of a sampling bias, significantly more T. unifasciatus originated from southern France and more T. parvulus from northern Italy (Chi-square = 13, p = 0.022). Nests were housed in plastic boxes containing a thick layer of plaster. The print of a microscope slide covered by a glass plate provided a nest site [48]. Colonies were fed twice a week with frozen fruit flies and a mixture of honey and apple. Rearing conditions were as follows: day/night: 14 h-24°C/10 h-17°C. Genus names comply with Bolton's new classification of Formicidae [49]. The experiments were performed in August and September 2003.
Pupae of T. unifasciatus and T. parvulus that were close to emergence were removed from their colonies (unparasitized) and isolated in glass tubes with either M. ravouxi (experimental batch A), C. muellerianus (experimental batch B) or homo-colonial (control batch C) workers. Each glass tube contained one to three pupae (from the same colony) and two to six adult workers. The small numbers of slave-maker workers available precluded larger worker/pupae ratios, but at least two adult workers were used for each pupa. Slave-maker workers of M. ravouxi and C. muellerianus were obtained from 20 and 27 colonies respectively. Pupae close to emergence were recognizable by pigmentation, and emergence took place within two days of uniting pupae with their respective conditioning workers. Amputation of the distal segments of one leg of adult Temnothorax workers from batch C allowed us to distinguish them unambiguously from the newly emerged test workers. Slave-makers from batches A and B were not subjected to this amputation. Workers were tested 10 ± 0.3 (mean ± SE) days after emergence.
Each test worker was introduced into a circular arena of 1.5 cm 2 where another worker was tethered by a nylon thread tied between the head and alitrunk. We were only interested in the behaviour of the test worker, thus we tethered the other worker to reduce the probability of interaction. Tethered workers could, however, still emit pheromones and engage in various other behaviours such as antennation, biting, stridulation etc. Tests started at least five minutes after tethering in order to let the tethered worker acclimate, but we cannot totally exclude the influence of pheromones on the behaviour of test workers. Duration of allogrooming and number of agonistic acts (mandibles opening, biting, stinging attempts) towards the tethered worker were recorded over 10 minutes (from the first encounter). Each test worker from batches A and B was tested successively against a slavemaker worker (M. ravouxi or C. muellerianus respectively) from its glass tube (hetero-colonial and familiar) and a worker from its colony of origin (homo-colonial and unfamiliar). Test order was controlled. Duration of allogrooming and number of agonistic acts were compared between the two tests using the Wilcoxon test for paired samples. Each test worker from batch C was tested successively against a worker from its glass tube (homo-colonial and familiar), a M. ravouxi worker, and a C. muellerianus worker (hetero-colonial, unfamiliar). All combinations of test order were equally represented. The test involving the homo-colonial and familiar worker was compared to each of the two tests involving a slave-maker worker using the Wilcoxon test for paired samples. Moreover, the number of agonistic acts towards the slave-makers was compared between T. unifasciatus and T. parvulus with a Mann-Whitney test for independent samples in order to test for a difference in levels of aggression between the two species. Differences in duration of allogrooming between the two species were similarly tested by comparing the performances of test workers from batch C in tests involving a homo-colonial and familiar worker. Tests were computed with Statistica 6.

Authors' contributions
RB conceived the study, carried out part of the experiments and data analysis, and drafted the manuscript. CS participated in the design of the study, carried out part of the experiments and data analysis. Both authors read and approved the final manuscript.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Expression of plasminogen activators in preimplantation rat embryos developed in vivo and in vitro

Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. The invasion of trophoblast cells into the endometrium during the implantation process can be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process. As in vitro developing embryos resulted in lower implantation rate than those developed in vivo we assume that a reduced PAs activity may lead to it. There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. The purpose of this study was to assess the expression of urokinase type (uPA) and tissue type (tPA) plasminogen activators in in vivo and in vitro preimplantation development in rat embryos using immunofluorescence confocal microscopy and computerized image analysis. Methods Zygotes, 2-cell, 4-cell, 8-cell, morula and blastocyst stages of development were flushed from the reproductive tract (control groups) of Wistar rats. Zygotes were flushed and grown in vitro to the above mentioned developmental stages and comprised the experimental groups. Immunofluorescence microscopy and computerized image analysis were used to evaluate both qualitative (localization) and quantitative expression of plasminogen activators. Results uPA and tPA were found to be expressed in rat embryos throughout their preimplantation development, both in vivo and in vitro. While uPA was localized mainly in the cell cytoplasm, the tPA was detected mainly on cell surface and in the perivitelline space. In blastocysts, both in vivo and in vitro, uPA and tPA were localized in the trophectoderm cells. Total uPA content per embryo was higher in the in vivo as compared with the in vitro developed embryos at all stages measured. Blastocyst uPA content was significantly low as compared with the four-cell, eight-cell, and morula stages. Total tPA content was higher in embryos developed in vivo than those developed in vitro except for the 4-cell and 8-cell stages. Conclusion In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing controls. The enzymes studied vary probably in the ratio of their active and inactive forms as there is no correlation between their content and the activity observed in our previous study. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs plays a role in the implantation process in rats.

Background
Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) have been implicated in mammalian gametogenesis [1], ovulation [2,3], fertilization [4,5], early stages of development and embryo implantation [6,7]. The PAs are serine proteases, which convert the inactive plasminogen to the potent protease plasmin. Plasmin can degrade directly or indirectly, through the activation of metalloproteinase zymogens, all components of the extracellular matrix [8,9]. There are two types of PAs, tissuetype plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Plasminogen, its activators and inhibitors, participate in the implantation process. Trophoblast cells of human blastocysts cultured in vitro produced PAs during the period corresponding to the in vivo invasion into the endometrium [10]. In embryos of the homozygous t w73 mouse mutant, PAs were reduced and was concomitantly associated with implantation failure [11]. The invasion of trophoblast cells during the implantation process could be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process [12,13]. In the human, embryo implantation following in vitro fertilization and embryo transfer (IVF-ET) is considered to play a major role in the success of the treatment. Only 12% of the transferred embryos are able to successfully implant [14].
In the implantation process, two major factors participate: the uterus undergoes changes that prepare it for the arrival and implantation of embryos, and the embryos undergo cellular reorganization that enables them to penetrate the endometrium and to form the placenta. We assume that one of the reasons for low implantation rate of embryos developed in vitro involves reduced PAs activity.
In a previous study we demonstrated differences in PAs activities between in vivo and in vitro preimplantation developed embryos. In both, uPA activity increased from the zygote towards the blastocyst stage while tPA activity remained relatively unchanged. However, tPA and uPA activities were lower in in vitro developed embryos as compared with in vivo developing ones, at all developmental stages, which may lead to a reduced implantation rate of in vitro developed embryos [15].
There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. Therefore, the purpose of this study was to investigate the PAs expression and localization during embryo development in vivo and in vitro by immunofluorescence confocal microscopy.

Methods
The following study was approved by the Institutional committee for animal care and ethics at Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Animals
Mature female Wistar rats 2-3 months old, weighing 180-230 g were used. The animals were kept in a temperaturecontrolled room maintained at 22-24°C with lighting regimen of 14 hours light 10 hours dark (light on 5:00 AM -7:00 PM). The rats were allowed free access to rat chow and tap water.
Daily vaginal smears were taken at 10:00 AM, and the stage of estrous cycle was determined. Overnight caging of a proestrous female with a male of proven fertility induced pregnancy. The next day, the presence of a vaginal plug or spermatozoa in the vaginal smear was designated as day 1 of pregnancy.

Collection of embryos
Zygotes, two-cell, four-cell, eight-cell embryos and morulae were flushed with rat 1-cell embryo culture medium (R1ECM) [16] from oviducts at days 1, 2, 3 and 4 of pregnancy, respectively, and blastocysts at day 5 from the uterine horns. All equipment and media used were sterile. Ovary-oviduct complexes were removed from anesthetized animals. The complexes were placed in R1ECM, and the oviducts were separated under a dissecting microscope. 30-gauge blunt-end needle attached to a syringe containing R1ECM was inserted through the oviductal end held by forceps surrounding the needle and tube. Embryos were gently flushed into 35-mm-diameter culture dish. Embryos were washed 3 times by transfer into fresh R1ECM to remove cell debris and any maternal factors present in the oviduct.
Zygotes in their cumulus mass were flushed and the cumulus cells were removed by gentle aspiration through a micropipette (diameter, 150-200 µm) several times in R1ECM containing 80 U/mL of hyaluronidase. Clean zygotes were washed 3 times by transfer into fresh R1ECM to remove traces of hyaluronidase. Flushed embryos were collected with a mouth-controlled micropipette (diameter, 150-200 µm).
Blastocysts were flushed from the uterine horns by insertion of a 23-gauge needle attached to a syringe containing R1ECM. Flushed, free-floating blastocysts were collected into a polypropylene tube inserted through the vagina and pushed gently to surround the cervical openings. Tubes were removed, and their contents were poured into Petri dishes. Blastocysts were washed and collected as described for zygotes and embryos. These embryos developed in vivo were the control groups.
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Embryo culture
As described for the in vivo embryos, clean zygotes were grown in vitro to the same developmental stages as controls; these were the experimental groups. Each group of embryos consisted of 25-35 embryos collected from six pregnant females. This was repeated three times for each developmental stage (total of about 90 embryos per stage). Groups of 25-35 zygotes were placed into 35-mmdiameter culture dishes (Nunc, Roskilde, Denmark) containing 50µL of R1ECM medium under a layer of mineral oil (previously equilibrated to the experimental conditions) and cultured at 37°C under 5% CO 2 in air. This medium was shown by Miyoshi et al. [16] to enable rat embryo culture to the blastocyst stage. In a comparison of various media at our laboratory, R1ECM was found to be the best medium to enable a synchronous development of embryos (95% of total) to the blastocyst stage. The developing embryos seemed to be normal in their morphology, with almost no fragmentation. At the end of incubation, embryos were washed 3 times with fresh R1ECM.

Embryo immunocytochemistry
The method used was basically that of Dubey et al. [17] with various modifications. Groups of 20-25 embryos at different developmental stages from the experimental and control groups were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) at room temperature and washed twice in PBS, PH 7.4 for 5 minutes. Five percent bovine serum albumin (BSA) in PBS was used for dilution of antibodies and washings (PBS-BSA). The embryos were washed four times in PBS-BSA before immunoreaction. Embryos, randomly chosen were either exposed to polyclonal rabbit anti rodent uPA or rabbit anti rat tPA (American Diagnostics, Pendelton, IN) at a concentration of 4µg/mL. Embryos were then incubated overnight in 50µL of each antibody solution under paraffin oil in a 35 mm culture plate in a moist chamber at 4°C. The embryos were then washed four times in PBS-BSA and incubated with Cy3-conjugated goat anti-rabbit IgG (Jackson Immu-noResearch Laboratories, Inc., West Grove, PA) at 37°C for 60 minutes. The conjugated antibody was used at a dilution of 1:300 in PBS-BSA. After incubation with the secondary antibody, the embryos were washed again in PBS-BSA and stained with DNA stain 4', 6-diamidino-2phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and mounted in Flouromount -G (Southern Biotechnology Associates, Inc. Birmingham, AL) to minimize quenching. To confirm that the fluorescence observed was neither attributable to nonspecific binding of the secondary antibody nor to formaldehyde-induced autofluorescence, negative controls (without primary antibody) were established during each immunoreaction procedure. The immunocytochemistry staining procedure was repeated three times for each stage of embryo development on different batches of embryos.

Image Analysis
The distribution and concentration of PAs in the embryos were visualized by fluorescent microscopy on a Zeiss laser scanning confocal microscope equipped with an X100 objective. Z-sections and XZ-sections were obtained from 3D scanning by using LSM510 software (Zeiss, Feldbach, Switzerland) The PAs density for each embryo was computed by image analysis based on the same principles as manual counting described elsewhere [17]. The embryos' fluorescent images were downloaded using an image analysis software, ImagJ (NIH, Bethesda, MD). These images were stored in the computer by use of pixels. All the slices obtained from 3D scanning were of 0.7µ and were analyzed by counting the number of pixels of the Cy3 (red color) in the whole slice. Total pixels in a whole embryo were calculated by summing the number of pixels in all the slices of an embryo. This method showed the total amount of PAs expression in each embryo. The number of pixels in an embryo represents the intensity of PAs staining (fluorescence), which is in turn proportional to the amount of PAs in that embryo. Each experimental group consisted of 8-10 embryos and the measurements repeated three times with different batches of embryos from each developmental stage stained at different times (total number of 24-30 embryos per stage).

Statistical analysis
Data are expressed as means ± SEM. Statistical analysis was performed with two-way analysis of variance, followed by the least significant differences test for multiple comparisons using computer software (Statistica 6.0, Statsoft, Inc. Tulsa, OK). P < 0.05 was defined as statistically significant difference.

PAs localization
Immunohistochemical staining for the location of tPA and uPA in preimplantation embryos developed in vivo and in vitro are shown in figure 1. The PAs were detected in all stages of embryo development, both in in vivo and in vitro (Fig. 1A-V). The uPA was expressed in the cell cytoplasm and plasma membrane (Fig. 1A-K) while tPA was detected on the cell membrane and in the perivitelline space (Fig. 1L-V). In blastocysts developed in vivo and in vitro PAs were localized mainly in the trophectoderm (Fig. 1F, K, Q, V). There was no difference in PAs localization comparing in vivo and in vitro developed embryos at the same stage.

Quantitative measurement of uPA
Quantitative measurement of total uPA in an embryo at each stage showed significantly lower expression (p < 0.01) in in vitro developed embryos from the 4-cell stage up to the blastocyst stage compared with the in vivo developed corresponding timepoint. The highest expression of  Fig. 3). At the 2-cell stage, morula and blastocyst, a significantly lower expression (p < 0.01) was found in in vitro developed embryos as compared with the in vivo ones.

Discussion
Our study demonstrates that uPA and tPA are expressed throughout all stages of preimplantation development of rat embryos. Zhang et al. [18] reported expression of uPA gene and uPA activity in preimplantation rat embryos developed in vitro and Khamsi et al. [19] reported the presence of mRNA for uPA in human blastocysts. However, information about the expression and immunolocalization of uPA and tPA has not been reported in in vivo nor in in vitro preimplantation developing rat embryos.
Quantitative measurement of uPA Figure 2 Quantitative measurement of uPA. Quantitative (Pixels/Embryo) uPA expression in preimplantation rat embryos developed in vivo and in vitro. Different letters represent statistically significant differences (P < 0.05). The results show localization of immunoreactive uPA in the embryonic cell cytoplasm and plasma membrane in all developmental stages both in vivo and in vitro, while tPA is detected on the cell membrane and in the perivitelline space. In the blastocyst stage, PAs are localized mainly in the trophectoderm. In our previous study [15] we showed that the activity of uPA was higher than that of tPA in the blastocyst. Its presence in the trophectoderm combined with its high activity in this stage, support the assumption that uPA is important for proper implantation. This assumption is supported by the study of Kubo et al. [20] who showed that inhibition of PAs activity prevents the adhesion of mouse embryos to decidual cells grown in vitro. In addition, trophoblast cells grown in vitro showed PAs activity at the time of their penetration into the endometrium in vivo and uPA was the major enzyme secreted from trophectoderm cells with the highest activity on days five to seven of pregnancy [7]. The exact source(s) of the immunoreactive PAs in in vivo developing embryos cannot be identified. The serum, oviduct and endometrium could be contributing sources as Quantitative measurement of tPA Figure 3 Quantitative measurement of tPA. Quantitative (Pixels/Embryo) tPA expression in preimplantation rat embryos developed in vivo and in vitro. Different letters represent statistically significant differences (P < 0.05). suggested in previous studies [10,[21][22][23]. Lack of the these source(s) in in vitro situation may lead to lower implantation ability of embryos as shown earlier [15].
Pro-uPA is synthesized as an inactive single chain that can be stored or secreted. The secreted pro-uPA can be cleaved to produce the two-chain active molecule, uPA, by the aid of limited proteolytic activity of plasmin [24]. The secreted pro-uPA or the active uPA can be found free in cytoplasm and extracellular matrix or bound to a membrane uPA receptor [25]. Whether the uPA identified in this study is the inactive pro-uPA or the active uPA associated with the embryonic cell membrane uPA receptor, is unknown. In our previous work we have shown an increase in uPA activity towards the blastocyst stage in in vivo and in in vitro developing embryos [15]. The results of the present study, showing lower expression of uPA in the blastocyst stage, may suggest a shift of uPA from the inactive form to the active form resulting in an increase of activity despite the reduction in its expression.
High tPA expression was detected at the zygote stage which is in accordance with high tPA activity found in this stage [15]. This is supported by the report of Zhang et al. [18] who showed presence of tPA mRNA in rat oocytes and two-cell embryos. The embryonic genome of rats and mice start to be expressed at the 2-cell stage [26] and the high tPA levels in the zygote are probably due to maternal mRNA expressed and accumulated in the oocyte [27].
The embryonic extracellular matrix is in a continuous turnover during the embryonic development. The 8-cell stage is characterized by structural changes taking place in the embryo during the compaction process. It is therefore very likely that such changes at the 8-cell stage could be associated with increased tPA expression and activity which is known to participate in tissue remodeling [8].
The high increase in tPA expression from the 4-cell stage to the 8-cell stage in in vitro developed embryos suggests de novo synthesis of tPA since there is no extraembryonic tPA source but the embryos in the culture.
Lower expression of uPA was observed in in vitro developed embryos as compared with in vivo ones from the 4cell up to the blastocyst stage while tPA expression was lower only in the morula and blastocyst stages. This could be explained by reduced metabolic activity in the in vitro developed embryos as suggested by Krisher et al. [28]. In addition, in vitro conditions may lead to a slower cell division rate which may result in a blastocyst comprised of fewer cells and decreased ability to hatch from the zona pellucida [28,29]. Carroll et al. [30] showed that the oviduct is also a source of PAs, which could attach to receptors on embryonic cell membrane, and this source is lacking in in vitro developing embryos. It should be noted that any culture media would lack maternal factors, known or yet unknown, which affect embryo development and thus, implantation rate, through their effect on the PA/plasmin system.
Additional studies addressing the regulation of PA/Plasmin system by adding exogenous factors may provide insights into its role in early embryo development and implantation.

Conclusions
The purpose of the study was to determine the relative importance of tPA and uPA in preimplantation embryo development. In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing ones. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs may play a role in the implantation process in rats.

Authors' contributions
EDA participated in the planning of the project, carried out the animal experimentation, immunohistochemistry and the image analysis studies. USM participated in the planning of the project, animal experimentation and participated in preparation of the manuscript. GP participated in preparation of the manuscript. IHV participated in the planning of the project, statistical analysis and in preparation of the manuscript.