Brian R Graziano

Amy Grace Dupage

Alphee Michelot

Dennis Breitsprecher

James B Moseley

Isabelle Sagot

Laurent Blanchoin

Bruce L Goode
email: goode@brandeis.edu

Fred Chang

Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor

Keywords: adenomatous polyposis coli, DAD, diaphanous autoregulatory domain, FH, formin homology, NPF, nucleation-promoting factor, WASp, Wiskott-Aldrich syndrome protein, WH, WASp homology

Formins are a conserved family of actin assembly-promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.

INTRODUCTION

A wide variety of processes in eukaryotic cells are driven by the dynamic assembly and remodeling of filamentous actin networks. One of the critical control points in regulating actin cytoskeleton dynamics in vivo is the nucleation of new filaments. To block spurious actin assembly, cells express a battery of actin monomer-binding proteins (e.g., profilin) and capping proteins, which collectively suppress spontaneous growth of filaments. These physiological barriers to actin assembly are overcome by localized activation of nucleation and elongation factors [START_REF] Chesarone | Actin nucleation and elongation factors: mechanisms and interplay[END_REF][START_REF] Dominguez | Actin filament nucleation and elongation factorsstructure-function relationships[END_REF]. Nucleators, such as the Wiskott-Aldrich syndrome protein (WASp)-Arp2/3 complex, formins, Spire, Cobl, Lmod, JMY, and adenomatous polyposis coli (APC), catalyze the initial formation of filament "seeds" (stable nuclei consisting of multiple actin subunits), whereas elongation factors, such as formins and Ena/VASP, increase the rate of monomer addition at the growing ends of filaments. Some nucleators also depend on cofactors, or nucleation-promoting factors (NPFs), for their activities (e.g., WASp for the Arp2/3 complex). In addition, there is mounting evidence for the direct interactions and synergy between pairs of actin nucleators, including Spire-Cappuccino, Spire-Fmn2, and APC-mDia1 [START_REF] Quinlan | Regulatory interactions between two actin nucleators, Spire and Cappuccino[END_REF][START_REF] Okada | Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1[END_REF][START_REF] Pfender | Spire-type actin nucleators cooperate with formin-2 to drive asymmetric oocyte division[END_REF][START_REF] Vizcarra | Structure and function of the interacting domains of Spire and Fmn-family formins[END_REF][START_REF] Zeth | Molecular basis of actin nucleation factor cooperativity: crystal structure of the Spir-1 KIND/formin-2 FSI complex[END_REF].

Formins are among the most widely expressed actin assemblypromoting factors and play essential roles in diverse cellular processes [START_REF] Goode | Mechanism and function of formins in the control of actin assembly[END_REF]. The C-terminal half of formins contains two conserved domains, formin homology 1 (FH1) and FH2, which directly facilitate actin nucleation and/or elongation. The dimeric FH2 domain is critical for nucleation and remains processively attached to the barbed end of the filament as it elongates, protecting it from capping proteins [START_REF] Pruyne | Role of formins in actin assembly: nucleation and barbed-end association[END_REF][START_REF] Sagot | An actin nucleation mechanism mediated by Bni1 and profilin[END_REF][START_REF] Zigmond | Formin leaky cap allows elongation in the presence of tight capping proteins[END_REF][START_REF] Kovar | Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces[END_REF][START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF][START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF]. The FH1 domain recruits profilin-actin complexes and increases the efficiency of actin subunit insertion at the FH2capped end to accelerate elongation [START_REF] Romero | Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis[END_REF][START_REF] Kovar | Control of the assembly of ATP-and ADP-actin by formins and profilin[END_REF]. Despite these advances, it is still not clear how formins catalyze the nucleation of an actin filament "seed." One earlier study hypothesized that the FH2 stabilizes spontaneously formed actin dimers or trimers [START_REF] Pring | Mechanism of formin-induced nucleation of actin filaments[END_REF]. However, this model does not explain how formins efficiently nucleate actin assembly in vivo, where most actin monomers are bound to other proteins such as profilin, which suppress spontaneous dimer and trimer formation. A more recent study revealed that the formin diaphanous autoregulatory domain (DAD), located C-terminal to the FH2 domain, binds to actin monomers to enhance nucleation activity [START_REF] Gould | The formin DAD domain plays dual roles in autoinhibition and actin nucleation[END_REF]. Furthermore, this and other studies suggest that the FH1 domain may contribute to actin nucleation through its interactions with profilin-actin [START_REF] Li | The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition[END_REF][START_REF] Paul | The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation[END_REF]. Although these mechanisms offer one explanation for how formins may overcome barriers to nucleation in vivo, they leave open the possibility of additional factors that interact with formins being involved in nucleation.

In the budding yeast Saccharomyces cerevisiae, the formin proteins Bni1 and Bnr1 share an overlapping, essential role in assembling actin cables, which serve as polarized tracks for myosin-dependent transport of secretory vesicles and other cargo delivered to the bud tip (Moseley and Goode, 2006). Bud6, an in vivo binding partner of Bni1, localizes to the bud tip and bud neck, and deletion of BUD6 results in a severe loss of actin cable staining [START_REF] Amberg | Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites[END_REF]. Similarly, deletion of BUD6 in Schizosaccharomyces pombe results in diminished staining of actin cables assembled by the formin for3p [START_REF] Feierbach | Regulation of a formin complex by the microtubule plus end protein tea1p[END_REF]. Bud6 also plays important roles in microtubule end capture to orient the mitotic spindle [START_REF] Segal | Kar9p-independent microtubule capture at Bud6p cortical sites primes spindle polarity before bud emergence in Saccharomyces cerevisiae[END_REF] and in maintaining endoplasmic reticulum and nuclear membrane diffusion barriers between mother and daughter cells [START_REF] Luedeke | Septindependent compartmentalization of the endoplasmic reticulum during yeast polarized growth[END_REF][START_REF] Shcheprova | A mechanism for asymmetric segregation of age during yeast budding[END_REF]. Whereas the N-terminal half of Bud6 directs its localization in vivo [START_REF] Jin | The secretory pathway mediates localization of the cell polarity regulator Aip3p/Bud6p[END_REF] and mediates microtubule capture [START_REF] Delgehyr | Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae[END_REF], the C-terminal half of Bud6 (C-Bud6; residues 489-788) binds to both the DAD region of Bni1 and G-actin [START_REF] Evangelista | Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis[END_REF][START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF]. Furthermore, C-Bud6 stimulates Bni1-mediated actin assembly in a concentration-dependent manner [START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF][START_REF] Moseley | Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6[END_REF]. However, these studies left open two critical questions about Bud6. First, they did not distinguish between effects of Bud6 on the nucleation versus elongation phases of actin filament assembly, which is an important mechanistic distinction. By 2006, it was discovered that formins catalyze not only the nucleation phase of actin filament assembly, but also elongation [START_REF] Kovar | Control of the assembly of ATP-and ADP-actin by formins and profilin[END_REF], making it critical to determine which phase of actin assembly is affected by each formin regulator. Profilin promotes the elongation of formin-capped filaments [START_REF] Kovar | Control of the assembly of ATP-and ADP-actin by formins and profilin[END_REF], but the effects of Bud6 on nucleation versus elongation are unclear. Second, the importance of Bud6 effects on formin activity was not tested in vivo. Bud6 is multifunctional (see earlier discussion), and due to the unavailability of separation-of-function bud6 alleles, it has been unclear whether the reduced levels of actin cable staining in bud6Δ cells stems from a loss of Bud6 effects on formins or the loss of another function of Bud6. To address these questions, we dissected the Bud6 mechanism in vitro, using bulk fluorescence and total internal reflection fluorescence (TIRF) analysis, generated separation-of-function bud6 alleles, and analyzed their effects in vivo.

RESULTS

Bud6 strongly enhances actin nucleation by Bni1 without affecting elongation

We first purified and compared C-Bud6 (489-788) effects on C-Bni1 (FH1-FH2-C)-induced actin assembly in the presence and absence of 5 μM profilin (Figure 1, A andB). In an earlier study, we showed that C-Bud6 and low concentrations (0.2 μM) of profilin have additive effects in stimulating C-Bni1 [START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF]. However, profilin is highly abundant in cells, present at concentrations well above monomeric actin, and inhibits nucleation. Thus the conditions used here, in which profilin suppresses spontaneous self-association of actin subunits, provide a more physiologically relevant test of the ability of Bud6 to stimulate Bni1-induced actin assembly. In these assays, we observed that C-Bud6 dramatically increased the rate of actin assembly by C-Bni1 both in the presence and absence of 5 μM profilin (Figure 1, A and B). Furthermore, these stimulatory effects were observed at different concentrations of actin (Figure 1C). As expected, C-Bud6 had no stimulatory effects on actin assembly in the absence of C-Bni1. These results suggest that Bud6 is capable of assisting Bni1 in stimulating actin filament assembly when challenged with a cellular inhibitor of nucleation, profilin.

Because bulk pyrene-actin assembly assays do not distinguish between nucleation and elongation effects on actin assembly, we next used TIRF microscopy to directly compare rates of barbed-end growth of individual actin filaments in the presence of C-Bni1 alone and C-Bni1 plus C-Bud6 or profilin (Figure 2A and Supplemental Movies S2-S4). Note that in these experiments, in which elongation rates were being measured, for the reactions containing C-Bud6 we used a lower concentration of C-Bni1 (1 instead of 2.5 nM) to control against filament crowding, which interferes with measurement of elongation. On the other hand, for the reactions containing profilin we used a higher concentration of C-Bni1 (5 nM) to counteract profilin's suppressive effects on nucleation. Consistent with previously reported effects [START_REF] Kovar | Control of the assembly of ATP-and ADP-actin by formins and profilin[END_REF], profilin increased the rate of elongation of C-Bni1-capped filaments by ∼2.5-fold compared with the actin/profilin control in the absence of C-Bni1 (Figure 2, A-C). By comparison, C-Bud6 showed no significant effect on rate of elongation of C-Bni1-capped filaments either in the presence or absence of profilin (Figure 2B). In a separate set of experiments, we used TIRF to quantify the number of filaments nucleated by C-Bni1 in the presence and absence of C-Bud6 (Figure 2D and red arrows in Figure 2E). This demonstrated that C-Bud6 increases the number of C-Bni1-nucleated filaments by approximately threefold. In these reactions, the spontaneously nucleated filaments (Figure 2E, white arrows), that is, those formed independent of C-Bni1 and C-Bud6, were identified by their slower speeds of elongation and brighter fluorescence intensities (see Materials and Methods).

Taken together, the TIRF data suggest that C-Bud6 stimulates the nucleation but not the elongation phase of Bni1-mediated actin assembly. This is further supported by data from seeded elongation assays (Figure 2F), in which unlabeled filamentous actin (F-actin) seeds were mixed with 0.5 μM pyrene-actin monomers, and C-Bud6 did not change the rate of barbed-end growth either in the presence or absence of C-Bni1. Furthermore, the ability of C-Bud6 to stimulate nucleation rather than elongation by Bni1 was demonstrated in two-step seeded nucleation/elongation assays (Figure 2G). This assay measures the concentration of barbed ends generated and therefore provides an index of nucleation independent of elongation. In the first step of the assay, monomeric actin (2 μM) is assembled in the presence or absence of C-Bni1 ± C-Bud6 (Figure 2G, inset). In the second step, samples of reactions from the first step are removed at 50% polymer mass assembly (dotted line, inset) and added to a second Molecular Biology of the Cell seeds generated by actin alone or actin + C-Bni1 in the first reactions. However, the elongation rate increased markedly upon addition of seeds produced from first reactions containing actin + C-Bni1 + C-Bud6, suggesting that C-Bni1 + C-Bud6 had amplified barbed ends in the first reaction. These results agree with our TIRF data, and together the results indicate that Bud6 promotes Bni1-mediated actin nucleation without significantly affecting rate of elongation.

Separation of C-Bud6 interactions with Bni1 and G-actin

C-Bud6 interacts with both the DAD-containing C-terminus of Bni1 and G-actin [START_REF] Amberg | Defining protein interactions with yeast actin in vivo[END_REF][START_REF] Evangelista | Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis[END_REF][START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF]; however, the lack of bud6 separation-of-function alleles has prevented direct tests of the relative contribution of each interaction to Bud6 activity. Using an alignment of C-Bud6 sequences from divergent fungal homologues, we identified conserved clusters of residues (Figure 3A) and generated five bud6 alleles, mutating a total of 15 conserved residues (alanine substitutions). Two of these alleles (Bud6-3 and Bud6-5) showed partial defects individually in the same assays, so we combined them into a single hybrid allele (Bud6-35) that exhibited stronger biochemical defects. Thus, for all further biochemical analyses discussed later, we compared wild-type C-Bud6 and four mutants (Bud6-1, , each expressed and purified from Escherichia coli (Figure 3B).

We first used bead pull-down assays to compare the abilities of soluble wild-type and mutant C-Bud6 to bind 6His-tagged C-Bni1 immobilized on Ni-NTA beads (Figure 3, C and D). In these assays, C-Bud6-8 displayed wild type-like binding to C-Bni1, C-Bud6-35 failed to interact with C-Bni1, and the two remaining mutants (C-Bud6-1 and C-Bud6-6) showed partial defects in binding.

Next we assessed C-Bud6 interactions with G-actin by comparing the abilities of wild-type and mutant C-Bud6 to inhibit spontaneous polymerization of actin monomers (Figure 4). As previously described [START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF], C-Bud6 binds specifically to G-actin (but not F-actin) and inhibits spontaneous self-association of monomers, suppressing actin polymerization in a concentration-dependent manner (Supplemental Figure S1, A andB). We tested mutant C-Bud6 polypeptides at concentrations of wild-type C-Bud6 that strongly suppress polymerization (4 μM C-Bud6 and 3 μM actin), where mutant effects would be readily apparent. This analysis revealed that Bud6-35 and Bud6-6 have wild type-like interactions with G-actin, whereas Bud6-1 and Bud6-8 show significantly weakened interactions with G-actin (Figure 4).

In summary, we obtained two Bud6 mutants with clear separationof-function defects in vitro (summarized in Figure 5D). Whereas Bud6-35 is defective specifically in binding Bni1 but not G-actin, Bud6-8 is defective specifically in binding G-actin but not Bni1. By comparison, Bud6-1 and Bud6-6 showed partial and/or mixed defects in binding Bni1 and G-actin. Thus Bud6-35 and Bud6-8 provided ideal tools for further dissecting Bud6 mechanism and cellular function.

Interactions of C-Bud6 with both Bni1 and G-actin are required for its NPF activity

We next used the mutants identified earlier to directly test the importance of Bud6 interactions with Bni1 and G-actin for its actin nucleation activity. Rates of Bni1-induced actin assembly were measured in pyrene-actin assays over a range of C-Bud6 concentrations (0-200 nM). Higher concentrations of C-Bud6 were not tested because they sequester free actin monomers and thus nonspecifically inhibit polymerization [START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF]. Wild-type C-Bud6 increased the rate of Bni1-mediated actin assembly in a concentration-dependent manner (Figure 5, A andC), whereas the reaction, which contains 1 μM actin monomers. Thus filaments generated in the first reaction act as "seeds" for polymerization in the second, and the rate of assembly in the second reaction is proportional to the concentration of barbed ends produced in the first reaction. We observed minimal effects in the second reactions upon addition of assembly. Furthermore, it has not been clear whether Bud6 function in actin cable assembly requires its direct interactions with Bni1 and/ or G-actin. To address these issues, we integrated our two separation-of-function alleles, bud6-35 (formin-binding defective) and bud6-8 (actin-binding defective) in haploid strains. Each construct included a 3xHA tag at the C-terminus, allowing verification of expression on immunoblots of whole-cell extracts (Figure 6A). A wildtype BUD6-HA allele was integrated in parallel and used as the control strain in all of the analyses that follow.

We first compared wild-type and mutant haploid strains, alongside an isogenic bud6Δ strain, for defects in cell growth at different temperatures after serial dilution and plating (Figure 6B). As previously reported, bud6Δ cells grew normally at these temperatures [START_REF] Jaquenoud | Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p)[END_REF]. Surprisingly, however, growth defects four mutants of C-Bud6 each showed severely impaired effects (Figure 5, B and C, and Supplemental Figure S2, A andB). The observation that Bud6-35 and Bud6-8 mutants failed to stimulate actin nucleation demonstrates that Bud6 NPF activity depends critically on its interactions with both Bni1 and G-actin. These findings suggest that Bud6 stimulates nucleation by recruiting actin monomers to Bni1.

In vivo mechanism of Bud6 in promoting actin cable assembly

Earlier studies showed that bud6Δ cells have greatly diminished levels of actin cable staining [START_REF] Amberg | Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites[END_REF]; however, until now it has not been possible to test whether this phenotype is due to the specific loss of Bud6 activity in stimulating formin-mediated actin Molecular Biology of the Cell were observed at 34 and 37°C for both the bud6-35 and bud6-8 strains, with bud6-35 showing a slightly stronger growth phenotype. We also compared F-actin organization in fixed wild-type and mutant cells by Alexa 488-phalloidin staining (Figure 6C). In wild-type cells, thicker actin cables filled the mother, and smaller cables were present in the bud but more difficult to detect due to the intense actin patch staining. In contrast, bud6Δ cells displayed a substantial loss of cable staining in the mother, and we rarely detected cables in the bud. These defects in F-actin organization were consistent with previous reports [START_REF] Amberg | Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites[END_REF][START_REF] Delgehyr | Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae[END_REF]. By comparison, bud6-35 and bud6-8 cells showed a stronger loss of actin cable staining and more depolarized actin patches than bud6Δ cells, in agreement with their more severe growth defects. These actin phenotypes were quantified by scoring populations of cells from each strain (Figure 6D), using similar criteria as described previously [START_REF] Delgehyr | Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae[END_REF].

Because of the unexpected nature of this observation-that the point mutations in bud6 cause stronger phenotypes than the gene deletion-we further tested whether bud6-35 and bud6-8 alleles are dominant or recessive in diploids. We generated heterozygous and homozygous diploid strains (BUD6/BUD6, BUD6/bud6Δ, bud6Δ/ bud6Δ, and compared their cell growth (Figure 7A) and actin organization (Figure 7, B andC), scoring the actin phenotypes as before (Figure 7D). This analysis indicated that the bud6-35 and bud6-8 alleles are recessive, as demonstrated by the heterozygotes (BUD6/bud6-35 and BUD6/bud6-8) growing as well as wild-type cells and having actin organization equivalent to BUD6/bud6Δ. On the other hand, the alleles showed some characteristics of dose-responsive toxicity. Specifically, homozygous bud6-35/ bud6-35 and bud6-8/bud6-8 strains showed stronger defects in cell growth and actin pronounced at 37°C (Figure 7D) and revealed a correlation between loss of cable staining and defective cell growth.

DISCUSSION

Bud6 and profilin have distinct functions in formin-mediated actin assembly

Until now, the molecular mechanism and cellular function of Bud6 as an actin assembly-promoting factor were unclear. Our findings provide important new insights. Biochemically, we showed that Bud6 stimulates the nucleation rather than the elongation phase of Bni1mediated actin assembly, defining it as an NPF. In addition, we demonstrated that the NPF activity of Bud6 requires distinct physical interactions with both G-actin and the DAD-containing C-terminus of Bni1. Together, these biochemical observations indicate that Bud6 stimulates filament nucleation by recruiting actin monomers to the DAD region of the formin. In contrast, profilin promotes filament elongation [START_REF] Romero | Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis[END_REF][START_REF] Kovar | Control of the assembly of ATP-and ADP-actin by formins and profilin[END_REF]. Therefore Bud6 and profilin have distinct, complementary mechanistic roles in promoting actin assembly, explaining the mechanistic basis for their previously reported synthetic lethal interaction [START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF]. This view of Bud6 and profilin making distinct contributions to actin assembly is also consistent with structural data. In the profilin-actin cocrystal structure, profilin associates with the "barbed end" of the actin monomer, leaving the pointed end free to associate with the barbed end of an actin filament (Figure 8A). As such, profilin-actin interactions are predicted to sterically interfere with the FH2 domain's ability to engage the barbed ends of actin subunits during nucleation. On the other hand, profilin is not predicted to interfere with elongation, because in the FH2 domain "open" state (when only half of the FH2 dimer is bound to actin subunits at the barbed end), profilin-actin can be readily added to the filament end (Figure 8C). In contrast to profilin, the Bud6-binding footprint on actin (Figure 8A) [START_REF] Amberg | Defining protein interactions with yeast actin in vivo[END_REF] predicts that Bud6 binds to the side and/or pointed end of the actin monomer, making it less likely to sterically interfere with the FH2 during nucleation. This agrees with our observation that Bud6 enhances Bni1dependent nucleation even in the presence of high levels of profilin (Figure 1).

What is the mechanism underlying Bud6 NPF activity? Because each FH2 dimer has two C-terminal extensions with DAD domains [START_REF] Nezami | Crystal structure of a complex between amino and carboxy terminal fragments of mDia1: insights into autoinhibition of diaphanous-related formins[END_REF][START_REF] Otomo | Crystal structure of the formin mDia1 in autoinhibited conformation[END_REF], we postulate that at least two Bud6 molecules bind to each formin (Figure 8C). As such, Bud6 would recruit and position multiple actin monomers in close proximity to each other to facilitate actin self-association. This would occur near the FH2 domain to increase the efficiency of barbed-end capture [START_REF] Otomo | Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain[END_REF]. After nucleation, the recruitment of profilin-actin subunits to the FH1 domain would accelerate elongation at the FH2-capped barbed end. It is interesting that Bud6 binds to Bni1 at a site very near or overlapping with the DAD domain, since we have recently shown that the DAD domain of mDia1 binds to G-actin and enhances nucleation [START_REF] Gould | The formin DAD domain plays dual roles in autoinhibition and actin nucleation[END_REF] and that the DAD-containing C-terminus of Bni1 binds G-actin and may contribute similarly to actin assembly. Future work will be required to determine whether Bud6 and Bni1 DAD can bind simultaneously to G-actin or instead Bud6 "replaces" the DAD, providing a higher-affinity G-actin recruitment site. Regardless of which is the case, our data show that Bud6 strongly stimulates nucleation of actin assembly by C-Bni1 (FH1-FH2-DAD), an effect that requires Bud6 interactions with DAD.

unifying properties of nucleation-promoting factors

Our results point to some interesting and unexpected parallels between Bud6 and WASp. Bud6 and WASp each have no effect on organization than bud6Δ/bud6Δ, bud6Δ/bud6-35, or bud6Δ/bud6-8 strains. The differences in actin organization of bud6-35/bud6-35 and bud6-8/bud6-8 compared with bud6Δ/bud6Δ were even more Molecular Biology of the Cell nucleation in the absence of other factors but have strong NPF activity when bound to their ligands-formins and Arp2/3 complex, respectively. Thus, for each nucleation system (Bud6-Bni1 and WASp-Arp2/3), robust activity requires both an actin monomerbinding protein and a high-affinity filament end-capturing protein. A second similarity between Bud6 and WASp is that they each use WASp homology 2 (WH2) domains to bind G-actin. Our bud6-8 allele, which abolishes G-actin binding, falls within a WH2-like sequence in Bud6 (residues 704-725) (Figure 8B). Similarly, it has been shown that WASp NPF activity depends on its WH2 domain-binding actin [START_REF] Higgs | Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization[END_REF]. WH2 domains are found in a variety of actin regulators [START_REF] Qualmann | New players in actin polymerization-WH2-domain-containing actin nucleators[END_REF] and consist of a short α-helix followed by a cluster of basic residues, with both elements contributing to G-actin binding [START_REF] Dominguez | Actin filament nucleation and elongation factorsstructure-function relationships[END_REF]. bud6-8 contains mutations of several conserved residues within the predicted α-helix (Figure 8C). The utilization of a WH2-like domain by Bud6 may also explain why Bud6 NPF activity is robust in the presence of profilin (Figure 1), since some WH2 domains and profilin can bind simultaneously to G-actin [START_REF] Ferron | Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP[END_REF]. Taken together, our findings draw intriguing links between these two nucleation systems (Arp2/3 and formins), which until now had been viewed as having unrelated mechanisms. Moreover, the observation that Bud6 uses a WH2-like domain to bind G-actin and stimulate nucleation reveals that it has common properties shared by a wide variety of nucleators. WH2related domains have been shown to play a critical role in six of seven eukaryotic actin nucleators (WASp-Arp2/3, Spire, Lmod, Cobl, JMY, and formin-Bud6) and many of their bacterial counterparts (VopF, VopL, and TARP) [START_REF] Dominguez | The beta-thymosin/WH2 fold: multifunctionality and structure[END_REF][START_REF] Qualmann | New players in actin polymerization-WH2-domain-containing actin nucleators[END_REF]. Thus the presence of WH2-like actin monomer-binding domains may be the most conserved feature among actin nucleators.

One important question raised by our observations is, how conserved is the nucleation cofactor strategy illustrated by Bud6-Bni1 in other formins? Recent genetic and/or biochemical studies point to a growing number of formins that directly collaborate with other nucleators or NPFs to promote actin assembly. These include Capu/ Fmn2-Spire [START_REF] Quinlan | Regulatory interactions between two actin nucleators, Spire and Cappuccino[END_REF], mDia1-APC [START_REF] Wen | EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration[END_REF][START_REF] Okada | Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1[END_REF], Daam1-flightless [START_REF] Higashi | Flightless-I (Fli-I) regulates the actin assembly activity of diaphanous-related formins (DRFs) Daam1 and mDia1 in cooperation with active Rho GTPase[END_REF], and now Bni1-Bud6. In addition, there is evidence indicating that Arp2/3 complex and formins collaborate to promote filopodia formation [START_REF] Yang | Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells[END_REF][START_REF] Lee | Self-assembly of filopodia-like structures on supported lipid bilayers[END_REF]. In each of these examples, the formin's processive capping and barbed-end protection activity is paired with an actin monomer-binding nucleator or NPF. This strategy of combining two factors with complementary activities may be highly beneficial in a cellular environment in which there are abundant suppressors of actin nucleation [START_REF] Chesarone | Actin nucleation and elongation factors: mechanisms and interplay[END_REF]. In principle, this scheme also provides cells with tighter control over actin assembly, since each nucleator or NPF can be regulated independently so that a convergence of signal inputs is required for actin assembly [START_REF] Prehoda | Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex[END_REF]. Thus, although Bud6 shares no sequence homology with other formin NPFs outside of having a WH2like domain, it may serve as a yeast functional counterpart. In this regard, it is interesting to note that Bud6 and Spire both interact with the C-terminal tail regions of formins [START_REF] Pechlivanis | Identification of a short Spir interaction sequence at the C-terminal end of formin subgroup proteins[END_REF] In vivo function of Bud6 as an NPF Until now it has not been possible to attribute specific phenotypes of bud6Δ cells (e.g., loss of actin cables and polarity) with the loss of individual activities of Bud6, since Bud6 is a multifunctional protein with roles in actin cable formation, polarized cell growth, capture of astral microtubule plus ends, and maintenance of endoplasmic reticulum and nuclear membrane diffusion barriers. We have overcome this barrier by introducing point mutations into the Cterminal half of Bud6 to produce separation-of-function alleles. These tools allowed us to directly test the in vivo importance of Bud6 interactions with G-actin and formins (and its NPF activity) for actin cable formation and polarized cell growth. Our data show that both of the interactions are critical for Bud6 function in actin cable assembly, strongly supporting the role of Bud6 as an NPF in vivo. These tools may also prove useful in future studies for testing the importance of Bud6 NPF activity in its other cellular functions.

Of interest, bud6-35 and bud6-8 alleles led to more severe defects in actin cable assembly and cell growth than a complete deletion of the BUD6 gene in haploids. These observations suggested that the alleles were dominant or recessive gain-offunction alleles. To distinguish between these two possibilities, we further generated mutant homozygous and heterozygous diploid strains and compared their cell growth and actin organization. This analysis revealed that bud6-35 and bud6-8 are recessive gain-of-function alleles. The alleles were never dominant over a wild-type copy of BUD6, and their toxicity in diploids was only observed when there were two copies of the mutant allele. At this point, it is not clear why the allele toxicity is dose dependent or why the alleles result in a gain of function, that is, they are more harmful to cell growth and actin organization than bud6Δ. One possibility for the latter is that a full deletion of BUD6 leads to changes in the expression levels of other proteins that partially compensate for the loss of Bud6 function. Future work is needed to address this and other possibilities.

Finally, although our data provide new clarity concerning the role of Bud6 in regulating Bni1 activity, they leave the function and mechanism of Bud6 in regulating Bnr1 to be determined. Previous studies showed that purified Bud6 (residues 447-788) binds directly to Bnr1 (residues 757-1374; [START_REF] Kikyo | An FH domain-containing Bnr1p is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae[END_REF], and in vivo the deletion of BUD6 in bni1Δ cells leads to further loss of actin cables and synthetic growth defects [START_REF] Tong | Systematic genetic analysis with ordered arrays of yeast deletion mutants[END_REF][START_REF] Delgehyr | Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae[END_REF]. Together, these observations strongly FIGuRE 6: Effects of bud6 alleles on cell growth and actin organization in haploids. (A) Immunoblot of whole-cell extracts from haploid strains, probed with anti-hemagglutinin and anti-tubulin antibodies (loading control). The control lane is from a wild-type strain expressing Bud6 with no tag. (B) Strains were serial diluted and grown at 25, 34, and 37°C on YEPD plates. (C) The strains were grown to log phase at 25°C, fixed, and stained with Alexa 488-phalloidin. Scale bar, 10 μm. (D) Quantification of F-actin phenotypes after fixation and actin staining as in C. For each strain, >200 budded cells were scored and categorized as follows: 1) Robust cables in mother; cables sometimes visible in bud; polarized patches. 2) Fewer and thinner cables in the mother, sometimes with a disorganized appearance; cables occasionally visible in bud; polarized patches. 3) Very few visible cables in the mother; no cables in bud; polarized patches. 4) No visible cables in the mother or bud; depolarized patches. and both regulate not only formin-mediated actin assembly, but also microtubule dynamics [START_REF] Theurkauf | Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes[END_REF][START_REF] Rosales-Nieves | Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino[END_REF][START_REF] Dahlgaard | Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte[END_REF].

4024 | B. R. Graziano et al.
Molecular Biology of the Cell dependent actin cable assembly in vivo by a mechanism related to that shown here for Bni1. However, functional effects of Bud6 on Bnr1 may require additional factors and/ or posttranslational modifications that are lacking in our purified system. Resolving this mechanism is an important future goal.

MATERIALS AND METHODS

Plasmids and strains

The vectors used in this study for expressing 6His-C-Bni1 (1227-1953) and GST-C-Bud6 (489-788) have been described [START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF][START_REF] Moseley | Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6[END_REF]. The C-Bud6 vector was modified further by sitedirected mutagenesis to generate plasmids used to express and purify Bud6-1, Bud6-3, Bud6-5, Bud6-35, Bud6-6, and Bud6-8 from E. coli. For in vivo mutant analyses, we first integrated an in-frame 3xHA tag (marked with HIS3) at the 3′ end of the BUD6 open reading frame (ORF; [START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF] in a wild-type strain of w303 background (BGY12: MATα;15;112;psi+;GAL+), producing a new strain, BGY1410. Bud6-3xHA was previously shown to complement Bud6 function in vivo [START_REF] Moseley | Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6[END_REF]. Next the wild-type BUD6-3xHA ORF plus 300 base pairs 5′ UTR and 200 base pairs 3′ UTR (including the HIS3 marker) was PCR amplified from genomic DNA and cloned into the pRS315 vector [START_REF] Sikorski | A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae[END_REF] using XhoI and NotI sites. This plasmid was used as a template for site-directed mutagenesis to generate integration vectors for bud6-35 and bud6-8. For integrations, DNA fragments encoding for amino acids 550-788 and the 3xHA tag, along with 200 base pairs 3′ UTR and the marker were amplified from the plasmids and transformed into BGY12, producing BGY1411 (bud6-35-3xHA::HIS3) and BGY1412 (bud6-8-3xHA::HIS3). All plasmids were verified by DNA sequencing, and all integrations were verified by sequencing the PCR-amplified ORFs. The haploid strains generated (BGY1411, BGY1412, and BGY1410) and BGY1413 (bud6Δ::TRP1, isogenic to BGY12) were each genetically crossed to BGY10 (MATa, isogenic to BGY12) to produce these heterozygous diploid strains: BGY1414 (BUD6/BUD6-3xHA::HIS3), BGY1415 (BUD6/ bud6Δ::TRP1), BGY1416 (BUD6/bud6-35-3xHA::HIS3), and BGY1417 (BUD6/bud6-8-3xHA::HIS3). These strains were sporulated to produce the following haploid strains: BGY1418 (MATa, bud6Δ::TRP1), BGY1419 (MATa,, and BGY1420 (MATa,. BGY1418, BGY1419, and BGY1420 were crossed to BGY1413, BGY1411, and BGY1412, respectively, to generate the following homozygous diploid strains: BGY1421 (bud6Δ::TRP1/ bud6Δ::TRP1), BGY1422 (bud6-35-3xHA::HIS3/bud6-35-3xHA::HIS3), and BGY1423 (bud6-8-3xHA::HIS3/bud6-8-3xHA::HIS3). The (D) Quantification of F-actin phenotypes. Cells were grown to log phase at 25°C, then fixed and stained with Alexa 488-phalloidin as in B and C, or shifted to 37°C for an additional 2 h before fixation. For each strain, >200 budded cells were scored and categorized as in Figure 6D.

suggest that Bud6 has some role in promoting Bnr1-dependent actin cable formation. However, our previous biochemical analysis of showed that it had no effect on C-Bnr1 actin assembly activity [START_REF] Moseley | Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6[END_REF]. Thus it waits to be seen whether and how Bud6 directly stimulates Bnr1 activity to promote cable formation. One clue from our data is that the bud6-35 and bud6-8 alleles, which disrupt DAD and G-actin binding, respectively, exhibit far stronger loss-of-actin cable phenotypes than bni1Δ. The specific nature of these alleles suggests that Bud6 promotes Bnr1- Volume 22 November 1,2011 Bud6 promotes actin nucleation in vivo | 4025 heterozygous diploid strains BGY1447 (bud6Δ::TRP1/bud6-35) and BGY1448 (bud6Δ::TRP1/bud6-8) were created by crossing BGY1413 to BGY1419 and BGY1420, respectively.

Protein purification

Rabbit skeletal muscle actin (RMA) and S. cerevisiae profilin were purified as described [START_REF] Spudich | The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin[END_REF][START_REF] Moseley | A conserved mechanism for Bni1-and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin[END_REF], and RMA was labeled with pyrenyliodoacetamide [START_REF] Pollard | Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation[END_REF]. C-Bni1 (1227C-Bni1 ( -1953) ) was expressed in S. cerevisiae strain BJ2168 as a 6His-fusion and purified as described (Moseley et al., 2006) with two minor modifications: expression was induced for 6-8 h, and proteins were gel filtered into HEKG 5 D (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 1 mM EDTA, 50 mM KCl, 5% [vol/vol] glycerol, and 1 mM dithiothreitol [DTT]). C-Bud6 (489-788) was expressed in E. coli strain BL21 DE3, grown to mid-log phase (OD 600 = 1.0) in 1 l of TB (12 g of tryptone, 24 g of yeast extract, 0.4% [vol/vol] glycerol, 17 mM KH 2 PO 4 , 72 mM K 2 HPO 4 ) at 37°C and induced with isopropyl-β-d-thiogalactoside for 4 h. Cells were harvested and frozen at -80°C, then later thawed and resuspended in lysis buffer (50 mM Tris, pH 8.5, 150 mM NaCl, 5 mM EDTA, 1.5% sarkosyl, 5 mM DTT, standard protease inhibitors). A small amount of lysozyme was added to cell suspensions, and they were incubated on ice for 5-10 min. DNase I was added, and lysates were incubated on ice for an additional 5-10 min. Cell lysates were then sonicated and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was harvested and mixed with Triton X-100, final concentration 3.3% (vol/ vol), and then added to 1 ml of glutathione FIGuRE 8: Model for Bud6 function as an NPF. (A) Cocrystal structure of profilin (yellow) bound to G-actin (gray) with the Bud6 binding footprint highlighted (red circle). Surfaces on actin essential for Bud6-actin interactions in two-hybrid assays [START_REF] Amberg | Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites[END_REF] are red; residues that make lesser contributions to the interaction are orange. (B) Alignment of WH2-like sequence in Bud6 homologues and WH2 sequences in other proteins. Yellow, conserved residues; red, residues mutated in bud6-8. (C) Model for Bud6 mechanism. C-Bud6 (red cylinders) uses its WH2-like domain to bind G-actin (gray) and a separate binding site to interact with the Bni1 DAD. This positions actin monomers near the FH2 (blue) to promote nucleation, catalyzing formation of actin dimers that are captured by the FH2. Bud6 dissociates, and elongation proceeds with the FH1-domain recruiting profilin (yellow)-bound actin monomers to accelerate barbed-end growth. (D) Arp2/3 complex (blue) interacts with two molecules of WH2 domain-containing WASp (red cylinders; [START_REF] Padrick | Hierarchical regulation of WASP/WAVE proteins[END_REF], which recruit actin monomers to promote nucleation by the filament end-capturing Arp2/3 complex. Although formins and Arp2/3 complex capture opposite ends of the filament, elongation proceeds in both cases by barbed-end addition.

Molecular Biology of the Cell idly mixing 1 μM actin (10% OG labeled, 0.5% biotinylated) in 1× TIRF buffer and transferring the reaction to a flow cell. Samples were imaged at 5-s intervals on a Nikon-Ti200 inverted microscope equipped with a 150-mW Ar laser (Mellot Griot, Carlsbad, CA), a TIRF objective with NA of 1.49 (Nikon Instruments, New York, NY), and an iXon EMCCD camera (Andor, Belfast, Northern Ireland). During measurements, optimal focus was maintained using the perfect focus system (Nikon Instruments). The pixel size corresponded to 0.27 μm. The time-lapse movies were analyzed with ImageJ (National Institutes of Health, Bethesda, MD). Elongation rates were determined by measuring the length increase of single filaments over time. Fluorescence intensities of filaments were measured using the ImageJ linescan tool. Bni1-generated filaments were distinguished from spontaneously generated filaments by their ∼threefold-faster elongation rates and their 50-70% decrease in fluorescence intensity due to the lower affinity of profilin for OGlabeled actin [START_REF] Kovar | Control of the assembly of ATP-and ADP-actin by formins and profilin[END_REF]. Bni1-elongated filaments were scored over a time course of 300 s, and each experiment was repeated three times.

Bead pull-down assays

Ni-NTA beads were incubated for 2 h with purified 6His-C-Bni1 and then washed twice with HEKD buffer. Binding reactions contained soluble untagged C-Bud6 and immobilized 6His-C-Bni1 (1 μM each) plus BSA (0.35 mg/ml). In control reactions, empty Ni-NTA beads (no Bni1) were mixed with 1 μM C-Bud6 plus 0.35 mg/ml BSA. Reactions were incubated for 10 min at room temperature, and then beads were pelleted and supernatants were harvested. Beads were washed twice with HEKD. Samples of pellets and supernatants were analyzed on SDS-PAGE gels, and Coomassie-stained bands were quantified using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). To determine the relative amount of C-Bud6 depleted from the supernatant, bands corresponding to soluble C-Bud6 in reactions containing Bni1 beads were compared with bands corresponding to soluble C-Bud6 in reactions containing empty beads.

Cell imaging

Yeast strains were grown in YEPD-A (yeast extract, peptone, 2% glucose, 0.15 mM adenine) at 25°C to early log phase and then fixed by incubation with 4% formaldehyde for 45-60 min. Cells were then resuspended in PBS, stained with Alexa 488-phalloidin (Molecular Probes, Eugene, OR), mixed 1:1 with mounting media (9.25 mM pphenylenediamine, 0.02 mg/ml DAPI, 13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na 2 HPO 4 , 0.15 mM KH 2 PO 4 , 90% [vol/vol] glycerol), and imaged on an Axioskop-2 mot plus microscope (Carl Zeiss, Thornwood, NJ) equipped with an ORCA-ER digital CCD camera (Hamamatsu Photonics, Bridgewater, NJ). Images were analyzed using OpenLab software (Improvision, Lexington, MA). All images are single planes from fields of view containing at least five cells. Actin phenotypes were scored for large numbers of cells (n > 200) in each strain.

agarose preswollen in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4). After 4 h of incubation at 4°C, the beads were washed four times with PBS and then twice with HEKD (20 mM HEPES, pH 7.5, 1 mM EDTA, 50 mM KCl, 1 mM DTT). Untagged C-Bud6 was released from beads by digestion with TEV protease for 2 h at room temperature and then aliquoted and snap-frozen in liquid nitrogen.

Pyrene-actin assembly assays

To prepare monomeric actin, pyrene-labeled RMA and gel-filtered unlabeled RMA were centrifuged in parallel for 1 h at 90,000 rpm in a TLA100 rotor (Beckman Coulter, Brea, CA). The upper half of each supernatant was carefully removed, actin concentrations were redetermined, and labeled and unlabeled RMA were mixed 1:19. Assembly reactions were 60 μl of final volume and contained final concentrations of 1-3 μM G-actin (5% pyrene labeled). The actin mixture was converted to Mg-ATP-actin ∼2 min before use, and then 42 μl of actin was mixed with 15 μl of proteins or control buffer, plus 3 μl of 20× initiation mix (40 mM MgCl 2 , 10 mM ATP, 1 M KCl) to initiate polymerization. Pyrene fluorescence was monitored at excitation 365 nm and emission 407 nm at 25°C in a fluorescence spectrophotometer (Photon Technology International, Lawrenceville, NJ) or an Infinite M200 plate reader (Tecan, Männedorf, Switzerland). For seeded elongation assays, 10 μM preformed F-actin was freshly sheared and mixed (0.3 μM final) with 0.5 μM monomeric actin (10% pyrene labeled) and 10 nM C-Bni1 ± 200 nM C-Bud6. Two-step seeded nucleation/elongation reactions were performed similarly (explained further in Results). Because low concentrations of C-Bni1 and/or C-Bud6 from the first reactions are introduced into the second reactions, we performed control second reactions containing the same final concentrations of C-Bni1 and/or C-Bud6 (without actin seeds from the first reactions). We subtracted the minimal levels of actin assembly observed in these control reactions in order to quantify the increase in actin assembly rate by C-Bni1 and C-Bud6 due to amplification of barbed ends in the first reactions (Figure 2E).

TIRF microscopy

For the TIRF analysis shown in Figure 2, A-C, actin was fluorescently labeled with Alexa 488 [START_REF] Isambert | Flexibility of actin filaments derived from thermal fluctuation-sEffect of bound nucleotide, phalloidin, and muscle regulatory proteins[END_REF]. Reactions contained 1.2 μM G-actin (30% labeled) ± 5 nM C-Bni1, ± 3.6 μM yeast profilin, ± 0.2 μM C-Bud6. To induce actin polymerization, reaction mixtures were diluted into freshly prepared fluorescence buffer containing 10 mM imidazole-HCl (pH 7.8), 50 mM KCl, 1 mM MgCl 2 , 100 mM DTT, 3 mg/ml glucose, 20 mg/ml catalase, 100 mg/ml glucose oxidase, and 0.5% methylcellulose. Samples were imaged at 30-s intervals on an Olympus IX-71 inverted microscope equipped with a 60× 1.45 numerical aperture (NA) Plan Apo objective (Olympus; Melville, NY) modified for TIRF illumination as described [START_REF] Amann | Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy[END_REF]. Samples were illuminated with an argon/krypton laser (CVI Melles Griot, Albuquerque, NM) emitting at 488 nm. Images were acquired with a Hamamatsu ORCA-ER camera (Hamamatsu Photonics Deutschland, Herrsching am Ammersee, Germany) running MetaMorph version 6.2r6 software (Universal Imaging, Media, PA).

For the TIRF analysis in Figure 2, E and F, actin was fluorescently labeled with Oregon Green (OG) [START_REF] Kuhn | Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy[END_REF]. Flow cells were first incubated with 20 mM Tris-HCl, pH 8.0, 1% bovine serum albumin (BSA) for 5 min, followed by washing with two chamber volumes 1× TIRF buffer (10 mM imidazole, 50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 10 mM DTT, 15 mM glucose, 20 μg/ml catalase, 100 μg/ml glucose oxidase, and 0.5% methylcellulose [4000 cP], pH 7.4). Reactions were initiated by rap-

FIGuRE 1 :

 1 FIGuRE 1: C-Bud6 strongly enhances Bni1-mediated actin assembly. (A, B) Actin monomers (2 μM, 5% pyrene labeled) were polymerized in the presence of 10 nM C-Bni1 and/or 200 nM C-Bud6, in either the presence (A) or the absence (B) of 5 μM profilin. (C) Rates of actin assembly were measured for reactions as before but in the presence of different concentrations of actin as indicated. Rates of assembly were determined from the slopes of the curves and averaged.

FIGuRE 2 :

 2 FIGuRE 2: C-Bud6 stimulates actin nucleation but not elongation. (A) Time-lapse TIRF microscopy comparing barbedend elongation rates. Reactions contained 1.2 μM monomeric actin, different concentrations of C-Bni1 (top, 2.5 nM; middle, 1 nM; bottom, 5 nM; explained in Results), and 200 nM C-Bud6 or 3.6 μM profilin. Time points indicated. (B) Average rates of elongation (n = 10 filaments). Colored bars correspond to reactions in A. Gray bars are controls. (C) Examples of individual filaments increasing in length over time, color coded as in B. (D) Average rates of elongation measured in seeded elongation assays (error bar for C-Bni1 + C-Bud6 is small). (E) Two-step nucleation/elongation assays. First reactions contained 2 μM actin monomers ± 20 nM C-Bni1 ± 200 nM C-Bud6. Samples were removed at 50% polymerization (dotted line, inset) and used to seed a second reaction containing 1 μM actin monomers (10% pyrene labeled). (F) Quantification of number of Bni1-generated filaments by TIRF. (G) Example images from these experiments, in which Bni1-generated filaments (red arrowhead) are distinguished from spontaneously generated filaments (white arrowhead) due their threefold-faster elongation rates and reduced fluorescent intensities (see Materials and Methods). Bottom, highlighted Bni1-generated filaments (red lines).

FIGuRE 3 :

 3 FIGuRE 3: Wild-type and mutant C-Bud6 interactions with Bni1. (A) Alignment of amino acid sequences in the C-terminal halves of fungal homologues of Bud6. Residues shaded in gray are conserved; those that were mutated to create the bud6 alleles are designated with a blue A. bud6-3 and bud6-5 were combined to produce bud6-35. (B) Coomassie-stained gel of purified

FIGuRE 4 :

 4 FIGuRE 4: Wild-type and mutant C-Bud6 interactions with G-actin. (A) Polymerization of 3 μM monomeric actin (5% pyrene labeled) in the presence of 4 μM wild-type or mutant C-Bud6 polypeptide. (B) Quantification of data as in A. Assembly rates for reactions containing actin + C-Bud6 were normalized to rates for reactions containing actin alone. Rates were determined from the slopes of the curves and averaged from three independent trials. Error bars, SD.

FIGuRE 5 :

 5 FIGuRE 5: Wild-type and mutant C-Bud6 effects on Bni1-mediated actin assembly. (A) Concentration-dependent effects of wild-type C-Bud6 on the assembly of 2 μM monomeric actin (5% pyrene labeled) by 10 nM C-Bni1. (B) Comparison of the effects of 200 nM wild-type or mutant C-Bud6 on actin assembly by 10 nM C-Bni1 as in

FIGuRE 7 :

 7 FIGuRE 7: Effects of bud6 alleles on cell growth and actin organization in diploids. (A) Diploid strains were serial diluted and grown at 25, 34, and 37°C on YEPD plates. (B, C) The strains were grown to log phase at 25°C, fixed, and stained with Alexa 488-phalloidin. Scale bars, 10 μm.(D) Quantification of F-actin phenotypes. Cells were grown to log phase at 25°C, then fixed and stained with Alexa 488-phalloidin as in B and C, or shifted to 37°C for an additional 2 h before fixation. For each strain, >200 budded cells were scored and categorized as in Figure6D.

ACKNOWLEDGMENTS

We thank Christopher Gould for technical and intellectual guidance throughout the work and Daqi Tu and Mike Eck for many helpful discussions and insights. This work was supported by National Institutes of Health Grant GM083137 to B.G.


C Marie-Etancelin

B Basso

S Davail

K Gontier

X Fernandez

Z G Vitezica

D Bastianelli

# M.-D E Baéza

‖ G Bernadet

Guy

J.-M Brun

A Legarra

Genetic parameters of product quality and hepatic metabolism in fattened mule ducks 1

Keywords: crossbred animal, duck, genetic parameter, hepatic metabolism, meat and fatty liver quality

ABSTRACT: Genetic parameters of traits related to hepatic lipid metabolism, carcass composition, and product quality of overfed mule ducks were estimated on both parental lines of this hybrid: the common duck line for the maternal side and the Muscovy line for the paternal side. The originality of the statistical model was to include simultaneously the additive genetic effect of the common ducks and that of the Muscovy ducks, revealing a greater genetic determinism in common than in Muscovy. Plasma metabolic indicators (glucose, triglyceride, and cholesterol contents) were heritable, in particular at the end of the overfeeding period, and heritabilities increased with the overfeeding stage. Carcass composition traits were highly heritable in the common line, with values ranging from 0.15 for liver weight, 0.21 for carcass weight, and 0.25 for abdominal fat weight to 0.32 for breast muscle weight. Heritabilities of technological outputs were greater for the fatty liver (0.19 and 0.08, respectively, on common and Muscovy sides for liver melting rate) than for the pectoralis major muscle (between 0.02 and 0.05 on both parental sides for cooking losses). Fortunately, the processing industry is mainly facing problems in liver quality, such as too high of a melting rate, than in meat quality. The meat quality appraisal criteria (such as texture and cooking losses), usually dependent on pH and the rate of decline of pH, were also very lowly heritable. This study demonstrated that genetic determinism of meat quality and ability of overfeeding is not similar in the common population and in the Muscovy population; traits related to fattening, muscle development, and BW have heritability values from 2 to 4 times greater on the common line than on the Muscovy line, which is relevant for considering different selection strategies.

INTRODUCTION

In France, about 95% of the fatty liver production comes from the mule duck, an infertile hybrid duck from a female common duck (Anas platyrhynchos) and a Muscovy drake (Cairina moschata). So, the genetic improvement of mule performances is done by selecting the parental populations for traits measured on their mule progenies. Therefore, it is necessary to know the genetic parameters (heritabilities and genetic correlations) in the pure breed parental strains for production traits measured in the mule crossbred populations [START_REF] Marie-Etancelin | Genetics and selection of mule ducks in France: A review[END_REF].

Genetic evaluation by BLUP is based on modeling the genetic means and the covariances among individu-als. This is complex in the case of crossbred animals, where founder populations have different variances. One theory to estimate the covariances between crossbred animals was proposed by [START_REF] Lo | Theory of modelling means and covariances in a two-breed population with dominance inheritance[END_REF], and then simplified for a 2-breed terminal cross [START_REF] Lo | Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance[END_REF] and used in swine data by [START_REF] Lutaaya | Genetic parameter estimates from joint evaluation of purebreds and crossbreds in swine using crossbred model[END_REF]. This model combines purebred and crossbred information, correctly accounting for additive and (if considered) dominance effects. In this study, the model of [START_REF] Lo | Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance[END_REF] was used without taking into account the dominance variation for the traits expressed only in the mule ducks.

Duck breeding today is directed mainly toward the fatty liver production, fat meat being a coproduct of fatty liver. As the quantity of product increased, the quality of products became a crucial point. However, meat and liver quality traits remain expensive and difficult to measure on a large number of overfed animals, and there are very few genetic variability studies that have been published in this field. This paper presents relevant genetic parameters of numerous foie gras and magret quality traits and of kinetics of plasma parameters during the overfeeding period, on common and Muscovy lines. The aim was to ascertain the percentage of the original traits determined by genes and also to ascertain the genetic links between these traits.

MATERIALS AND METHODS

All experimental procedures were performed in accordance with the French National Guidelines for the care and use of animals for research purposes (Certificate of Authorisation to Experiment on Living Animals No. 7740, Ministry of Agriculture and Fish Products).

Animals

From 2005 to 2006, 800 male mule ducks were hatched each year in 2 pedigree batches of 400 ducklings. These mule ducks were hybrids between 2 experimental populations: the dams were 382 back-cross common ducks (Anas platyrhynchos) and the sires were 56 Muscovy drakes (Cairina moschata). Pedigrees of these 1,600 male mule ducks are described in Figure 1. Four out of the 7 F 1 families of mule ducks were produced in 2005 and the other 3 in 2006. For each year and hatch (a 3-wk gap between annual hatches), mule ducklings were bred in 8 batches of 50 animals. They were fed ad libitum from 0 to 6 wk of age with a starting diet (2,820 kcal of ME/kg and 17.5% CP) and then feed restricted from 6 to 10 wk of age (230 g/d with a growing diet, 2,850 kcal of ME/kg and 15.5% CP). With the same growing diet, the pre-overfeeding period started at 10 wk of age with 5 d of restriction (200 g/d) and 6 d of gradual increase of feed amount (from 220 to 320 g/d). At 12 wk of age, ducks were overfed for 12 d in 2 successive series of 200 animals, with a gap of 2 d between series started at 80 and 82 d of age, with 2 different crammers. Full-brother mule ducks were equally distributed between the 2 annual hatching series, the 8 breeding batches and the 2 cramming series. During this period, animals were bred in collective cages of 4 or 5 individuals and were overfed twice each day with a mix of 35% corn-flour, 25% corn-grain, and 40% water; the average feed amount ingested by animal and meal varied from 410 to 825 g. At the end of the overfeeding period, animals were slaughtered at 92 and 94 d of age, respectively. The animals were bled after electronarcosis and plucked. The carcasses were refrigerated 24 h at 4°C. Then, they were eviscerated: fatty liver, breast muscles, legs, and abdominal fat were removed.

Traits

Three groups of traits were recorded on all these 1,600 mule ducks, including plasma metabolites traits during overfeeding period with a kinetic approach, overfeeding ability traits by weighing carcass pieces, and quality traits of fatty liver and fattened breast muscle.

Plasma Metabolite Traits

Five milliliters of blood was collected by puncture in the duck occipital venous sinus. Blood was collected in a vacuum tube added with EDTA (0.8 g/L) and sodium azide (0.1 g/L). Plasma samples were immediately separated by centrifugation at 3,000 × g for 10 min at 5°C, and frozen at -20°C until further biochemical analyses. Blood samples for measurement of plasma metabolite quantities were taken 3 h after the meal, 3 times during the overfeeding period, at the beginning (after the second meal), at the middle (after the 10th meal) and at the end (after the 20th meal) of the overfeeding period. Plasma triglycerides and total cholesterol were quantified by colorimetric enzymatic methods using kits provided by Bio-Mérieux (Marcy-l'Etoile, France) according to [START_REF] Fossati | Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide[END_REF] and [START_REF] Richmond | Preparation and properties of a cholesterol oxydase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum[END_REF]. Plasma glucose was quantified by the colorimetric enzymatic methods of [START_REF] Trinder | Determination of blood glucose using 4-amino phenazone as oxygen acceptor[END_REF] using a kit provided by Sobioda (Montbonnot St Martin, France). These data were used as markers of liver metabolism for glucose and lipids and bird nutritional status.

Overfeeding Ability Traits

The overfeeding ability of the ducks was measured by weighing the animals at the beginning and at the end of the overfeeding period. The individual feed consumption was also estimated during the period, by recording for each of the 25 overfeeding meals if the crammer gave to ducks the whole dose, one-half the dose, or no dose. After slaughtering, the carcass, the fatty liver, the thighs + shanks, the abdominal fat, the pectoralis major (PM) muscle, and the skin with subcutaneous fat covering the PM muscle were weighed for each animal. The PM muscles and PM skin + subcutaneous fat as a whole constitute the magret, a typical duck meat cut.

Quality Traits of Products

Quality traits of fatty liver and fattened breast muscle were measured. Color descriptives L* (lightness), a* (redness), and b* (yellowness) of the liver and the PM muscle were recorded with a Chroma Meter (CR 300 Minolta, Minolta Corporation USA, Ramsey, NJ). Lipid content and protein content (only for the liver) were estimated by near-infrared spectroscopy on a NIR-System 6500 spectrophotometer (FOSS NIRSystems, Silver Spring, MD) on samples of ground muscle and liver. The technological properties of fatty liver and magret were measured by a cooking test: the liver melting rate (percentage of fat release after sterilization of 60 g of liver for 50 min at 105°C) and the muscle cooking losses (15 min at 85°C in a water bath). For muscle, temperature and pH at 20 min and 24 h postmortem were measured in the PM muscle. Drip losses of PM muscles wrapped under a plastic film were measured after 6 d storage at 6°C. Last, tenderness of raw meat was measured with the Warner-Bratzler test; from the force deformation curve, maximal shear force and energy at the maximum were obtained. For liver, the total quantity of collagen content was determined according to [START_REF] Woessner | The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid[END_REF].

Statistical Analysis

Preliminary least squares analyses were performed using the GLM procedure (SAS Inst. Inc., Cary, NC) to determine the fixed effects which should be taken into account in the following analyses. The model for all traits included the fixed effect of year, batch, and crammer, and the additive breeding value for all animals as a random effect. Genetic parameters were estimated combining pedigree information from both parental populations (common and Muscovy) and from mule duck performances [START_REF] Lo | Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance[END_REF], with the following model:

y X b Z Z u u e AC BC AC BC C C C C ( ) = ( )( ) + ( ) ae è ç ç ç ç ç ö ø ÷ ÷ ÷ ÷ ÷ + ( ) ,
where y C is a vector of observations for crossbreds (mule ducks); b C is a vector of fixed effects corresponding to the combination of year, batch, and crammer effects (12 levels); u AC (u BC ) is the additive genetic effects of the common dams (Muscovy sires) of mule ducks, as expressed in the phenotypes of the mule ducks (population C); e C is a vector of residual effects; X and Z are incidence matrices relating records corresponding effects. We stress that e C contains the remaining genetic effects of crossbred animals (i.e., the Mendelian sampling), as well as the "true" residual environmental effects. In short, the model substitutes the genetic effect of the crossbred animals with the genetic effect of their parents, plus a Mendelian sampling that is confounded with the residual. Pedigrees were traced back up to 5 generations of ancestors on both parental lines and consisted on 596 animals in the common line and 201 animals in the Muscovy line. Only additive genetic effects were considered because to obtain accurate estimates for dominance variation large families, as well as purebreed phenotypes, are required. Also, because all animals with phenotype are mule ducks, average dominance effects are the same for all animals and need not be modeled explicitly. Multiple trait genetic parameters estimations were performed by REML using the program remlf90 [START_REF] Misztal | Complex models, more data: Simpler programming[END_REF]. Standard errors of these estimations were computed by Gibbs sampling using the program gibbsf90 [START_REF] Misztal | Complex models, more data: Simpler programming[END_REF]. A total chain length of 100,000 iterations was run, and 20,000 samples were discarded as burn-in. In both cases, a full multiple-trait analysis was run.

RESULTS

Zootechnic Results

Tables 1,2, and 3 show summary statistics for the 3 groups of studied traits, and the level of significance for year, batch, and crammer. Plasma contents of glucose, cholesterol, and triglyceride increased (P < 0.0001) with overfeeding progress. Glucose and cholesterol increase was about 40% from the 2nd to the 20th meal, whereas triglyceride content was more stable with an increase of 15% during the same period. For these 3 blood variables, the variability of measurements (CV ranging from 16 to 28% on average) increased with overfeeding stage. For almost all plasma traits, the level of significance of the 3 fixed effects was less than 0.001.

Despite a low carcass weight of 4.9 kg, due to their dam line, mule ducks produced a fatty liver of about 570 g and a magret of 408 g (37% of the weight corresponded to the skin). Phenotypically, the carcass weight was more strongly related to the thigh + shank weight (+0.66) than to the liver weight (+0.43). Otherwise, the abdominal fat weight was logically linked to the PM skin and fat weight (+0.49). Among these overfeeding traits, the crammer effect was very high for the carcass weight and the fatty liver weight.

Concerning foie gras quality, the melting rate, with a mean value of 38.7%, had a particularly high variability (CV of 32%). The liver lipid content was logically high (52% on average) and not very variable, whereas the liver protein content was very low (8%) but quite variable. The collagen content in the liver was low (about 1.27 mg/g). We showed that melting rate was more correlated with liver protein and lipid contents (-0.80 and +0.76, respectively), than with the liver weight (+0.65), and not much linked with the collagen content (-0.27). The liver lipid and protein contents seemed also to affect on the liver lightness (+0.49 and -0.54, respectively) and redness (-0.37 and +0.40, respectively). The melting rate and the lipid and protein contents in liver were significantly influenced by the crammer effect.

Concerning magret quality, the value of the pH at 24 h postmortem was 5.7, but 20 min postmortem the pH has already a low value of 6.0, suggesting a fast postmortem decline of the duck meat pH. The magret was also characterized by decreased drip losses (1.6%) and quite large cooking losses (22%). The average muscle lipid content was about 4.9% and presented a low variability. A low and positive correlation (+0.34) connected the cooking losses and the shearing force. No clear links could be seen between both pH and other traits describing muscle quality, except a low correlation (-0.24) between pH 20 min postmortem and the shearing force. No syndrome of PSE meat was observed in this experiment. Lastly, the most variable chromatic color traits were the liver redness and the muscle yellowness. The muscle lipid content was significantly correlated with the lightness (+0.43), the redness (+0.38), and logically the yellowness (+0.50) of the muscle. For the liver, the lightness and redness were linked with the protein and lipid content (-0.54 and +0.40, respectively, for protein; +0.49 and -0.37, respectively, for lipid), and to a lesser extent with the melting rate (+0.39 and -0.32, respectively). The crammer effect was highly significant (Table 3) on all meat quality traits except the pH 20. Last, there was no significant correlation between plasmatic contents and overfeeding ability or products quality traits.

Genetic Results

Tables 4, 5, 6, and 7 give the genetic parameters for the 2 parental lines for the plasma traits, the overfeeding ability traits, the liver quality traits, and the muscle quality traits, respectively. Note that the part of vari-Table 1. Means (in g/L), phenotypic SD, and level of significance for fixed effects of triglyceride (TG), cholesterol (CHO), and glucose (GLU) plasma contents after the 2nd, the 10th, and the 20th overfeeding meals (M) ances due to the parental lines showed in the tables (call heritability on common vs. Muscovy lines) does not account for Mendelian sampling variation in the progeny. However, an estimate of a pseudo-heritability in the mule duck can be obtained as follows: let s s 2 be the part of additive variation in the mule ducks due to the sire, s d 2 be the part of additive variation in the mule ducks due to the dam, and s e 2 the remaining variation in the mule duck. This remaining variation contains environmental variation as well as genetic variation due to Mendelian sampling and dominance. In the absence of variation of dominant effects, Mendelian sampling in the progeny would be equal to s s s d 2 2

+ [START_REF] Lo | Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance[END_REF]. Thus, the pseudo-heritability in the mule ducks is equal to

h s d s d e 2 2 2 2 2 2 2 = + ( ) + + ( ) s s s s s
, which is twice the sum of the elements in the diagonal in Tables 4 to 7, or 4 times the average of the 2 parts of variance. Nevertheless, this pseudo-heritability in the mule duck has no meaning because of the sterility of the hybrid.

Plasma glucose, triglyceride, and cholesterol contents that were measured at the 2nd, 10th, and 20th meals have low to moderate heritabilities. For each of these 3 plasmatic traits, the greatest heritability was obtained at the 20th meal; the heritabilities on the 2nd meal were always less than 0.06. Whatever the plasmatic trait considered, the estimates obtained on the paternal side of the mule duck were definitely less than those estimated on the common maternal side. Likewise, the BW had greater heritabilities on the common maternal side than on the Muscovy paternal side. On the common side, the heritability of BW was smaller at the end of the overfeeding period than at the beginning, despite a strong genetic correlation of +0.95 between the traits at both stages, which is in accordance with the small heritabilities of the BW gain and of the daily feed intake.

After slaughter, the heritabilities of carcass weight and of weights of carcass pieces were moderate to high in the common line, ranging from 0.15 to 0.32. The PM muscle weight was more heritable than the PM skin + fat weight, which had a similar heritability to fatty liver weight. The weight of abdominal fat, despite the difficulty of measurement, showed a suggestive heritability. In the Muscovy line, the heritabilities were again low (values ranging from 0.05 to 0.10) with few variations. Regarding genetic correlations, fatty liver weight was slightly opposed to the PM muscle weight in both parental lines. The link between fatty liver weight and PM skin and fat weight was variable according to the parental lines. In addition, PM muscle weight appeared to be linked to the PM skin and fat weight and the thigh + shank weight, in the same direction in the common line and in the opposite direction in the Muscovy line. On both lines, the carcass weight was genetically the same trait (correlations greater than 0.99) as the BW at the end of the overfeeding period. Nevertheless, the carcass weight was genetically linked with the PM muscle weight in the common line and not with the fatty liver weight in the Muscovy line. The carcass weight was more dependent on the fatty liver weight than to the PM muscle weight.

Concerning fatty liver quality, the melting rate had an intermediate heritability on the maternal line and a small one on the paternal line. Nevertheless, the genetic correlation between fatty liver weight and melting rate was high, positive and similar for both parental lines (+0.80). The liver lipid and protein contents have a smaller genetic variability than melting rate, but lipid and protein contents were highly correlated to the melting rate. The liver collagen content had the smallest heritability either on the maternal than on the paternal lines, and was less negatively linked with the fatty liver weight and the melting rate than the protein contents. For the 3 color traits (L*, a*, and b*), heritabilities were moderate on the common line and slightly weaker on the Muscovy line; only the yellowness had a comparable heritability on both parental lines. The link between lightness and yellowness seems to be different according to the parental line.

Regarding the muscle quality, the muscle lipid content had the greatest genetic variability. The 3 color traits had moderate heritabilities with, once again, greater values on the maternal side. The muscle redness (as for the liver yellowness) had similar heritability estimates on both lines. We have shown that muscle lipid content was genetically linked with drip losses (about -0.80) and with muscle yellowness (about +0.88). For other muscle quality traits (ultimate pH, drip losses, cooking losses, and shearing force), whatever the lines studied, heritability estimates were very low, and not different from zero. The parental lines have a different genetic link between the pH 20 min postmortem and the lipid content in the muscle. Likewise the link between muscle tenderness (muscle force maximum by Warner-Bratzler test) and yellowness of the meat was negative in the common line and null in the Muscovy line.

All genetic correlations between plasmatic traits at the end of overfeeding period and overfeeding ability traits (fatty liver weight, abdominal fat weight, PM skin + subcutaneous fat weight, and PM muscle weight) or quality traits (liver melting rate, liver lipid contents, liver protein contents, muscle cooking losses, liver lightness, and muscle lightness) were not different from 0.

DISCUSSION

In our approach, the total genetic variability of mule traits was divided into the paternal and the maternal lines, as done by a "sire-dam" model in a pure population, the sire and the dam accounting each for one-quarter of the additive variance of the trait. The comparison of estimates obtained on each parental line reveals that the values were systematically greater in the common maternal line. This difference of heritability values according to the transmission way had already been highlighted for carcass and muscle weights by [START_REF] Chapuis | How to estimate simultaneously genetic parameters in parental Pekin and Muscovy duck lines using overfed mule ducks performances[END_REF]. It became widespread to all the traits in our study.

We provide 3 hypotheses for this disequilibrium between genetic determinism in parental lines. First, the greater heritability SD from the Muscovy line is simply due to scarcity of data (there were only 56 Muscovy sires against 382 common dams), which could induce that estimates of heritabilities are poor and less accurate. Second, although the common population is a crossbreeding between 2 different common lines, we assumed that these 2 lines formed a single population. Thus, extra between-lines variation due to, for example, fixation of alternative QTL alleles of QTL in each line is included in the heritability. Nevertheless, regarding the results, we hypothesize that our Muscovy population is more genetically homogeneous than our common population. Third, we hypothesize that the greater heritability estimates on the common line result from a maternal effect absorption in the additive genetic effect of the female duck. That is, the heritability is then artificially overestimated on the maternal common line. Nevertheless, [START_REF] Larzul | Meat quality in an intergeneric factorial crossbreeding between Muscovy (Cairina moschata) and Pekin (Anas platyrhyncos) ducks[END_REF] estimated maternal effects on overfeeding ability and meat quality traits with a duck factorial crossbreeding study; only fatty liver weight and overfeeding BW gain presented a favorable Pekin maternal effect of low magnitude, whereas muscle temperature 20 min postmortem and shearing force show a favorable Muscovy maternal effect. However, we have a huge difference of heritabilities between the 2 parental lines for BW at the beginning and at the end of the overfeeding period and [START_REF] Larzul | Meat quality in an intergeneric factorial crossbreeding between Muscovy (Cairina moschata) and Pekin (Anas platyrhyncos) ducks[END_REF] showed no significant maternal effects on both traits. Thus, the hypothesis of a maternal effect bias does not seem adequate for most traits. Moreover, [START_REF] Chapuis | How to estimate simultaneously genetic parameters in parental Pekin and Muscovy duck lines using overfed mule ducks performances[END_REF] showed the same trend of greater heritabilities in the common line than in the Muscovy line, with a large number of data. Thus, we could conclude that it seems more efficient to select mule ducks traits (i.e., overfeeding ability traits or liver and muscle quality traits) on the maternal common line rather than on the paternal Muscovy line.

To our knowledge, these are the first genetic parameters published for some plasma variables during overfeeding: the glucose, triglyceride, and cholesterol contents were heritable, in particular at the end of the overfeeding period, and the level of heritabilities increased with the overfeeding stage. With our data set, the genetic correlations between these metabolic indicators and overfeeding ability traits or product quality traits were not significant, and lead to the conclusion that these metabolic variables are not relevant genetic predictors of overfeeding ability traits or quality of product traits. Nevertheless, because the SE of correlations are large, the study of these links must be deepened.

Our estimate of fatty liver weight heritability of 0.18 on the maternal line was greater than that of [START_REF] Larzul | Genetic parameters for overfed mule duck traits[END_REF] and [START_REF] Chapuis | How to estimate simultaneously genetic parameters in parental Pekin and Muscovy duck lines using overfed mule ducks performances[END_REF], which amounted respectively to 0.10 and 0.06, but strictly conformed to that estimated by [START_REF] Poujardieu | Paramètres génétiques de croissance et de gavage de la cane commune[END_REF]. We confirmed the moderate heritability of the melting rate (0.19), as well as the strong genetic correlation between fatty liver weight and melting rate already estimated at +0.88 by [START_REF] Poujardieu | Paramètres génétiques de croissance et de gavage de la cane commune[END_REF]. Moreover, we showed that this correlation was comparable in both parental lines. The PM muscle weight and, to a lesser extent, the PM skin and fat weight were heritable, but our estimates appeared to be slightly greater than those previously published [START_REF] Poujardieu | Paramètres génétiques de croissance et de gavage de la cane commune[END_REF]. On the common line, our correlation between the fatty liver weight and the PM muscle weight was similar to that published by these same authors. Last, the abdominal fat weight and the thigh + shank weight were proved to be also heritable. Thus, it is thus also possible to genetically improve them.

Among product quality traits, the visual appearance was one of the most important traits. We have shown, for the first time in overfed waterfowl, that the 3 color traits either for the fatty liver or for the PM muscle were heritable on the common line, as the liver yellowness and the muscle redness on the Muscovy line. These original results are strengthened by published estimates on broiler meat (heritabilities ranging from 0.25 to 0.35; [START_REF] Debut | Analyse en Composantes Principales (ACP) de la qualité technologique de la viande de poulet en relation avec le génotype et le stress avant abattage[END_REF] and on turkey meat (heritability slightly less than 0.20, [START_REF] Renand | L'amélioration génétique de la qualité de la viande dans les différentes espèces: Situation actuelle et perspectives à court et moyen terme[END_REF] heritabilities ranging from 0.10 to 0.32, Le [START_REF] Bihan-Duval | Genetic parameters of meat technological quality traits in a grand-parental commercial line of turkey[END_REF].

Finally, we obtained singular results on the PM muscle quality. First of all, muscle lipid contents were highly heritable. It could be interesting to estimate the genetic correlation between muscle lipid contents and meat gustative quality. Otherwise, the pH 20 min or 24 h postmortem of red duck meat was not heritable, which conflicts with the poultry literature in which estimates range from 0.30 to 0.49 [START_REF] Bihan-Duval | Estimation of the genetic parameters of meat characteristics and of their genetic correlations with growth and body composition in an experimental broiler line[END_REF][START_REF] Debut | Analyse en Composantes Principales (ACP) de la qualité technologique de la viande de poulet en relation avec le génotype et le stress avant abattage[END_REF], but is similar to estimates on bovine red meat (0.11 ± 0.05 for pH 24 h; [START_REF] Renand | Genetic parameters of French beef breeds used in crossbreeding for young bull production II-Slaughter performance[END_REF]. The result in duck is in accordance with the genetic determinism of the red meat pH variation. Unlike the white muscle fibers in which energetic metabolism is only glycolytic and strongly related to the pH variation, the red muscle fibers have a more complex metabolism, which could explain the low genetic determinism of the pH postmortem. The meat quality appraisal criteria (such as texture and cooking losses), usually partly dependent on the pH and its rate of decline, were also not very heritable. This aspect remains to be deepened to confirm the original genetic determinism of duck meat.

We outlined some opposing correlations among traits according to the parental lines (carcass weight and fatty liver weight; fatty liver lightness and yellowness; pH 20 min postmortem and muscle lipid contents; tenderness and yellowness of the meat). These results demonstrated that genetic determinism of meat quality aptitude and ability of overfeeding is not similar in the common population and in the Muscovy population. According to the trait, the parental population with the highest heritability must be chosen to perform the more efficient selection.

Conclusions

Based on the [START_REF] Lo | Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance[END_REF] model, this study points out differences in the genetic determinism between mule duck parental lines. Most overfeeding ability traits and fatty liver or muscle quality traits are more heritable in the common line than in the Muscovy line. Thus, except for meat and liver color which have similar heritabilities on common and Muscovy lines, traits related to fattening (fatty liver weight, liver lipid content, PM skin and subcutaneous fat weight, abdominal fat weight), to muscle development (PM muscle weight, thigh and shank weight, and carcass weight) and above all to BW have heritability values from 2 to 4 times greater on the common line than on the Muscovy line. This study also demonstrated that liver quality traits are more heritable than meat quality traits (apart from the muscle lipid contents). Fortunately, the processing industry is mainly facing problems in liver quality, such as too high of a melting rate, than in meat quality. Our experimental design allowed the genetic parameter estimates of physiological indicators of metabolism. If the overfeeding stage is sufficient, these indicators are heritable on the common side, but because they are not correlated with quality traits they cannot be used as quality predictors.

Figure 1 .

 1 Figure 1. Experimental genetic design. BC = backcross.

Table 2 .

 2 Means, phenotypic SD, and level of significance for fixed effects of overfeeding traits

	Item 1	n	Mean	SD	Year	Batch	Crammer
	DFI, g/d	1,498	1,325	74	**	***	*
	BWbeg, g	1,501	3,830	300	ns	ns	**
	BWend, g	1,498	5,805	378	*	**	ns
	BWG, g	1,498	1,974	247	*	***	**
	CW, g	1,474	4,902	329	ns	**	***
	FLW, g	1,476	569.6	113.5	ns	ns	***
	pmMW, g	1,476	256.5	23.6	ns	***	**
	pmSFW, g	1,476	152.4	20.3	*	**	**
	TSW, g	1,476	481.9	45.1	ns	*	ns
	AFW, g	1,476	175.0	28.8	*	ns	*

1 DFI: daily feed intake during overfeeding; BWbeg: BW at beginning of overfeeding period; BWend: BW at end of overfeeding period; BWG: BW gain; CW: bled-plucked carcass weight; FLW: fatty liver weight; pmMW: pectoralis major muscle weight; pmSFW: pectoralis major skin + subcutaneous fat weight; TSW: thigh + shank weight; AFW: abdominal fat weight.

***P < 0.001; **P < 0.01; *P < 0.05; ns: not significant.

Table 3 .

 3 Means, phenotypic SD, and level of significance for fixed effects of liver and magret quality traits

	Item 1	n	Mean	SD	Year	Batch	Crammer
	MR, %	1,472	38.7	12.4	**	***	***
	LLipC, %	1,476	52.4	4.3	*	ns	***
	LProtC, %	1,476	7.7	1.1	***	ns	***
	LColC, mg/g	1,436	1.27	0.27	ns	*	ns
	LL*	1,476	72.4	2.36	*	ns	*
	La*	1,476	9.17	1.77	***	*	*
	Lb*	1,476	31.2	2.9	***	ns	ns
	MExu, %	1,462	1.58	0.84	ns	ns	*
	MCookL, %	1,437	22.10	3.84	***	***	***
	MLipC, %	1,476	4.93	0.70	***	**	*
	MpH20	1,476	6.01	0.18	ns	ns	ns
	MpHu	1,476	5.72	0.14	***	***	***
	MFmax, n	1,443	42.49	7.65	***	ns	***
	ML*	1,476	47.3	3.4	***	ns	***
	Ma*	1,476	20.4	2.5	***	**	*
	Mb*	1,475	7.62	1.46	***	***	***
	1 MR: liver melting rate; LLipC: liver lipid contents; LProtC: liver protein contents; LColC: liver collagen
	contents; LL*: liver lightness, La*: liver redness; Lb*: liver yellowness; MExu: muscle exudation; MCookL:
	muscle cooking losses; MLipC: muscle lipid contents; MpH20: muscle pH 20 min postmortem; MpHu: muscle
	ultimate pH; MFmax: muscle force maximum (Warner-Bratzler test); ML*: muscle lightness; Ma*: muscle red-
	ness; Mb*: muscle yellowness.
	***P < 0.001; **P < 0.01; *P < 0.05; ns: not significant.

Table 4 .

 4 Genetic parameters (heritabilities ± SE on the diagonal, genetic correlations ± SE above and below the diagonal) for triglyceride (TG), cholesterol (CHO), and glucose (GLU) plasma contents after the 2nd, the 10th, and the 20th overfeeding meals (M): common duck line above and Muscovy duck line below the diagonal 1

	Item TG 2nd M TG 10th M TG 20th M CHO 2nd M CHO 10th M CHO 20th M GLU 2nd M GLU 10th M GLU 20th M	TG 2nd M 0.05 ± 0.02 +0.66 ± 0.21 +0.76 ± 0.18 -0.12 ± 0.37 +0.07 ± 0.31 +0.33 ± 0.27 +0.40 ± 0.32 +0.61 ± 0.53 +0.60 ± 0.24	0.04 ± 0.02	TG 10th M +0.38 ± 0.44 0.11 ± 0.03 +0.86 ± 0.11 +0.28 ± 0.33 +0.35 ± 0.19 +0.41 ± 0.23 +0.37 ± 0.31 +0.77 ± 0.24 +0.76 ± 0.13	0.03 ± 0.02	TG 20th M +0.91 ± 0.17 +0.29 ± 0.51 0.13 ± 0.03 +0.15 ± 0.24 +0.28 ± 0.23 +0.48 ± 0.18 +0.16 ± 0.41 +0.62 ± 0.34 +0.85 ± 0.14	0.03 ± 0.03	CHO 2nd M +0.21 ± 0.33 -0.18 ± 0.37 +0.21 ± 0.37 0.06 ± 0.03 +0.67 ± 0.22 +0.77 ± 0.16 -0.43 ± 0.30 +0.18 ± 0.54 +0.22 ± 0.23	0.05 ± 0.02	CHO 10th M -0.25 ± 0.31 -0.39 ± 0.33 -0.16 ± 0.39 +0.87 ± 0.11 0.11 ± 0.03 +0.81 ± 0.14 -0.08 ± 0.30 +0.53 ± 0.36 +0.59 ± 0.21	0.05 ± 0.02	CHO 20th M -0.01 ± 0.31 -0.14 ± 0.33 +0.01 ± 0.41 +0.75 ± 0.17 +0.67 ± 0.21 0.12 ± 0.03 -0.31 ± 0.30 +0.42 ± 0.36 +0.59 ± 0.15	0.03 ± 0.02	GLU 2nd M +0.53 ± 0.38 +0.68 ± 0.28 +0.63 ± 0.24 -0.01 ± 0.34 -0.17 ± 0.42 -0.02 ± 0.44 0.04 ± 0.02 +0.59 ± 0.32 +0.28 ± 0.32	0.02 ± 0.02	GLU 10th M +0.07 ± 0.29 -0.26 ± 0.45 +0.26 ± 0.28 -0.02 ± 0.36 +0.09 ± 0.30 -0.35 ± 0.30 +0.01 ± 0.39 0.01 ± 0.01 +0.75 ± 0.48	0.04 ± 0.02	GLU 20th M -0.24 ± 0.39 -0.44 ± 0.39 -0.01 ± 0.35 -0.29 ± 0.29 -0.03 ± 0.30 -0.39 ± 0.29 -0.13 ± 0.62 +0.89 ± 0.11 0.12 ± 0.04	0.06 ± 0.03	1 TG 2nd M: triglyceride contents after 2nd meal; TG 10th M: triglyceride contents after 10th meal; TG 20th M: triglyceride contents after 20th meal; CHO 2nd M: cholesterol contents after 2nd	meal; CHO 10th M: cholesterol contents after 10th meal; CHO 20th M: cholesterol contents after 20th meal; GLU 2nd M: glucose contents after 2nd meal; GLU 10th M: glucose contents after 10th	meal; GLU 20th M: glucose contents after 20th meal.

Table 5 .

 5 Genetic parameters (heritabilities ± SE on the diagonal, genetic correlations ± SE above and below the diagonal) for overfeeding ability traits: common duck line above and Muscovy duck line below the diagonal 1

	AFW	+0.21 ± 0.29		+0.40 ± 0.09		+0.44 ± 0.10		+0.15 ± 0.16		+0.49 ± 0.09		+0.03 ± 0.15	+0.25 ± 0.10	+0.55 ± 0.10	+0.39 ± 0.10	0.25 ± 0.04	0.09 ± 0.04
	TSW	+0.38 ± 0.20		+0.86 ± 0.06		+0.84 ± 0.07		-0.01 ± 0.21		+0.84 ± 0.05		-0.31 ± 0.15	+0.74 ± 0.07	+0.76 ± 0.11	0.20 ± 0.04	0.05 ± 0.03	+0.19 ± 0.29
	pmSFW	+0.51 ± 0.19		+0.59 ± 0.09		+0.58 ± 0.09		+0.01 ± 0.22		+0.60 ± 0.09		-0.29 ± 0.14	+0.48 ± 0.11	0.16 ± 0.03	0.10 ± 0.04	+0.56 ± 0.22	+0.41 ± 0.21
	pmMW	+0.19 ± 0.19		+0.89 ± 0.04		+0.84 ± 0.05		-0.07 ± 0.20		+0.82 ± 0.05		-0.17 ± 0.14	0.32 ± 0.04	0.07 ± 0.04	-0.09 ± 0.30	-0.08 ± 0.38	-0.01 ± 0.30
	FLW	-0.28 ± 0.29		-0.10 ± 0.13		+0.08 ± 0.16		+0.55 ± 0.16		+0.11 ± 0.16		0.15 ± 0.03	0.08 ± 0.03	-0.05 ± 0.25	+0.39 ± 0.23	-0.13 ± 0.27	+0.41 ± 0.24
	CW	+0.13 ± 0.60		+0.93 ± 0.03		+0.99 ± 0.01		+0.28 ± 0.22		0.21 ± 0.04	0.05 ± 0.03	+0.44 ± 0.24	+0.15 ± 0.28	+0.79 ± 0.13	+0.58 ± 0.16	+0.63 ± 0.19
	BWG	-0.39 ± 0.28		-0.09 ± 0.19		+0.24 ± 0.17		0.06 ± 0.03	0.04 ± 0.03	+0.65 ± 0.23		+0.67 ± 0.17	-0.09 ± 0.36	+0.39 ± 0.34	+0.31 ± 0.43	+0.22 ± 0.35
	BWend	+0.12 ± 0.22		+0.95 ± 0.03		0.23 ± 0.04	0.05 ± 0.03	+0.65 ± 0.23		+0.99 ± 0.05		+0.47 ± 0.28	+0.14 ± 0.45	+0.82 ± 0.11	+0.59 ± 0.21	+0.60 ± 0.25
	BWbeg	+0.25 ± 0.19		0.34 ± 0.04	0.05 ± 0.03	+0.84 ± 0.11		+0.15 ± 0.38		+0.84 ± 0.16		+0.13 ± 0.35	+0.25 ± 0.46	+0.80 ± 0.14	+0.55 ± 0.29	+0.63 ± 0.24
	DFI	0.06 ± 0.03	0.05 ± 0.02	+0.62 ± 0.29		+0.55 ± 0.31		+0.14 ± 0.34		+0.58 ± 0.45		+0.03 ± 0.29	+0.53 ± 0.23	+0.19 ± 0.30	+0.34 ± 0.20	+0.66 ± 0.33
	Item	DFI		BWbeg		BWend		BWG		CW		FLW	pmMW	pmSFW	TSW	AFW

1 DFI: daily feed intake during overfeeding; BWbeg: BW at beginning of overfeeding period; BWend: BW at end of overfeeding period; BWG: BW gain; CW: bled-plucked carcass weight; FLW: fatty liver weight; pmMW: pectoralis major muscle weight; pmSFW: pectoralis major skin + subcutaneous fat weight; TSW: thigh + shank weight; AFW: abdominal fat weight.

Table 6 .

 6 Genetic parameters (heritabilities ± SE on the diagonal, genetic correlations ± SE above and below the diagonal) for fatty liver quality traits: common duck line above and Muscovy duck line below the diagonal 1

	Item	FLW	MR	LLipC	LProtC	LColC	LL*	La*	Lb*
	FLW	0.18 ± 0.03	+0.80 ± 0.07	+0.86 ± 0.06	-0.93 ± 0.04	-0.66 ± 0.12	+0.68 ± 0.11	-0.69 ± 0.12	-0.27 ± 0.18
		0.09 ± 0.03
	MR	+0.79 ± 0.13	0.19 ± 0.04	+0.87 ± 0.05	-0.86 ± 0.05	-0.56 ± 0.14	+0.48 ± 0.15	-0.61 ± 0.16	-0.06 ± 0.18
			0.08 ± 0.03
	LLipC	+0.79 ± 0.12	+0.93 ± 0.06	0.16 ± 0.03	-0.96 ± 0.03	-0.68 ± 0.15	+0.50 ± 0.16	-0.61 ± 0.19	-0.05 ± 0.19
				0.06 ± 0.03
	LProtC	-0.81 ± 0.10	-0.89 ± 0.08	-0.89 ± 0.08	0.17 ± 0.03	+0.78 ± 0.16	-0.66 ± 0.11	+0.69 ± 0.15	+0.15 ± 0.18
					0.09 ± 0.03
	LColC	-0.77 ± 0.20	-0.51 ± 0.28	-0.68 ± 0.36	+0.52 ± 0.55	0.09 ± 0.03	-0.71 ± 0.17	+0.73 ± 0.21	-0.03 ± 0.28
						0.02 ± 0.02
	LL*	+0.80 ± 0.13	+0.52 ± 0.20	+0.52 ± 0.26	-0.61 ± 0.16	-0.60 ± 0.39	0.11 ± 0.03	-0.87 ± 0.09	-0.49 ± 0.17
							0.05 ± 0.03
	La*	-0.64 ± 0.16	-0.35 ± 0.24	-0.45 ± 0.24	+0.47 ± 0.22	+0.76 ± 0.21	-0.77 ± 0.14	0.10 ± 0.03	+0.35 ± 0.22
								0.06 ± 0.03
	Lb*	-0.19 ± 0.23	-0.18 ± 0.24	-0.22 ± 0.24	+0.43 ± 0.21	+0.05 ± 0.55	+0.01 ± 0.25	-0.01 ± 0.26	0.13 ± 0.04
									0.12 ± 0.04
	1 FLW: fatty liver weight; MR: liver melting rate; LLipC: liver lipid contents; LProtC: liver protein contents; LColC: liver collagen contents; LL*:
	liver lightness, La*: liver redness; Lb*: liver yellowness.

This work was supported by the French National Research Agency GENANIMAL, by the Scientific Group of avian genome analysis AGENAVI, and the Midi-Pyrénées and Aquitaine regional councils.

2 Corresponding author: Christel.Marie-Etancelin@toulouse.inra. fr Received April 16, 2010. Accepted October 26, 2010.

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Genetic variability of meat quality in duck

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Genetic variability of meat quality in duck