Krzysztof Rogowski

François Juge

Juliette Van Dijk

Dorota Wloga

Jean-Marc Strub

Nicolette Levilliers

Daniel Thomas

Marie-Hélène Bré

Alain Van Dorsselaer

Jacek Gaertig

Carsten Janke
email: carsten.janke@crbm.cnrs.fr

Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation

Keywords: Tubulin, microtubules, glycylation, axoneme, flagella Summary

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Polyglycylation is a posttranslational modification that generates side chains of glycine on the -carboxyl groups of specific glutamate residues of target proteins (Bré et al., 1998;[START_REF] Vinh | Structural characterization by tandem mass spectrometry of the posttranslational polyglycylation of tubulin[END_REF]. Similarly to polyglutamylation [START_REF] Eddé | Posttranslational glutamylation of alpha-tubulin[END_REF], this posttranslational modification was discovered on tubulin, where it occurs within the carboxy-terminal tail domains [START_REF] Redeker | Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules[END_REF]. While polyglutamylation was observed on many different types of microtubules (MTs), polyglycylation is particularly prominent in cilia and flagella and is mostly found in cells that possess these organelles [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF][START_REF] Levilliers | Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin[END_REF].

Functional experiments on tubulin glycylation were difficult to perform since the modifying enzymes were unknown. Initial insights into the potential function of polyglycylation were obtained using modification-specific antibodies. For example, anti-glycylation antibodies affected axoneme beating in vitro, suggesting that tubulin polyglycylation could regulate ciliary dynein [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF]. In Tetrahymena, the elimination of the major glycylation sites on -tubulin was lethal or led to severe axonemal defects, including short axoneme size, absence of the central pair MTs, and incomplete outer doublet MTs [START_REF] Redeker | Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha-and beta-tubulin and affect the ciliary matrix in Tetrahymena[END_REF][START_REF] Thazhath | Polyglycylation domain of beta-tubulin maintains axonemal architecture and affects cytokinesis in Tetrahymena[END_REF][START_REF] Xia | Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila[END_REF]. Thus, tubulin glycylation could be an important regulator of both the assembly and functions of axonemal MTs. The recent observations that non-tubulin proteins are also substrates of polyglycylation [START_REF] Ikegami | TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1[END_REF][START_REF] Lalle | The Giardia duodenalis 14-3-3 Protein Is Post-translationally Modified by Phosphorylation and Polyglycylation of the C-terminal Tail[END_REF]Xie et al., 2007) indicate that glycylation is a posttranslational modification of general importance.

Here we describe the discovery of a group of enzymes that catalyze tubulin glycylation and show that some of these enzymes have non-tubulin substrates. We demonstrate that glycylases are members of the tubulin tyrosine ligase-like (TTLL) family, which already contains three types of known amino acid ligases, the tubulin tyrosine ligase (TTL; [START_REF] Ersfeld | Characterization of the tubulin-tyrosine ligase[END_REF], polyglutamylases [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF][START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF][START_REF] Wloga | Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila[END_REF] and an enzyme that ligates glycine to nucleosome assembly protein 1 (NAP1; [START_REF] Ikegami | TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1[END_REF]. We show that in mouse, TTLL3 and TTLL8 proteins are initiating glycylases with different substrate specificities, whereas TTLL10 is the elongating polyglycylase. Cooperation of these two types of enzymes generates polyglycine side chains in mammals, whereas in Drosophila, bifunctional enzymes catalyze both reactions. We also demonstrate that the previously reported absence of long glycine side chains on human sperm tubulin [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF] is caused by two inactivating amino acid substitutions in human TTLL10. Finally, we show by RNA interference (RNAi) that depletion of one of the two glycylases in Drosophila leads to strong reduction in polyglycylation, which causes defects in sperm maturation and results in male sterility. Moreover, expression of interfering RNA under a stronger promoter is lethal in early developmental stages. This suggests that polyglycylation is important for whole-organism development in Drosophila. The identification of new substrates of glycylation indicates that this modification could have a broad range of functions.

Results

Identification of murine polyglycylases

Our study was initiated with the hypothesis that tubulin glycylases, like two other types of tubulin amino acid ligases, TTL [START_REF] Ersfeld | Characterization of the tubulin-tyrosine ligase[END_REF] and polyglutamylases [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF][START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF], are members of the TTLL protein family.

Within the mammalian TTLL family, TTLL3, 8, 10 and 12 remained uncharacterized.

According to phylogenetic studies, the TTLL12 gene is present in species that appear to lack glycylation [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF][START_REF] Schneider | Subpellicular and flagellar microtubules of Trypanosoma brucei are extensively glutamylated[END_REF], and murine TTLL12 has no glycylation activity in vivo and in vitro (KR, unpublished observations). In contrast, the presence of orthologs of TTLL3, 8 and 10 in genomes of diverse species [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF] correlates with the known presence of tubulin glycylation, and a recent study showed that one of these three genes, TTLL10, encodes a glycylase for NAPs [START_REF] Ikegami | TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1[END_REF].

To test whether mouse TTLL3, 8 and 10 have a tubulin glycylase activity, we expressed the respective cDNAs [START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF] in HEK293 cells and tested the cell extracts in an in vitro MT glycylation assay. To distinguish between two possible types of activities, initiation (addition of the first glycine to a -carboxyl group of the target glutamate residue) and elongation of glycine side chains, we used substrates differing in the levels of tubulin glycylation: MTs polymerized from brain tubulin (that lack detectable glycylation) and strongly glycylated MTs of ciliary axonemes of Tetrahymena thermophila. Both, TTLL3 and 8 promoted incorporation of [ 3 H]-glycine into brain MTs, but had very low activity on axonemal MTs. In contrast, TTLL10 was almost inactive on brain MTs, but promoted efficient [ 3 H]-glycine incorporation into axonemal MTs (Fig. 1A). These observations suggest that TTLL3 and 8 are initiating glycylases, whereas TTLL10 is an elongating glycylase (polyglycylase) for tubulin. A similar functional specialization has been previously observed among polyglutamylases [START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF][START_REF] Wloga | Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila[END_REF].

We also examined the selectivity of the enzymes for -and -tubulin. TTLL3 and 8 modified both, -and -tubulin of brain MTs, with TTLL8 showing preference for -tubulin (Fig. 1A). For TTLL10, which modified only axonemal MTs, two-dimensional electrophoresis was used to separate axonemal -and -tubulin. Using anti--or tubulin antibodies as well as TAP952 (an antibody that recognizes monoglycylated sites on tubulins; [START_REF] Bré | Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in Paramecium[END_REF] we found that both tubulin subunits of Tetrahymena axonemes are monoglycylated, and thus are potential substrates for the elongation reaction. However, TTLL10 incorporated [ 3 H]-glycine mainly into -tubulin (Fig. 1B).

Next, we expressed TTLL3, 8 and 10 in U2OS cells and labeled the cells with antibodies that are specific to either mono-or polyglycylation: TAP952 and polyG (polyG specifically recognizes polyglycine chains; [START_REF] Duan | Both carboxy-terminal tails of alpha-and betatubulin are essential, but either one will suffice[END_REF], respectively (Fig. 1C). Non-transfected U2OS cells show no reactivity with either of these two antibodies (arrowheads). After expression of TTLL3 or 8, we detected specific labeling of MTs with TAP952, but not with polyG. Thus, TTLL3 and 8 mediate initiation of glycine side chains on MTs in vivo, but not side chain elongation. In contrast, cells expressing TTLL10 alone showed no labeling with TAP952, but had a diffuse labeling with polyG that did not correspond to MTs. This suggests that TTLL10 adds polyglycine chains directly onto proteins that are not associated with MTs, while it cannot modify MTs without prior monoglycylation.

Two-step glycylation mechanism in mammals

To test whether the two types of mammalian glycylases can function in a cooperative manner to create long side chains on MTs, we co-overexpressed either TTLL3 or 8 together with TTLL10 in U2OS cells. In both cases, we observed a strong labeling of MTs with the polyG antibody (Fig. 2A). These results indicate that MTs have acquired polyglycine side chains, which was not the case when TTLL3, 8 or TTLL10 were expressed alone (Fig. 1C).

On immunoblots, the monoglycylation-specific antibody TAP952 detected strong signals in the tubulin region upon expression of TTLL3 or 8 alone. Additional protein bands were labeled after TTLL8 expression, suggesting that TTLL8 can mediate glycylation of other non-tubulin proteins (Fig. 2B). TTLL3 increased glycylation of mostly -tubulin in U2OS cells, while it promoted modification of both -and tubulin in the in vitro assay (Fig. 2B,1A). Thus, it appears that the substrate preferences of the glycylating enzymes change depending on additional factors. Neither TTLL3 nor TTLL8 alone generated polyG-reactive epitopes, indicating that they are indeed restricted to reactions that generate monoglycine side chains (Fig. 2B). In contrast, no TAP952 labeling was detected in extracts of cells expressing TTLL10. However, a strong polyG signal was induced on two distinct protein bands, one of them the size of -tubulin (Fig. 2B). We demonstrate that the substrate in the tubulin region corresponds to NAPs. First, two-dimensional immunoblot analysis shows that the polyglycylated 50 kDa protein band superimposes with NAPs, but not with tubulins (Fig. 2C), and second, ectopically expressed NAP1-EYFP and NAP2-EYFP were strongly polyglycylated by TTLL10 (Fig. S1). This is in agreement with the results of [START_REF] Ikegami | TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1[END_REF] and indicates that in contrast to tubulins, NAPs can be polyglycylated directly by TTLL10.

After co-expression of TTLL3 or 8 with TTLL10, no TAP952 labeling was detected, whereas several protein bands were labeled with the polyG antibody. Virtually all protein bands labeled with TAP952 after expression of TTLL3 or 8 alone became labeled with polyG when TTLL10 was co-expressed (Fig. 2B), suggesting that following monoglycylation by TTLL3 or 8, TTLL10 elongates glycine side chains of all major substrates of the initiating enzymes. The efficiency of this process is illustrated by the reduction of monoglycylated epitopes below detectable levels. This further indicates that the substrate specificity of polyglycylation in mammals is predetermined by the specificity of the initiating glycylases rather than by the elongating enzyme, except for proteins that are directly modified by TTLL10, such as the NAPs.

Loss of elongating activity in humans

Human sperm axonemes, unlike sperm axonemes of several other mammalian species studied so far, have side chains limited to monoglycylation [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF].

We confirmed this by comparing the status of glycylation in protein extracts prepared from mouse, rhesus monkey and human sperm using the TAP952 and the polyG antibody (Fig. 3A). Several mouse and rhesus protein bands were strongly labeled with polyG, while only weak signals were detected with TAP952. In contrast, human sperm proteins were not labeled with polyG, but strongly with TAP952. No differences between the three species were found for the related tubulin modification, polyglutamylation (Fig. 3A, panel polyE).

To determine the molecular basis for the lack of elongated polyglycine chains in human sperm, we investigated the only known elongating glycylase in mammals, TTLL10. First, we confirmed the presence of a full-length mRNA by cloning TTLL10 from human testes cDNA. We then expressed human, mouse and rhesus TTLL10 in U2OS cells. The mouse and rhesus TTLL10 generated polyglycylation that could be specifically detected with polyG, whereas the human protein was inactive (Fig. 3B).

Accordingly, when cell extracts from HEK293 cells overexpressing the respective TTLL10 proteins were used for in vitro assays, only the mouse and rhesus, but not the human TTLL10 mediated incorporation of [ 3 H]-glycine into axonemal MTs (Fig. 3C). Domain-swapping experiments localized the inactivating mutations to the central region of human TTLL10, which corresponds to the highly conserved core TTL domain (Fig. S3, S4A, B;[START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF]. Within this domain, human and rhesus TTLL10 differ by 11 amino acids, out of which 4 are not evolutionarily conserved. Thus, we analyzed the remaining 7 amino acid changes by introducing them separately into the rhesus TTLL10. Two of these mutations led to a complete loss of polyglycylase activity (Fig. S3, S4C), suggesting that the corresponding amino acids are essential for activity.

When these two mutations in human TTLL10 were both reverted to the conserved amino acids found in rhesus and mouse, the enzyme recovered polyglycylase activity (Fig. 3D,E). This was not the case if only one mutation was reverted. As neither of the two mutations has been found in the genomes of the great apes (not shown), it appears that humans are unique in having lost the polyglycylation activity of TTLL10. One of the two inactivating mutations (mut3) is polymorphic in the human population, leading to a high incidence (55.0% heterozygote and 16.7% homozygote) of TTLL10 alleles without mut3 in the sub-Saharan African population, whereas 91.7% of the European population are homozygote for the presence of mut3 (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=1320571). No polymorphism was found for mut5. To investigate if the mut3 polymorphism could affect the enzymatic activity of TTLL10, we compared polyglycylation of sperm proteins from two homozygote individuals either with or without mut3, but both bearing mut5 (Fig. S5A).

The complete absence of polyglycylation on sperm proteins from both individuals (Fig. S5B) confirmed that the presence of one of the two inactivating mutations is sufficient for the complete loss of enzymatic activity of human TTLL10 (Fig. S4C).

In conclusion, we show that the human elongating polyglycylase TTLL10 is functionally inactive due to two mutations, resulting in the absence of polyglycylation in sperm. Considering that all known amino acid ligases that modify tubulin are members of the TTLL family, and that all TTLLs are expressed in testes [START_REF] Ersfeld | Characterization of the tubulin-tyrosine ligase[END_REF][START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF], it is likely that TTLL10 is the only polyglycylating enzyme in mammals. Thus, humans could be a unique mammalian species that lacks protein polyglycylation.

Cancer-associated mutations inactivate the TTLL3 enzyme

In a recently published screen for putative cancer-associated genes, TTLL3 obtained a relatively high score as a potential cancer gene [START_REF] Sjöblom | The consensus coding sequences of human breast and colorectal cancers[END_REF]. It was striking that the two mutations identified in the screen were localized in the vicinity of the ATP-binding residue E662, which is essential for enzymatic activity in all TTLLs (Fig. 4A; [START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF]. To test the potential impact of these mutations, we introduced them separately into mouse TTLL3, which is identical to human TTLL3 in this region (Fig. 4A), and tested the enzymatic activity of the mutated enzymes. Each cancer-related mutation led to a complete loss of glycylase activity of TTLL3 in vivo and in vitro (Fig. 4B-D), suggesting that loss of monoglycylating activity could be related to cancer.

A divergent polyglycylation mechanism in Drosophila

In mammals, TTLL10 is the only enzyme able to elongate glycine side chains. However, in Drosophila, an organism with polyglycylated MTs [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF], only two orthologs of the TTLL3/8 group are present, whereas no TTLL10 gene has been found [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF]. To establish the enzymatic mechanism that leads to the elongation of glycine side chains in the absence of TTLL10, we investigated the activity of the Drosophila melanogaster (dm) enzymes dmTTLL3A (CG11323) and dmTTLL3B (CG11201) in U2OS cells. Both enzymes induced monoglycylation on MTs (Fig. 5A, panel TAP952). Some cells expressing dmTTLL3B showed a restricted localization of this enzyme to the nucleus, and accordingly, only the nucleus was labeled with TAP952 in those cells (Fig. 5A,#). However, in contrast to the mammalian orthologs (Fig. 1C), dmTTLL3A also generated polyglycylated MTs as detected with polyG, while dmTTLL3B polyglycylated nuclear proteins, but not MTs (Fig. 5A, panel polyG). Immunoblots revealed that dmTTLL3A initiates and elongates glycine side chains on -and -tubulin, whereas dmTTLL3B initiates glycylation on number of proteins including -tubulin, but functions as an elongating polyglycylase only for nontubulin substrates (Fig. 5B). These polyglycylated substrates of yet unknown identity are likely to be responsible for the observed nuclear staining (Fig. 5A). Thus, Drosophila has a polyglycylation mechanism that utilizes bifunctional initiating/elongating polyglycylases.

dmTTLL3B is essential in Drosophila

Polyglycylation is particularly abundant in testes where it is associated with the axonemes of the sperm flagella [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF][START_REF] Bressac | A massive new posttranslational modification occurs on axonemal tubulin at the final step of spermatogenesis in Drosophila[END_REF]. In Drosophila, one of the polyglycylase genes, dmTTLL3B, is highly expressed in testes (Fig. S6), suggesting that dmTTLL3B is the major polyglycylase in this organ. Thus, we attempted to deplete dmTTLL3B by RNA interference (RNAi), using a transgenic fly carrying inverted repeats that correspond to dmTTLL3B under the control of UAS sequences inducible by Gal4 (UAS-IR-dmTTLL3B transgene). Strikingly, 50% of the embryos carrying UAS-IR-dmTTLL3B and the ubiquitous da-Gal4 driver (dmTTLL3B da- RNAi ) did not survive to adulthood (Fig. 6A).

RT-PCR analysis of adult dmTTLL3B da-RNAi flies demonstrated that dmTTLL3B inverted repeats were expressed and dmTTLL3B mRNA levels were strongly decreased.

In contrast, mRNA levels of the closest homolog of this gene, dmTTLL3A, were unchanged (Fig. 6B), demonstrating the specificity of the dmTTLL3B RNAi.

Immunoblots of whole-protein extracts showed a strong reduction of polyG-labeling on a range of protein bands in the dmTTLL3B da-RNAi flies (Fig. 6C), whereas polyglutamylation levels were slightly increased in males but not in females (Fig. 6C, panel GT335). These data provide strong evidence that the majority of the protein polyglycylation in Drosophila is catalyzed by dmTTLL3B. Almost all adult male dmTTLL3B da-RNAi flies were sterile, as only 2.4% were able to generate offspring (Fig. 6D), although with 20 times less progeny as compared to wild type. This suggests that polyglycylation is important for spermatogenesis.

The process of spermatogenesis in Drosophila can be divided into three major steps: (i) the generation of 64 haploid, interconnected spermatocytes from a stem cell, (ii) the formation of the axonemes and the condensation of the sperm nuclei in the still interconnected spermatids and (iii) the individualization that resolves the syncytial sperm bundle into 64 separate sperm cells. This last step is achieved by a group of synchronously moving individualization complexes that remove excess cytoplasm and place the membranes around each cell. To determine which of the three steps were affected in dmTTLL3B da-RNAi flies, we analyzed the testes by immunofluorescence and transmission electron microscopy (TEM). In immunofluorescence (Fig. 6E) of wild type testes, the polyG antibody stained prominent bundles of fibers that correspond to the sperm tails. The actin staining visualized groups of regularly arranged individualization complexes, which were always associated with the polyG-reactive MT bundles, confirming that sperm polyglycylation appears at the onset of individualization [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF][START_REF] Bressac | A massive new posttranslational modification occurs on axonemal tubulin at the final step of spermatogenesis in Drosophila[END_REF]. In contrast, in dmTTLL3B da-RNAi testes, polyG staining was strongly reduced and actin cones were scattered along the tails, indicating a sperm individualization defect [START_REF] Fabrizio | Genetic dissection of sperm individualization in Drosophila melanogaster[END_REF]. Moreover, we observe that the tips of the testes were often enlarged in dmTTLL3B da-RNAi male flies (Fig. 6E).

To better understand the observed defects we compared cross-sections of sperm cysts at different stages of maturity using TEM. In the preindividualization stage, sperm cysts of both control and dmTTLL3B da-RNAi flies appeared similar, as they contained the expected 64 sperm cells with an axoneme as well as a minor and a major mitochondrial derivate (Fig. 6F). Upon maturation, sperm cysts become more compact due to strongly reduced cytoplasmic ground substance. At this late stage, control cysts were highly organized as each axoneme was positioned in the close proximity of a major mitochondrial derivate (Fig. 6G, panel control). In contrast, most of the mature cysts from dmTTLL3B da-RNAi flies were lacking the axoneme structures, whereas the mitochondrial derivates were still present (Fig. 6G, panels dmTTLL3B da-RNAi ). In some cases, disorganized axonemes composed of singlet MTs were found. We also observed rare cases of disorganized cysts that still contained axonemes, but had reduced or absent mitochondria. Together our findings indicate that polyglycylation plays a role in individualization and is essential for the maintenance of the axoneme structure at later stages. However, it is not required for the early steps of Drosophila spermatogenesis, including the assembly of the axonemes.

Finally, we hypothesized that the initially observed partial lethality of the dmTTLL3B da-RNAi flies indicates an essential function of dmTTLL3B in Drosophila, and that the flies that reached adulthood survived due to the incomplete suppression of dmTTLL3B under the da-Gal4 driver. To test this hypothesis, we increased the efficiency of the RNAi by introducing the UAS-Dcr-2 transgene [START_REF] Dietzl | A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila[END_REF] into the dmTTLL3B da-RNAi flies. Strikingly, we observed 100% lethality at the larval stage. This phenotype is not due to side effects of the UAS-Dcr-2 transgene [START_REF] Dietzl | A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila[END_REF], since the expression of UAS-IR-dmTTLL3B under the control of the very strong and ubiquitous Act5C-Gal4 driver also led to 100% lethality (not shown). Thus, in addition to an important role in spermatogenesis, dmTTLL3B-mediated polyglycylation could be essential for Drosophila development.

Identification of potential new substrates of glycylation

The observation that mouse TTLL8 as well as Drosophila TTLL3B glycylate a range of non-tubulin proteins (Fig. 2B, 5B, 6C) raised the possibility that glycylation is a posttranslational modification with a broad range of substrates. Following the observation that the two most prominent substrates of polyglutamylation, tubulins [START_REF] Eddé | Posttranslational glutamylation of alpha-tubulin[END_REF] and NAPs [START_REF] Regnard | Polyglutamylation of nucleosome assembly proteins[END_REF], are also modified by polyglycylation [START_REF] Redeker | Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules[END_REF][START_REF] Ikegami | TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1[END_REF], we hypothesized that additional substrates of glycylation are among the previously identified substrates of polyglutamylation [START_REF] Van Dijk | Polyglutamylation Is a Post-translational Modification with a Broad Range of Substrates[END_REF]. We expressed 12 potential substrates of polyglutamylation together with either TTLL8 or an enzymatically inactive (dead) version of TTLL8 in HEK293 cells. Five proteins (ANP32A, ANP32B, SET, RanGAP and nucleolin) were found to be glycylated in the presence of active, but not inactive TTLL8 (Fig. 7). Seven other proteins tested (NF45, B23, GRP78, NASP, RNP-K, NCT, EB1, data not shown) were not modified. To uncover additional substrates by a more direct approach, we separated proteins from fly testes using two-dimensional electrophoresis and analyzed polyG reactive proteins by mass spectrometry. We selected six of the identified proteins (Table S1; see supplementary information for more details) and co-overexpressed them with active or inactive TTLL8 in HEK293 cells. Among these proteins, troponin T was found to be specifically glycylated by ectopic TTLL8 (Fig. 7). The identification of a range of potential substrates of glycylation suggests that this modification could regulate the function of many proteins.

Thus, the lack of glycylation of several proteins, and not only tubulin, could have contributed to the defects observed in the dmTTLL3B depleted flies.

Discussion

Polyglycylation was first described as a polymodification that generates glycine side chains of variable length on -and -tubulins [START_REF] Redeker | Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules[END_REF]. Similarly to polyglutamylation, it targets the C-terminal tails of tubulins, which are the major interaction sites for MT-associated proteins and molecular motors [START_REF] Nogales | Structural insights into microtubule function[END_REF].

Thus, polyglycylation could be an important regulator of MT functions.

We have identified a group of enzymes that mediate the generation of glycine side chains on proteins. The glycylating enzymes are members of the TTLL family, which also contains other enzymes that catalyze amino acid ligations: tubulin tyrosine ligase [START_REF] Ersfeld | Characterization of the tubulin-tyrosine ligase[END_REF] and diverse polyglutamylases [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF][START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF][START_REF] Wloga | Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila[END_REF]. Initiation and elongation of the glycine side chains are catalyzed by separate glycylases in mammals. Two initiating enzymes, TTLL3 and TTLL8, both generate monoglycylation on tubulin, whereas only TTLL8 modifies additional proteins. In agreement with our results, TTLL3 proteins in the ciliate Tetrahymena and in zebrafish are also initiating tubulin glycylases [START_REF] Wloga | TTLL3 protein generates tubulin glycylation and regulates ciliogenesis[END_REF].

The subsequent elongation of glycine side chains in mammals is catalyzed by the elongating polyglycylase TTLL10.

The discovery of a strict two-step mechanism for MT polyglycylation in vertebrates raised two questions related to previous descriptions of polyglycylation patterns in different organisms. First, elongated glycine side chains are completely absent in human sperm tubulin [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF]. We show that this phenotype is directly related to human TTLL10, which lacks enzymatic activity due to two point mutations in the conserved core TTL domain. It is striking that these mutations appear to be unique to human TTLL10, which suggests that the loss of polyglycylation was an event important for human evolution. Furthermore, the existence of a species devoid of polyglycylation suggests that the "poly" character of glycylation is not essential for protein functions, and that the functions of glycylation could be efficiently fulfilled by monoglycylation, which is present in all cilia in mammals [START_REF] Dossou | Mammalian cilia function is independent of the polymeric state of tubulin glycylation[END_REF].

Another surprising observation was that Drosophila, an organism with reported polyglycine side chains on tubulin [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF], has no homologs of TTLL10. We show that in Drosophila, glycylases from the TTLL3/8 group act as bifunctional enzymes that are able to catalyze both, initiation and elongation of glycine side chains, explaining why polyglycine side chains are present despite the absence of a TTLL10 ortholog. The two distinct glycylation mechanisms in mammals and Drosophila correlate with the previously described order of appearance of polyglycylation during sperm maturation. In mouse, polyglycine side chain elongation is delayed, appearing only after initial monoglycylation [START_REF] Kann | Expression of glycylated tubulin during the differentiation of spermatozoa in mammals[END_REF], whereas in Drosophila, monoand polyglycylation occur simultaneously [START_REF] Bré | Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility[END_REF].

In Drosophila the depletion of dmTTLL3B induced a significant decrease in glycylation, accompanied by decreased viability and an almost complete sterility of the surviving males. Detailed analysis of the dmTTLL3B da-RNAi flies revealed that the lack of dmTTLL3B affects late stages of spermatogenesis. This coincides with the timing of appearance of glycylation in Drosophila sperm development [START_REF] Bressac | A massive new posttranslational modification occurs on axonemal tubulin at the final step of spermatogenesis in Drosophila[END_REF]. We observed that although axonemes were assembled, they become unstable and disassemble in final stages of spermatogenesis. The disassembly of the axonemes is likely to proceed through a stage in which MT doublets are reduced to singlets, followed by the complete disappearance of all MT structures. Thus, one of the roles of glycylation in spermatogenesis appears to be the maintenance of the axonemal structure, whereas the modification is not required for the assembly.

Due to their sensory role in cells, cilia are centers of signal transduction pathways involved in embryonic development and tissue homeostasis in adults. Therefore, defects in cilia can be related to cancer [START_REF] Mans | All along the watchtower: is the cilium a tumor suppressor organelle?[END_REF][START_REF] Michaud | The primary cilium in cell signaling and cancer[END_REF]. Our finding that two cancer-related mutations in TTLL3 (identified by [START_REF] Sjöblom | The consensus coding sequences of human breast and colorectal cancers[END_REF] both completely inactivate this enzyme, suggests that the lack of TTLL3 activity could affect cilia, which in turn could contribute to cancer progression.

The complexity of signals generated by polyglutamylation and polyglycylation on

MTs could allow for different modes of regulation. Besides the possibility that both polymodifications generate signals for different target proteins, glycylation could also function as a counter-regulator of polyglutamylation, since both modifications compete for similar modification sites on tubulin [START_REF] Redeker | Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha-and beta-tubulin and affect the ciliary matrix in Tetrahymena[END_REF]. In agreement with this hypothesis, we show that tubulin polyglutamylation increases upon depletion of polyglycylation. Such a function could be sufficiently fulfilled by monoglycylation, which would help explaining the apparent lack of phenotypes related to the loss of elongated glycine side chain in humans.

Most of the past work on glycylation has been focussed on the role of this modification in MT functions; however, several recent studies have shown that other proteins are substrates of glycylation [START_REF] Ikegami | TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1[END_REF][START_REF] Lalle | The Giardia duodenalis 14-3-3 Protein Is Post-translationally Modified by Phosphorylation and Polyglycylation of the C-terminal Tail[END_REF]Xie et al., 2007). Together with our results, this opens the possibility that glycylation targets a broad range of substrates.

To conclude, we have identified a new group of tubulin modifying enzymes and two evolutionarily divergent mechanisms for glycyl side chain initiation and elongation.

Functional studies in Drosophila show that glycylation is important for sperm maturation, and that it might also be essential in early developmental stages in flies.

Finally, the discovery that several important proteins could undergo glycylation raises the possibility that this modification acts as a general regulator of cellular functions.

Experimental procedures

Cloning and mutagenesis

Mouse TTLL3, 8 and 10 have been identified and cloned before [START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF].

The rhesus monkey (Macaca mulatta) TTLL10 cDNA sequence was reconstructed from fragments found by NCBI Blast (http://www.ncbi.nlm.nih.gov/BLAST/) using known TTLL10 sequences as templates. The predicted sequence was amplified from testis cDNA (BioChain Institute Inc. Hayward, CA, USA), cloned and the DNA was sequenced. PCR-related mutations were excluded by sequencing of multiple clones (complete coding sequence: Fig. S2A). For human TTLL10, a splice variant (Fig. S2C) of the sequence AK124125 has been amplified from testis cDNA (BioChain Institute Inc.) and verified by DNA sequencing. Drosophila dmTTLL3A (CG11323) and dmTTLL3B (CG11201) were cloned from cDNA from adult flies.

Point mutations were introduced by a PCR-based quick-change method. The presence of the mutation was verified by DNA-sequencing.

Drosophila genetics

Drosophila was grown on standard medium at 25°C. The w 1118 , UAS-IR-TTLL3B transgenic line was obtained from the Vienna Drosophila RNAi Center (www.vdrc.at).

The transgene, localized on the X chromosome, expresses an inverted repeat from the dmTTLL3B gene (CG11201) under the control of UAS sequences. UAS-IR-TTLL3B flies were crossed with strains carrying the da-Gal4 or the Act5C-Gal4 driver. For amplification of the RNAi-effect, the da-Gal4 transgene was associated with the UAS-Dcr-2 transgene [START_REF] Dietzl | A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila[END_REF], and the resulting strain was crossed with UAS-IR-TTLL3B flies. Total RNA was extracted from adult flies with TRIZOL reagent (Sigma), and reverse transcription was performed using the First-Strand cDNA synthesis kit (GE Healthcare) with 5 µg of RNA and random hexamers as primers. 1/60 of this cDNA was used for PCR amplification. Primer pairs used for the detection of different gene transcripts can be provided on request. Number of PCR-cycles used for RT-PCR: RNAi, dmTTLL3B: 30, dmTTLL3A: 35, rp49: 25. Male fertility was tested in individual crosses with w 1118 females, and the presence of progeny was scored after 2 weeks.

Isolation of axonemes from Tetrahymena cilia

CU427 Tetrahymena cells were grown in SPP medium (1% proteose peptone, 0.2% glucose, 0.1% yeast extract, 0.003% EDTA ferric sodium salt) at 30°C to mid log phase (4-5x10 5 cells/ml). Cilia were collected as described [START_REF] Brown | Kinesin-II is preferentially targeted to assembling cilia and is required for ciliogenesis and normal cytokinesis in Tetrahymena[END_REF]) and stored at -80°C. For the preparation of axonemes, cilia were demembranated using 0.2% NP40 in 1x ERB buffer (50 mM Tris/HCl, pH 8.0, 10% glycerol, 5 mM MgCl 2 , 1 mM EGTA). To remove the endogenous glycylase activity, axonemes were further treated with 1 M NaCl in 1x ERB for 30 min, followed by an incubation at 60°C for 20 min.

Axonemes were spun down (12,000 g, 30 min, 4°C), and the supernatant was discarded.

The pellet was then washed in 1x ERB and resuspended in 1x ERB at a final concentration of 5 mg/ml.

Tubulin polyglycylation assay

The enzymatic activity of glycylase was determined based on incorporation of Enzymatic activity was measured using cells extracts prepared in 1x ERB containing 0.2% NP-40. 20 µg of axonemal or taxotere-stabilized MTs (prepared from adult mouse brains as described; [START_REF] Regnard | Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells[END_REF] were resuspended in the reaction mixture and immediately transferred to 30°C, followed by 4 h of incubation. The samples were then subjected to SDS-PAGE (10% gels) and electrotransferred onto nitrocellulose membrane. The tubulin bands were visualized by staining with Ponceau S, cut out and placed in 5 ml of scintillation liquid (Ultima Gold, Perkin Elmer) for quantification.

Alternatively, SDS-PAGE gels were stained with Coomassie Blue, incubated in Amersham Amplify TM (GE healthcare), and [ 3 H]-glycine incorporation was visualized by autoradiography.

Affinity-purification of EYFP-tagged proteins

EYFP-tagged potential substrates were co-expressed with TTLL8 in HEK293 cells. After lyses of the cells in PBS, 0.2% NP40, the supernatant (100,000 g, 30 min, 4°C) was incubated with GFP-TRAP ® agarose beads (ChromoTek, Martinsried, Germany) for 4 h at 4°C. Beads were washed twice in PBS, 0.2% NP40 and boiled in Laemmli sample buffer for 5 min. The samples were spun at 20,000 g for 5 min and supernatants were analyzed by SDS-PAGE and immunoblot.

Protein electrophoresis and immunoblot

One-dimensional SDS-PAGE was performed using standard protocols, or a special protocol for the separation of mammalian -and -tubulin [START_REF] Eddé | Control of isotubulin expression during neuronal differentiation of mouse neuroblastoma and teratocarcinoma cell lines[END_REF]. Two-dimensional electrophoresis consisted of an isolelectric focusing step (IPGphor Isoelectric Focusing System; GE Healthcare) and a SDS-PAGE. For the resolution of the tubulin in Fig. 1B, 40 µg of tubulin were loaded on 18 cm pH 4.5-5.5 Immobiline strips (GE Healthcare). Cell extracts shown in Fig. 2C were prepared directly in the loading buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer (GE Healthcare), 40 mM DTT) and resolved in 8.5 cm pH 3.0-5.6 Immobiline strips (GE Healthcare).

Membranes were incubated with the following antibodies: polyG (1:6,000), polyE (1:1,000; both courtesy of M. Gorovsky), TAP952 [START_REF] Bré | Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in Paramecium[END_REF]; ascitic fluid, 1:2,000), 12G10 [START_REF] Thazhath | Polyglycylation domain of beta-tubulin maintains axonemal architecture and affects cytokinesis in Tetrahymena[END_REF]1:250), E7 [START_REF] Chu | The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus[END_REF]; 1:20), 12CA5 (1:10), anti-GFP (1:5,000; Torrey Pines Biolabs), anti-NAP 4A8 [START_REF] Fujii-Nakata | Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity[END_REF][START_REF] Ishimi | Rapid purification of nucleosome assembly protein (NAP-I) and production of monoclonal antibodies against it[END_REF]1:20). Antibodies against TTLL8 were raised in rabbits with bacterially expressed protein, and then purified on recombinant TTLL8 and used at 1:500 dilution.

Protein bands were visualized with HRP-labeled donkey anti-rabbit or anti-mouse IgG 1:10,000 (GE healthcare) followed by detection with ECL immunoblot detection kit (GE Healthcare).

Immunofluorescence and microscopy

U2OS cells were cultured on glass coverslips under standard conditions and fixed using a protocol for preservation of cytoskeletal structures [START_REF] Bell | Improved methods for preserving macromolecular structures and visualizing them by fluorescence and scanning electron microscopy[END_REF]. Fixed cells were incubated with TAP952 (hybridoma supernatant 1:200), polyG (1:3,000), anti-TTLL8 (1:1,000) or anti-GST (1:1,000) antibody for 1 h, followed by 30 min with anti-mouse or anti-rabbit Alexa 555 or Alexa 488 antibody (1:1,000; Molecular Probes). DNA was visualized by DAPI staining (0.02 µg/ml). Coverslips were mounted with MOWIOL. Drosophila testes were fixed for 30 min with 3,7% paraformaldehyde in PBS, permeabilized in PBS containing 0.5% Triton X100, and incubated with polyG (1:9,000) for 1 h, followed by 30 min anti-rabbit Alexa 647.

Actin was visualized with TRITC-conjugated phalloidin (1:10,000; Sigma). We used DMRA microscopes (Leica, Germany) and Metamorph (Universal Imaging Corp., USA) software.

For ultrastructural analysis, testes were isolated from 3-4 days old male flies and fixed in 3.5% glutaraldehyde in 100 mM phosphate buffer, pH 7.4 overnight at 4°C. The samples were rinsed in phosphate buffer and post-fixed in 1% osmic and 0.8% potassium ferrocyanide for 2 h in the dark at RT. After rinsing in phosphate buffer, samples were dehydrated in a series of ethanol solutions (30-100%) and embedded in EmBed 812 DER 736. 85 nm sections were cut on a Leica-Reichert Ultracut E microtome and counterstained with uranyl acetate and lead citrate. Images were aquired with a Hitachi 7100 transmission electron microscope at the Centre de Ressources en Imagerie Cellulaire de Montpellier (France). Xie, R., Clark, K.M., and Gorovsky, M.A. (2007). Endoplasmic reticulum retention signal-dependent glycylation of the hsp70/grp170-related pgp1p in tetrahymena.

Eukaryot Cell 6, 388-397. EYFP-tagged mouse or Drosophila proteins were co-expressed with active (A) or inactive ("dead"; D) mouse TTLL8 in HEK293 cells. After EYFP affinity purification proteins were detected with anti-GFP antibody, and the level of glycylation was determined with TAP952. The total extract was probed for TTLL8 expression with anti-TTLL8 antibody. -----------------------mmTTLL10 1 MALHPQAGRPHRDGSEAQAEAAAQDLGRLPSPSKVGAAVCRIQGLGHRAARRPRRGIGTTSASRVPRPGALMPATRNRPRFIHCRGQPPR hsTTLL10 60 -------------APGHCPV-GPAHERPMGSSQEEGLRCQPSQPDHDADGHCGPDLEGAERASATPGPPGLLNSHRPADSDDTNAAGPSA macTTLL10 40 ---------------------GPAHERPMGSSQEEGLPCQPSQPDHDTD----------------------------- notably between mouse and the two primates. For the generation of chimeras (Fig. S4A) the central domains of rhesus and human TTLL10 were exchanged using the Sbf I and Eco RI restriction sites. Within this region, only 7 specific amino acid alterations between rhesus and human TTLL10 that could be responsible for the loss of activity of the human enzyme are present (green). 4 additional alterations between the sequence of rhesus and human TTLL10 correspond to non-conserved amino acids as they were also different in the active mouse TTLL10. Thus, they were not investigated. The essential ATP-interacting residue E511 (hsTTLL10; *) is indicated in red. Only rhesus TTLL10 and chimera N°1 increased the polyG signal on NAPs, indicative of enzymatic activity of the proteins. This revealed that the loss of activity of human TTLL10 is related to amino acid changes in the central domain (amino acid 334-513; see Fig. S3) that corresponds approximately to the core TTL domain.

-MDHSCTRFIHRRGPPTRTRAGFK-RGKRP----------RIQ-------QRPR--------ARVSGT---IPASRLHPAPASQPGPCP-macTTLL10 -MAHSCTRFVHRRGPPTRTQAGSK-RGKRP----------KIQ-------RRPR--------ARVP-

(C) To map which of the single amino acid changes lead to a loss of TTLL10 activity, the 7 conserved mutations found in human TTLL10 (mut1-7; see Fig. S3) were separately introduced into rhesus TTLL10. Note that only two mutations, mut3 and 5 led to a complete loss of activity of rhesus TTLL10, as indicated by the absence of a polyG signal on immunoblot (WT: wild type).

  [ 3 H]-glycine into MTs. The assay was performed in 50 µl of 1x ERB supplemented with 2 mM ATP and 250 pmol (5 µl) [ 3 H]-glycine (20.0 Ci/mmol, GE Healthcare).

Figure 2 Figure 3

 23 Figure 2The cooperative mechanism of murine polyglycylases.

Figure 4

 4 Figure 4 Loss of enzymatic activity of TTLL3 in colon cancer.

Figure 5 Figure 6

 56 Figure 5The enzymatic mechanism of polyglycylation in Drosophila.

Figure 7

 7 Figure 7 New substrates of glycylation.

Figure S2 :

 S2 Figure S2: Sequences of Macaca mulatta and Homo sapiens TTLL10.

  hsTTLL10

Figure S3 :

 S3 Figure S3: Sequence alignment and features of TTLL10 proteins.

Figure S4 :

 S4 Figure S4: Mapping of the mutations that lead to the loss of enzymatic activity of human TTLL10. (A) Schematic representation of chimeras generated by domain exchange between rhesus (red) and human (blue) TTLL10. The sequence positions of the exchange point are shown in Fig. S3. (B) To test the enzymatic activity of the respective constructs, rhesus and the chimeric TTLL10 proteins were expressed in U2OS cells.

Figure S5 :

 S5 Figure S5: The polymorphism of the human TTLL10 gene has no impact on the enzymatic activity of TTLL10 protein (A) Alignment of DNA sequences of human genomic DNA from two individuals with European and central African origins. The mutation A1343G (mut3; nucleic acid count of the TTLL10 open reading frame), which introduces a N448S amino acid change into TTLL10 in individuals of European origin is absent in the TTLL10 gene of an individual of central African origin. In contrast, the mutation C1400A (T467K) is present in both individuals. Statistic data on the distribution of this polymorphism in the human population can be found at: http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=1320571 (B) Immunoblot of mouse and human (European and central African) sperm protein with polyG (polyglycylation), TAP952 (monoglycylation) and 12G10 (α-tubulin). Despite the polymorphism in mut3 (A), both human individuals have no polyglycylation (polyG), instead strong monoglycylation was detected on αand β-tubulin (TAP952).

Figure S6 :

 S6 Figure S6: Tissue-specific expression of the Drosophila melanogaster TTLL3A and TTLL3B genesRT-PCR analysis of dmTTLL3A and dmTTLL3B in wild type flies. In male flies, testes were dissected from remaining body tissue and analyzed separately. Similarly, the ovaries and the body were analyzed from female flies. dmTTLL3A is expressed at moderate levels in the bodies of both, male and female flies, but very low expression levels are detected in testes and ovaries. In contrast, dmTTLL3B is highly expressed in testes, but at very low levels in other tissues (body). No dmTTLL3B expression was detected in ovaries.The results are in agreement with published microarray expression data: dmTTLL3A: http://flyatlas.org/atlas.cgi?name=FBgn0031854 dmTTLL3B: http://flyatlas.org/atlas.cgi?name=FBgn0031853.(* contaminating PCR product from genomic DNA)

Table S1 : Summary of the proteins identified in the major polyG-reactive protein spots after two- dimensional gel electrophoresis

 S1 (proteins chosen for further analysis are in bold letters)

	spot	protein name	accession	gene	molecular	N° of	RMS score sequence
	N°		N°		weight (Da)	pep-			coverage
						tides
	1	aldehyde	gi|20129399 CG3752	56,983	25	5 1797	57%
		dehydrogenase
		amino peptidase	gi|21355725 CG6372	61,474	11	3	729	24%
		RPT1	gi|17137738 CG1341	48,525	7	4	398	18%
		ERp60	gi|45445579 CG8983	55,235	10	2	482	28%
		TCP1η	gi|23170738 CG8351	54,835	3	4	176	7%
		NADH-ubiquinone	gi|17933656 CG6343	46,868	2	3	156	9%
		reductase
		aldehyde	gi|24650465 CG31075	52,573	4	3	188	9%
		dehydrogenase
		enolase	gi|7946	CG17654	46,534	3	2	145	11%
		EF-1α	gi|7915	CG8280	50,250	2	4	99	4%
	2	HSP60C	gi|21064097 CG7235	61,570	28	6 2024	59%
		TCP1β	gi|18858175 CG7033	58,047	20	3 1515	52%
		α1 tubulin	gi|17865841 CG9476	49,890	10	3	687	26%
		FK506-binding protein gi|24583150 CG4535	48,765	7	4	306	17%
		homolog of lipase	gi|24642748 CG18258	51,135	2	8	141	4%
		homolog of leucine	gi|24661038 CG32351	61,333	2	4	144	5%
		amino-peptidase 3
		hu-li tai shao protein	gi|157747 CG9325	127,873	2	6	114	3%
		homolog of	gi|21356607 CG3534	61,115	3	2	113	6%
		xylulokinase
	3	paramyosin	gi|10959	CG5939	102,322	46	4 3291	61%
		HSP60C	gi|21064097 CG7235	61,520	3	3	263	10%
		HSC70Cb	gi|21357475 CG6603	88,484	4	2	191	6%
	4	HSP60B	gi|15010456 CG2830	68,620	35	3 2680	64%
	5	HSP60A	gi|33636453 CG12101	60,778	43	6 1643	76%
		esterase 6	gi|1832142 CG6917	61,109	10	4	646	23%
		CG4546p	gi|24647265 CG4546	52,111	4	6	275	10%
	6	ATP synthase β	gi|24638766 CG11154	54,091	26	6	877	76%
		β2 tubulin	gi|158743 CG9359	49,826	6	6	361	52%
		β3 tubulin	gi|158749 CG3401	50,882	4	5	234	36%
		β4 tubulin	gi|21428640 CG4869	51,220	2	7	164	10%
		loopin-1	gi|20130057 CG4750	56,686	3	3	259	7%
		upheld	gi|38570263 CG7107	47,431	4	2	259	12%
		(troponinT)

Table S2 : Complete mass spectrometry data of selected proteins; see attached file Rogowski_suppl_tableS2.xls

 S2

Acknowledgements:

This work was supported by the CNRS, the Universities Montpellier 2 and 1, the Association pour la Recherche sur le Cancer (ARC) awards CR504/7817 and 3140 (to CJ) and 4894 (to FJ), the French National Research Agency (ANR) award JC05_42022 to CJ and by the Ligue contre le Cancer fellowship JG/VP-5454, the EMBO long-term fellowship ALTF 546-2006 to KR and NSF grant MBC-033965 to JG. We thank J.-M.Donnay, G. Herrada-Aldrian, B. Lacroix, L. Linarès, J.-C. Mazur, J. Miro, D. Moustapha Abba Moussa, Y. Thomas (CRBM, Montpellier, France), V. Bäcker, J. Cau, S. DeRossi, P. Travo (RIO Imaging facility at the CRBM) and U. Rothbauer (ChromoTek GmbH, Martinsried, Germany) for technical assistance, and C. Cazevieille (CRIC, Montpellier, France) for technical assistance and helping us interpreting ultrastructural data. We are grateful to M. Bosch-Grau, A. Burgess, B. Eddé, B. Lacroix, J. Miro, A. C. Seixas (CRBM), M. Bettencourt-Dias (Instituto Gulbenkian de Ciência, Oeiras, Portugal), M. Knop, I. Mattaj (EMBL, Heidelberg, Germany) and C. Sardet (IGMM, Montpellier, France) for instructive discussions. The authors want to thank the following researchers for providing essential reagents: C. Dehay (Institute of Brain and Stem Cell Research, INSERM, Bron, France) for proving us with Rhesus sperm samples, M. Gorovsky (University of Rochester, Rochester, NY, USA) for his generous gift of the polyG and polyE antibodies, and Y. Ishimi (Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan) for kindly providing mAb 4A8. The authors

Supplementary text:

To identify new substrates of polyglycylation form Drosophila testes, we resolved testes protein extracts using two-dimensional gel electrophoresis. The gel was stained with Coomassie brilliant blue, and in parallel immunoblots with polyG were performed from similar gel. Comparing the immunoblots to the stained gel, we identified 6 prominent spots that were labeled with polyG, and submitted these spots to mass spectrometry for protein identification. From the proteins that were identified in each spot (Table S1), we selected in most cases proteins with a high score for further analysis. Different paralogs of HSP60 were identified in several spots. We focused on HSP60B, since it has been the only protein detected in spot n°4.

From spot n°1, we decided to select only RPT1, based on the consideration that the function of this protein as a subunit of the 26S proteasome could be important for sperm maturation (Zhong and Belote, 2007). From spot n°2 we chose TCP1β. From spot n°3 we chose all proteins except for the HSP60 paralog, whereas we decided not to analyze any protein from spot n°5. Spot n°6 was rich in β-tubulins, suggesting that the detected polyG signal was generated by polyglycylated forms of tubulin. Nevertheless we decided to investigate the modification of upheld, a homolog of troponin T, because this protein has an extended glutamate-rich C-terminal tail that could serve as a potential site of modification.

The proteins chosen for further analysis, RPT1, TCP1β, paramyosin, HSC70Cb, HSP60B and troponin T were cloned into mammalian expression vectors bearing a C-terminal (or, in the case of troponin T, a Nterminal) EYFP-tag. Expression in HEK293 cells confirmed that proteins of the correct size were expressed as EYFP-fusion proteins.

Supplementary materials and methods:

Sequencing of human genomic DNA Genomic DNA was purified from human blood or sperm using the REDExtract-N-Amp TM Tissue PCR kit (Sigma, USA). Each DNA was amplified with the primer pair hTTLL10_seq_F1263 (5'-CATGCAGAAGAAGAGCCCTCTGTAC-3') hTTLL10_seq_introRa (5'-CTGCCAAGTGGAGGATCTGAAAGG-3') in two independent polymerase chain reactions (PCRs). The resulting PCR products of approximately 500 bp were purified and sequenced with both primers. No PCR-related mutation was detected.

Protein identification by mass spectrometry

Protein preparation for in gel digestion. The gel pieces were cut out from a Coomassie brilliant blue stained gel and successively washed with 50 µl of 25 mM NH 4 HCO 3 and 50 µl of acetonitrile (three times), and dehydrated with 100 µl of acetonitrile before reduction in the presence of 10 mM DTT in 25 mM NH 4 HCO 3

(1 h at 57°C) and alkylation in the presence of 55 mM iodoacetamide in 25 mM NH 4 HCO 3 . For tryptic digestion, the gel pieces were resuspended in 2 volumes of trypsin (12.5 ng/µl; V5111 Promega, USA) freshly diluted in 25 mM NH 4 HCO 3 and incubated overnight at 37°C. The digested peptides were then extracted from the gel in a buffer containing 34.9% H 2 O, 65% acetonitrile, and 0.1% HCOOH, and directly analyzed by nanoLC-MS/MS.

Chromatography conditions on NanoAcquity. The analysis was performed on a nanoACQUITY Ultra-Performance-LC (UPLC, Waters, Milford, USA). The samples were trapped on a 20х0.18 mm, 5 µm Symmetry C18 precolumn (Waters, Milford, USA), and the peptides were separated on a ACQUITY UPLC ® BEH130 C18 column (Waters, Milford, USA), 75 µm x 200 mm, 1.7 µm particle size. The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Trapping was performed during 3 min at 5 µl/min with 99% of solvent A and 1% of solvent B. Elution was performed at a flow rate of 400 nl/min, using 1-40% gradient (solvent B) over 35 min at 45°C followed by 65% (solvent B) over 5 min. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes (MS 0.5 s/scan on m/z range [250;1500] and MS/MS 0.7 s/scan on m/z range [50;2000]). The 3 most abundant peptides (intensity threshold 60 counts/s), preferably doubly and triply charged ions, were selected on each MS spectrum for further isolation and CID fragmentation with 2 energies set using collision energy profile. Fragmentation was performed using argon as the collision gas. Protein identifications were validated when at least two peptides were identified with Mascot ion score greater than 35 for each MS/MS spectra.

MS and MS/MS

For the estimation of the false positive rate in protein identification, a target-decoy database search was performed (Elias & Gygi, 2007). The false positive rate was close to 0.5%.

Criteria used for protein identifications followed the general guidelines for reporting proteomic experiments (MIAPE; http://www.psidev.info)

Supplementary references:

Elias, J.E., and Gygi, S.P. ( 2007


Christophe Candolfi
email: christophe.candolfi@cpfs.mpg.de

Bertrand Lenoir

Anne Dauscher

Michael Marek Koza

Marc De Boissieu

Malgorzata Sternik

Krzysztof Parlinski

Generalized

Keywords: number: 78, 70, Nx, 63, 20, -e

come

I. Introduction

Inelastic neutron scattering (INS) constitutes a powerful tool to probe elementary excitations in magnetic and non-magnetic solids. INS experiments enable to investigate in detail the lattice dynamics of crystalline materials, the superconducting order parameter of unconventional superconductors as well as to obtain a deeper insight into the symmetries and driving forces of magnetic ground states. [1][2][3][4] The Mo3Sb7 compound has recently attracted attention due to the coexistence of type-II superconductivity below Tc ~ 2.3 K and low dimensional magnetism related to antiferromagnetically coupled molybdenum dimers that lead to the formation of a spin gap below T * = 53 K. 5,6 Several studies with the aim at determining the symmetry of the order parameter of the superconducting state have been undertaken. While specific heat analysis as well as muon spin spectroscopy measurements have suggested a single isotropic energy gap, 7,8 further experimental investigations using muon spin relaxation appeared to be consistent with the existence of two superconducting BCS-like gaps, even though these results are still under debate. [9][10][11] In addition, Wiendlocha et al. 12 have successfully estimated the experimental electron-phonon coupling constant from combined first-principle electronic band structure and lattice dynamical calculations.

The formation of the spin gap in Mo3Sb7 is accompanied by a structural transition breaking the cubic symmetry of the crystalline lattice. 13,14 Uncommon temperature dependences of the transport properties, i.e. of the electrical resistivity, thermopower and thermal conductivity were reported. These investigations have provided insights into the coupling between the different degrees of freedom of the system i.e. between phonons, electrons and magnetic dimers. 15 In this regard, strong interactions of phonons with localized magnetic dimers have been suggested as the prominent scattering mechanism controlling the thermal transport. 15 Further experimental evidence of this interplay was provided by detailed studies of the transport properties of the ternary Mo3Sb7-xTex and Mo3-xRuxSb7 systems. [16][17][18][19] Diluting the magnetic interactions by increasing the Te and Ru contents was then found to progressively restore a conventional temperature dependence of the thermal conductivity.

Recently, muon spin relaxation combined with inelastic neutron scattering experiments performed on the Mo3Sb7 and Ru3Sn7 compounds were reported further confirming the formation of a spin gap in Mo3Sb7. 20 In this context, a thorough investigation and analysis of the lattice dynamics of this compound may provide additional information to help understand the anomalous thermal transport of the binary compound and on its behaviour upon alloying.

Here we report inelastic neutron scattering measurements of the generalized density of states (GDOS) of Mo3Sb7 and of the non-magnetic and isostructural Mo3Sb5.4Te1.6 compound in the 2 -300 K temperature range. These experimental results are confronted with a phonon density of states (PDOS) and temperature dependence of the specific heat of Mo3Sb7 derived by lattice dynamical calculations and discussed in light of the aforementioned unconventional behaviour of the thermal conductivity.

II. Experimental detail

Polycrystalline Mo3Sb7 and Mo3Sb5.4Te1.6 samples have been prepared by reaction of stoichiometric amounts of high purity Mo powders (99.999%), Sb and Te shots (99.999%) in quartz ampoules sealed under an inert atmosphere (He-H2, 95-5%). The ampoules were then heated up to 750°C and kept at this temperature for 10 days. The obtained ingots were ground and further annealed at the same temperature for 15 days to ensure a good chemical homogeneity. X-ray and neutron powder-diffraction studies have been performed on both samples and have shown that Mo3Sb7 contained a negligible amount (< 2% vol.) of antimony as a secondary phase while Mo3Sb5.4Te1.6 did not exhibit any secondary phases. 21 These results were further corroborated by electron probe microanalysis. The Table I summarizes the lattice parameters of both compounds together with the data on the low-temperature phase of Mo3Sb7 inferred from X-ray powder diffraction. Standard data corrections for empty container scattering and detector calibration using an incoherent standard (vanadium) have been applied. Using the incoherent approximation that implies an averaging of the inelastic signal over the accessed momentum space, the data were converted into the generalized density of states applying established mathematical relations. [23][START_REF] Lovesey | Theory of Neutron Scattering from Condensed Matter[END_REF][START_REF] Squires | Introduction to the Theory of Thermal Neutron Scattering[END_REF][START_REF] Mutka | [END_REF] All presented in this paper are normalized to 30 phonon modes in the 0.5-36 meV energy range for both spectrometers.

An estimation of a potential multiphonon contribution by a self-consistent technique indicates the presence of an additional weak background in the recorded spectra. We assume that a minute amount of hydrogen as a remnant of the sample preparation is present in our samples. Note that in terms of scattering power (see Table IV) a single hydrogen atom scatters more than 10 3 times more neutrons than any of the sample constituents. It is worth mentioning that this contribution neither affects our analysis nor our conclusions.

) (w G ) (w G
Lattice dynamical calculations were carried out using the Vienna Ab-Initio Simulation Package (VASP) and the direct phonon method within the sofware package PHONON. Some data are discussed in detail elsewhere. 12,27 In the present paper, total PDOS together with the temperature dependence of the specific heat, , calculated within the framework of harmonic solids, are presented.

III. Results

A. Generalized density of states

C ) (w G ) (w G < w  ) (w G ) (w G » w  D q B D k E 3 4 = q B k ò = w w w d G E ) ( E
Mo3Sb7 and Mo3Sb5.4Te1.6, respectively, we then obtain respective of 255 and 272 K.

These values are in fair agreement with those derived from low temperature specific heat analyses on both samples, and neutron diffraction experiments along with ultrasound velocity measurments for Mo3Sb7 (Table II). 7, 16,[START_REF] Candolfi | [END_REF] The careful reader might notice that the low energy region tends to deviate from a harmonic Debye behavior for which . As shown in Figure 2, this feature is due to a dispersive band coming from acoustic phonons which originate from a Bragg reflection located above the highest angle that could be attained in the present experiment. In other words, the incoherent approximation is not fully fulfilled for the intensity distribution at the lowest energies and high values in the IN6 data. However, the prominent peaks at higher energies are not affected by this deviation as they originate from rather localized, nondispersive modes (i.e. modes for which the group velocity vanishes ) detectable in a vast region of the phase space.

The PDOS of Mo3Sb7 derived from lattice dynamical calculations has been previously published in Ref. 12. In this paper, the simulated PDOS displays one phonon peak close to 4 meV. This low-energy excitation does not show up in the measured density of states, shown in Figure 1. This low energy mode originates in the simulation from a longitudinal excitation which becomes soft at the high symmetry point H. Using similar Hellmann-Feynman forces as those used in Ref. 12, we have introduced a dispersion of the previously flat phonon curve originating from a longitudinal acoustic branch as seen in Figure 3. To derive these dispersion curves, it was sufficient to change only six diagonal independent force-constant parameters of the Mo-Mo and Sb-Sb pairs by unexpected small magnitudes of 0.17% and 0.70%, respectively, all calculated with respect to the xx component of the Mo-Mo on-side force constant (Table III). The remaining 344 independent force-constant parameters remained practically unchanged (within numerical noise which was less than 0.005 %).

D q 2 ) ( w w µ G q 2 Q 0 / Q ¶ ¶w ) (w Z
Intriguing is the fact that so small changes of the force-constant parameters are able to renormalize the phonon to distinctly higher energies. 12 Using these new force constants, we have calculated the total and partial PDOS shown in Figure 4a. Note that in order to allow for a direct comparison with experiments, has been convolved with a gaussian function whose width approximates the energy resolution of the instrument. 22 A good agreement between the simulation and our measurements can be observed.

Even though Sb and Te display similar atomic radii, molar masses and total scattering power (Table IV), modifications arise from the substitution of Sb by Te (Figure 1). A prominent effect of the substitution on the inelastic response is the shift of the full phonon spectrum towards higher energies. This observation is qualitatively in agreement with the unit cell contraction as the Te concentration increases. 16,21 Beyond the overall shift of , the distribution of the modes is not preserved by the partial substitution of Sb by Te. As Figure 1 shows, the presence of Te influences some distinct eigenmodes. It has been shown from powder neutron diffraction experiments together with electronic band structure calculations that Te is exclusively located on the Sb1 site. 21 As shown in Figure 4b which depicts the partial density of states of Mo and Sb, the Sb1 and Sb2 sites display different contributions. It is thus likely that this partial substitution may only influence some distinct phonon modes resulting in slight differences between the two spectra.

Based upon the lattice dynamical calculations, the specific heat of Mo3Sb7 has been computed and compared with experimental data in Figure 5a, whereby we assume the difference , where is the volumic thermal expansion coefficient, the bulk modulus, the specific volume and the absolute temperature, to be negligible. 30 An overall satisfactory match between computed and measured data can be

) (w Z ) (w G ) (w Z ) (T C V ) (T C p BVT C C V p 2 a = - a B V T
observed. The slight discrepancy above 150 K is likely related to the anharmonicity of the crystalline lattice which is not captured by the calculations. Below the characteristic temperature T * , the position of the peak in the data is well reproduced by the calculations. The more pronounced discrepancy between the two sets of data (Figure 5b) may raise the question whether or not the cubic-to-tetragonal distortion of the crystal structure (see Table I) 13,14 can be held responsible of this difference.

On a more quantitative level, the lattice contribution to the specific heat can be inferred from the experimental INS data via the relation

(1)
As shown in Figures 6a and6b, the INS data reproduce well the temperature dependence of the experimental for both compounds. Note that in the case of the Te-substituted sample, the somewhat stronger discrepancy between the two sets of data towards room temperature may be due to a higher experimental uncertainty in the measurement of the values above ~ 250 K. Prior investigations have revealed that the magnetic properties of the binary compound lead to a substantial contribution to the specific heat at low temperatures which progressively disappears with increasing the Te content. 6,16 The data of Mo3Sb7 can be thus decomposed into three terms

where is the electronic contribution with the Sommerfeld coefficient and is the magnetic contribution originating from the

3 /T C p ) ( , T C ph V w w w w w d T k T k T k G k T C B B B B ph V ò ¥ ÷ ÷ ø ö ç ç è ae - ú û ù ê ë é ú û ù ê ë é ÷ ÷ ø ö ç ç è ae = 0 2 2 , 1 exp exp ) ( 3 ) (    ) (T C p p C ) (T C p [ ] 2 2 , ) / exp( 3 1 ) / exp( ) / ( ) ( ) ( ) ( ) ( ) ( T k T k T k T T C T C T C T C T C B B B ph V mag e lattice p D + D D + + = + + = g T T C e g = ) ( g [ ] 2
2

) / exp( 3 1 ) / exp( ) / ( ) ( T k T k T k T C B B B mag D + D D =
antiferromagnetically-coupled Mo dimers in which is the width of the spin gap which opens below T * . In Eq.( 2), we make the reasonable assumption that the anharmonic contribution within the quasiharmonic approximation is negligible in the temperature range investigated i.e. . Tran et al. have shown that the magnetic contribution can be described by a spin gap model of isolated dimers. 6 In the present case, can be determined by subtracting the lattice and electronic contributions from the experimental data. As a first approximation, we have assumed the parameter to be temperatureindependent ( ~ 34.5 mJ.mol -1 .K -2 [11,16]). The temperature dependence of obtained by this approach (see Figure 6a) is in good agreement with that derived by Tran et al. 6 using an isostructural and non-magnetic compound as a phonon reference. In the Tesubstituted sample, the subtraction of the lattice contribution leads to the temperature dependence of the electronic contribution . As can be seen in Figure 6b, no significant magnetic contribution can be identified in agreement with the low-temperature transport property and magnetic susceptibility data indicating a strong weakening of the magnetic interactions upon alloying on the Sb or the Mo sites.

T C T C ph V lattice = ) (T C mag ) (T C p g g ) (T C mag ) (T C e ) (w G
suppressed below 5 meV. Even though Mo3Sb7 undergoes a spin gap formation concomitant with a cubic-to-tetragonal structural phase transition at 53 K, no significant changes are captured in the inelastic response of the compound. This observation is consistent with the small magnitude of the structural distortion as concluded from low temperature x-ray diffraction studies (Table I). 13,14 In addition, no clear signature of spin excitations above and/or below T * could be unraveled in the present measurements. Of course, this statement holds only within the accuracy of the performed experiments. The phase-space limit towards low-values (see Figure 2) and the strong elastic signal up to 5 meV might obscure dynamic fingerprints of a spin gap at low temperatures. Further investigations should therefore be carried out at lower values or with another technique such as neutron polarized experiments which might enable a separation of the magnetic and phonon contributions. As

shown by the low-energy region of obtained at IN4 using a wavelength of 1.8 Å (Figure 8), increasing temperature from 2 to 300 K leads to a progressive softening of the GDOS for both compounds. Such a temperature dependence can usually be understood within the quasiharmonic model which assumes that variations in phonon frequency originate from the thermal expansion. 30 In this context, the variation of the average phonon energy, , can be quantified through the relation or equivalently, where is the Grüneisen parameter which can be expressed in terms of thermodynamic quantities 30

where and are the isothermal and isentropic bulk moduli, respectively. A positive Grüneisen parameter, as typically observed in most materials, then results in a decrease of with increasing temperature following the thermal expansion of the unit cell. To

Q Q ) (w G E V d E d G ln / ln - = g 0 0 / ) ( / ) ( V T V E T E G D - = D g G g p S V T G C VB C VB a a g 3 3 = = T B S B E
determine whether or not the temperature dependence of the PDOS of both compounds can be adequately described by this model, the temperature dependence of the average phonon energy and of the three main peaks in are plotted in Figure 9. The positions of these peaks, listed in Table V, were estimated by using both measured spectra at 1.22 and 1.80 Å in a similar fitting procedure as described above (Figure 9). The theoretical temperature dependence of of Mo3Sb7 was calculated according to the quasiharmonic model using the thermal expansion coefficient inferred from neutron diffraction experiments and the Grüneisen parameter and bulk modulus derived from ultrasound velocity measurements. 21 These experiments yielded = 0.95, = 72 GPa and = 25x10 -6 K -1 . Note that the experimental value of the bulk modulus is in very good agreement with the theoretical value of 73.6 GPa derived from the lattice dynamical calculations described in Section A. As a first approximation, the same values were used to determine the dependence of on temperature for the Te substituted sample. As can be seen in Figure 9a, the parent compound shows a systematic decrease in energy of all the modes upon heating from 2 up to 300 K by a comparable amount. This gradual softening is however stronger in Mo3Sb7 than in the Te substituted sample (Figure 9b). The shift in energy induced by increasing the temperature affects the entire spectra as reflected by a similar amount of softening whatever the phonon mode is (Figure 9c). While the Mo3Sb5.4Te1.6 compound follows the behaviour predicted by the quasiharmonic model, this theory fails to capture the temperature dependence of the phonon spectrum of Mo3Sb7. Thus, the latter exhibits a higher degree of anharmonicity which falls beyond the quasiharmonic approximation. Another evidence of the anomalous behaviour of Mo3Sb7 is related to the contraction of the unit cell upon alloying with Te. At 300 K, this effect is expected to result in a stiffening of of ~ 0.12% ( ~ 0.0012) based on our experimental Grüneisen parameter. The observed stiffening of ~ 7% thus largely exceeds the

) (w G E G g B a E E E E / D
value expected for a pure volume contraction. These results are in agreement with low temperature heat capacity analyses revealing that the relation , with the the lattice contribution, holds in a significantly broader temperature range in Mo3Sb5.4Te1.6

with respect to Mo3Sb7. 16 This conclusion is also consistent with the thermal conductivity which follows the same trend i.e. lower values are observed in Mo3Sb7 than in Mo3Sb5.4Te1.6. 16 The stronger anharmonicity of Mo3Sb7 may also explain the discrepancy between the Debye temperatures derived from low-temperature specific heat and INS measurements (see Table II).

IV. Discussion

Non-harmonic effects in solids are usually related to phonon-phonon interactions or to interactions of phonons with electronic excitations. 30 The presence of magnetic dimers in Mo3Sb7 and their absence in Mo3Sb5.4Te1.6 raises the question whether a strong interplay between phonons and dimers may be responsible for the strong phonon softening in Mo3Sb7.

As already mentioned, it has been suggested that strong phonons-dimers interactions are responsible for the unusual thermal transport in both Mo3Sb7 and in the ternary Mo3Sb7-xTex and Mo3-xRuxSb7 systems. 15,16,18 An increase in the Te and Ru content results in a progressive suppression of the magnetic interactions as revealed by magnetic susceptibility data which in turn, leads to an increase in the thermal conductivity values. The strong anharmonicity observed in Mo3Sb7 and its absence in Mo3Sb5.4Te1.6 might therefore corroborate this scenario.

Alternatively, softer phonons than expected from a pure volume expansion can arise in materials whose Fermi level is located close to sharp features in the electronic DOS such as in Cr, Mo, Nb or in the V3Si and V3Ge superconductors. The electron-phonon interaction exhibited by these materials was found to substantially affect the phonon energies leading to b g + = 2 T b an excess phonon softening even at temperatures well above the Debye temperature. 32,33 In particular, in the above-mentioned superconductors, the departure from the quasiharmonic model was correlated to the strength of the electron-phonon coupling which causes a stiffening of the PDOS upon increasing the temperature. 32 Electronic band structure calculations carried out on Mo3Sb7 have shown that the Fermi level lies near sharp features suggested to be at the core of the nearly zero thermopower observed below ~ 25 K. 12,15 Thus, the Mo3Sb7 compound shares some similarities with the aforementioned elements and alloys which might be the origin of the extra softening observed. Since the partial substitution of Sb by Te results in both a shift of the Fermi level towards the band gap and a lift of the sharp features, this could also explain the absence of a departure from the quasi-harmonic model in the Te susbtituted sample as well as the strong stiffening of the PDOS upon alloying. 16 Since superconductivity and magnetism coexist in Mo3Sb7 and are progressively suppressed in Mo3Sb7-xTex and Mo3-xRuxSb7 with increasing x, both electronic properties might be at the heart of the observed behaviour. At low Te concentration, low temperature transport properties measurements have shown that the electron-phonon coupling is significantly decreased whereby destroying the superconductivity while the magnetic properties remain practically unperturbed. 16 Thus, further experiments performed at small Te substitution levels should help to disentangle the role played by these interactions on phonons in Mo3Sb7 and in the ternary derivatives.

Conclusion

  To determine the GDOS, , INS experiments were carried out at room temperature on the high resolution time-of-flight spectrometer IN6 and extended down to 2 K using the thermal time-of-flight spectrometer IN4 of the European neutron source, Institut Laue-Langevin in Grenoble, France. IN6 experiments were performed on ~ 10 g of powdered Mo3Sb7 and Mo3Sb5.4Te1.6 placed in a cylindrical aluminum can with a radius of 4 mm ) (w G resulting in a transmission of ~ 89 %. Data were recorded at 300 K with an incident neutron wavelength of 4.14 Å in the inelastic time-focusing mode to attain a high energy resolution (~200 -400 µeV in the 0 -20 meV energy range). 22 An elastic Q range of up to the (4.0.0) zone center has been covered with this setup. Since the IN6 spectrometer exploits the anti-Stokes line of scattering processes, this setup enabled us to probe the low temperature inelastic response of the compounds down to ~ 100 K in the entire energy range of vibrational excitations. To access lower temperatures, IN4 experiments were carried out on ~40 g of Mo3Sb7 and Mo3Sb5.4Te1.6 powders. A flat, thin-walled aluminum sample holder was utilized resulting in a sample layer thickness of 6 mm. The transmission of the samples was ~ 90 %. Data on both compounds were recorded in the neutron-energy-loss mode (Stokes side) at 220, 100, 70, 50 and 2 K using two distinct incident neutron wavelengths of 1.22 and 1.80 Å. A maximum elastic Q range of up to the (8.8.8) and (14.0.0) zone centers have been covered with the 1.22 Å setup.

  Temperature dependence of the generalized density of states of Mo3Sb7 and Mo3Sb5.4Te1.6 was measured by inelastic neutron scattering in the 2 -300 K temperature range. The experimental of Mo3Sb7 is in good agreement with the phonon density of ) (w G ) (w G states derived by lattice dynamical calculations. Within the accuracy of the experiments, no magnetic excitations could be observed in the temperature range investigated. The structural transition concomitant to the spin gap formation does not result in marked differences of the measured spectra below and above the characteristic temperature T * . The partial substitution of Sb by Te leads to a shift of the entire towards higher energy consistent with the decrease of the unit cell volume. Upon cooling, the of Mo3Sb7 revealed a strong softening which is more pronounced than expected from the volume thermal expansion within the quasi-harmonic approximation. Since substituting Sb by Te suppresses this strong softening as it suppresses the magnetic interactions, the interplay of phonons and antiferromagnetically coupled molybdenum dimers is conjectured to be the origin of the enhanced anharmonicity. This interplay may be as well made responsible for the anomalous thermal transport in those materials. To further probe the magnetic behaviour in Mo3Sb7 below T * may require investigations on high-quality single crystal by phonon spectroscopy techniques and/or by neutron polarized experiments. Acknowledgement C.C. greatly thanks M. Amiet and P. Maigné, and financial support of DGA (Déléguation Générale pour l'Armement, Ministry of Defence, France) and of the European Network of Excellence CMA (Complex Metallic Alloys). Lattice parameters, , of the Mo3Sb7 and Mo3Sb5.4Te1.6 compounds. The possible low-temperature crystal structure of Mo3Sb7 based on X-ray powder diffraction refinements is also given.

Figure 1 :

 1 Figure 1: (Color online) Generalized density of states of Mo3Sb7 (blue open symbols)

Figure 2 :

 2 Figure 2: maps of Mo3Sb7 recorded at IN6 at 300 K and IN4 at 220 K. For

Figure 3 :

 3 Figure 3: Phonon dispersion curves of Mo3Sb7 along high symmetry directions. Upper panel:

Figure 4 :

 4 Figure 4: (Color online) Phonon density of states of Mo3Sb7 as obtained from ab initio

Figure 5 :

 5 Figure 5: (Color online) (a) Theoretical (○) and experimental (□) temperature dependence of

Figure 6 :

 6 Figure 6: (Color online) Temperature dependence of the experimental specific heat

Figure 7 :

 7 Figure 7: (Color online) at the different temperatures of 2, 100 and 300 K. Left,

Figure 8 :

 8 Figure 8: (Color online) Temperature dependence of of the Mo3Sb7 and Mo3Sb5.4Te1.6

Figure 9 :

 9 Figure 9: (Color online) Temperature dependence of the mean phonon energy for the (a)

  Figure 4

Table III .

 III Values of the on-side force constants (xx component) modified leading to the disppearance of the mode centred around 2 THz. The relative changes are given with respect to the (0 0) force constant which amounts to 122 990. The remaining 344 force-constant parameters are only changed by 0.0013 to 0.018 % which is within the numerical accuracy.Te atoms. For comparison purposes, the data for hydrogen have been added.

	temperature specific heat analyses of Mo3Sb7 and Mo3Sb5.4Te1.6. The values obtained via
	neutron diffraction and ultrasound velocity measurements for Mo3Sb7 are also given.
	Element
	H				1.76				80.26	Mo3Sb7	Mo3Sb5.4Te1.6 1.01	82
	Mo				5.67	INS	0.040	255	272 95.94	0.059
	Sb				3.90				0.007	121.76	0.032
										310	275
	Te				4.23				0.090	127.60	0.034
					Neutron diffraction		300	/
					Ultrasound velocity		285	/
					a
	Atom 1	Atom 2			Distance (Å)	Force constant prior model (Ref. 12)	Force constant new model	Relative change (%)
	Mo	Mo						0.0		122 990	123 204	0.174
	Sb	Sb						0.0	Mo3Sb7	Mo3Sb7 80797	Mo3Sb5.4Te1.6 80797	0.755
	Mo	Mo	T (K)			3.045	298	-25472	10	298 -25686	0.174
	Sb	Sb						3.408		-1373	-1930	0.689
		Space group				Im3m	I4/mmm	Im3m
	Mo	Mo						4.664		5258	4707	0.164
				a (Å)				9.5688	9.5611	9.5648
	Sb	Sb						4.820		-1771	-1484	0.682
				c (Å)				/	9.5388	/
		Volume at 300 K (Å 3 )		876.14	871.98	875.04
	Table IV. Coherent (	s	coh				s	inc
	Table II. Debye temperature,	q	D	, inferred from inelastic neutron scattering (INS) and low-M M inc coh t / ) ( / s s s + =

) and incoherent ( ) neutron scattering cross sections given in barns, atomic mass and total scattering power (

) of the Mo, Sb and p C

Table V .

 V Temperature dependence of the three main peaks characterizing lower part of the GDOS of Mo3Sb7 and Mo3Sb5.4Te1.6 as inferred from IN4 data. Room temperature data obtained from IN6 experiments are also included.

		s	coh	s	inc	M	s	t /	M
			Figure captions
				Mo3Sb7
	2 K	8.80	11.45		13.23	15.28			18.95
	70 K	8.71	11.47		13.33	15.26			18.81
	220 K	8.44	11.51		12.96	15.05			18.81
	300 K	8.41	11.27		12.83	14.88			18.64
				Mo3Sb5.4Te1.6
	2 K	9.33	12.02		14.68	18.54			20.60
	70 K	9.36	12.07		14.66	18.45			20.55
	220 K	9.29	11.98		14.74	18.40			20.38
	300 K	9.16	11.95		14.38	18.26			20.57

C. Candolfi, B. Lenoir, A. Dauscher, J. Hejtmanek, E. Santava, and J. Tobola, Phys. Rev. B,