Michale Bouskila

Noor Esoof

Laurie Gay

Emily H Fang

Maria Deak

Mike Begley

Lewis C Cantley

Alan Prescott

Kate G Storey

Dario R Alessi
email: d.r.alessi@dundee.ac.uk

Weiss Dando

Colucci

TTBK2 kinase substrate specificity and the impact of spinocerebellar ataxiacausing mutations on expression, activity, localisation and development

Mutations that truncate the C-terminal, non-catalytic moiety of the Tau tubulin Kinase 2 (TTBK2) cause the inherited, autosomal dominant inherited spinocerebellar ataxia type 11 (SCA11) movement disorder. In this study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate priming-groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localisation. We generate an SCA11-mutation-carrying knock-in mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that in homozygosity, the SCA11 mutation causes embryonic lethality at E10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11causing mutations impact on protein expression, catalytic activity, localisation and development. We hope these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Introduction

Recent studies have revealed that mutations within the Tau tubulin Kinase 2 (TTBK2) cause a serious autosomal dominant inherited movement disorder termed spinocerebellar ataxias type 11 (SCA11) [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF][START_REF] Bauer | Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds[END_REF]. SCA11 is characterised by progressive cerebellar ataxia, pyramidal features, peripheral neuropathy, and on occasion dystonia, with age of onset from the early teens to the mid 20s [START_REF] Houlden | Spinocerebellar Ataxia Type 11[END_REF]. TTBK2 is closely related to a neuronal specific protein kinase termed TTBK1, which was discovered in 1995 as a protein kinase in bovine brain extract that phosphorylated tau and tublin [START_REF] Takahashi | A novel tau-tubulin kinase from bovine brain[END_REF]. TTBK2 consists of 1244 residues and apart from an N-terminal serine/threonine protein kinase domain (residues 20 to 280) possesses no distinctive functional domains or motifs.

Thus far, four distinct SCA11 families have been identified, each possessing a mutation leading to premature termination of the TTBK2 protein at around residue 450. This leaves the kinase catalytic domain intact, but eliminates most of the noncatalytic portion of the enzyme (Fig 1A). One of the affected families from Devon, UK, stretches over eight generations (family 1 in Fig 1A) [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. All affected individuals of this family displayed progressive cerebellar ataxia, abnormal eye signs and pyramidal features, as well as cerebellar atrophy visible upon magnetic resonance imaging [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. Neuropathological examination of the brain blocks of one affected family-1 individual, revealed gross atrophy of the cerebellum accompanied by severe and near-complete loss of Purkinje cells, with a marked preservation of the basket cells, and a significant loss of cerebellar granule cells [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. There were also changes consistent with pathological aging, with some tangles and -amyloid-positive plaques observed in the neocortex, hippocampus, transentorhinal, entorhinal and insular cortex [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF].

TTBK2 belongs to the casein kinase CK1 group of eukaryotic protein kinases composed of CK1 isoforms (CK1 , CK1 2, CK1 , CK1 , CK1 1, CK1 2, CK1 3), Vaccinia Related Kinase (VRK) isoforms (VRK1, VRK2 and VRK3) and TTBK isoforms (TTBK1 and TTBK2) [START_REF] Manning | The protein kinase complement of the human genome[END_REF]. CK1 differs from most other protein kinases by the presence of the sequence S-I-N motif instead of A-P-E in kinase domain region VIII [START_REF] Hanks | Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification[END_REF]. TTBK1 and TTBK2 possess a P-P-E motif at this site. TTBK2 possesses 38% identity with CK1 within the kinase domain, but there is no obvious homology beyond the catalytic moiety of TTBK to CK1 or VRK isoforms. TTBK1 and TTBK2 share 84% identity within the catalytic domain and also display significant homology in the non-catalytic region.

TTBK isoforms have been found in many species including, mouse, zebrafish, C.elegans and Drosophila [START_REF] Manning | Evolution of protein kinase signaling from yeast to man[END_REF]. TTBK1 is mainly expressed in neuronal tissues [START_REF] Sato | Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation[END_REF], whereas TTBK2 is more widely expressed and has been observed in tissues including heart, muscle, liver, thymus, spleen, lung, kidney, testis and ovary [START_REF] Takahashi | Involvement of tau protein kinase I in paired helical filament-like phosphorylation of the juvenile tau in rat brain[END_REF][START_REF] Tomizawa | Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau[END_REF]. In situ hybridization showed that TTBK2 mRNA was present in all brain regions in human, rat and mouse tissue [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. Highest expression of TTBK2 mRNA was observed in the cerebellum Purkinje cells, granular cell layer, hippocampus, midbrain and substantia nigra [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. Lower expression was seen in the cortex of human, rat and mouse brains [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF].

Biochemical investigations initially identified a proteolytic fragment of the catalytic domain of TTKB2 encompassing residues 1-316, in various brain extracts that was capable of phosphorylating tau at two sites (Ser208 and Ser210) [START_REF] Takahashi | Involvement of tau protein kinase I in paired helical filament-like phosphorylation of the juvenile tau in rat brain[END_REF][START_REF] Tomizawa | Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau[END_REF]. Phosphorylation of these residues primes Tau for the phosphorylation by isoforms of Glycogen Synthase Kinase-3, an enzyme that has also been suggested to modulate tau pathology [START_REF] Noble | Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo[END_REF]. TTBK1 was reported to phosphorylate tau at four residues (Ser198, Ser199, Ser202 and Ser422) [START_REF] Sato | Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation[END_REF]. More recently overexpression of TTBK1 in mouse, was reported to enhance phosphorylation and oligomerisation of tau in the brain [START_REF] Xu | Tau-tubulin kinase 1 enhances prefibrillar tau aggregation and motor neuron degeneration in P301L FTDP-17 tau-mutant mice[END_REF]. Whether endogenous TTBK1 and/or TTBK2 regulate phosphorylation of endogenous A c c e p t e d M a n u s c r i p t tau has not been established. Recent genetic studies have also implicated TTBK1 in Alzheimer's disease and in tangle formation [START_REF] Vazquez-Higuera | Genetic variations in tau-tubulin kinase-1 are linked to Alzheimer's disease in a Spanish case-control cohort[END_REF][START_REF] Yu | Tau-tubulin kinase-1 gene variants are associated with Alzheimer's disease in Han Chinese[END_REF]. Thus far only one brain from a SCA11 family-1 patient has thus far been analysed and it displayed some tau deposition [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. This may suggesting that TTBK2 could be involved in regulating tau, but much further investigation is required to substantiate this conclusion.

Despite this previous work, little is understood about TTBK2 substrate specificity and how SCA11 truncating mutations impact on protein expression, kinase activity, stability and localisation. We have therefore undertaken an initial analysis of TTBK2 substrate specificity and shown that it has a conspicuous preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. We exploit this information to develop an optimised peptide substrate to assess TTBK2 catalytic activity. Furthermore, we demonstrate that SCA11 truncating mutations markedly enhance TTBK2 protein expression, lead to inhibition of TTBK2 kinase activity and promote nuclear localisation. We generate TTBK2 knock-in mice expressing an SCA11 disease causing mutation and confirm that this results in the inhibition of endogenous protein kinase activity. We find that in homozygosity the SCA11 mutation causes embryonic lethality. These findings will help with further investigations into the function and regulation of TTBK2 as well as its role in SCA11. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Materials and Methods

Reagents and general methods

Lysis Buffer contained 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 1 mM sodium orthovanadate, 10 mM sodium--glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM benzamidine and 2 mM phenylmethanesulphonylfluoride (PMSF), supplemented with either 1% (v/v) Triton X-100 or 0.5% (v/v) NP-40 and 150 mM NaCl as indicated. Buffer A contained 50 mM Tris/HCl, pH 7.5, 50 mM NaCl, 0.1 mM EGTA and 0.27 M sucrose. Tissueculture reagents were from Life Technologies and [ -32P]-ATP was from Perkin Elmer. P81 phosphocellulose paper was from Whatman. The Flp-in T-REx system was purchased from Invitrogen and stable cell lines were generated following the manufacturer instructions by selection with hygromycin.

The coding region of full length human TTBK2 was amplified by PCR from Image clone 4829013 (NCBI acc. BC071556 ) and subcloned as a Not1-Not1 fragment into several expression vectors. Site directed mutagenesis was carried out using the QuickChange® method (Stratagene) with the KOD polymerase (Novagen) in the presence of DMSO. All DNA constructs were verified by DNA sequencing, which was performed by The Sequencing Service, School of Life Sciences, University of Dundee, Scotland, UK.

Antibodies

A fragment of the mouse TTBK2 fusion protein encompassing residues 314-385 with an N-terminal GST (glutathione S-transferase) tag was expressed in bacteria and purified by glutathione-Sepharose chromatography. After cleavage of the GST tag, TTBK2 (314-385) was used as an immunogen to raise a sheep polyclonal antibody (S572C). Antibodies were affinity purified from antisera using the TTBK2 314-385 protein immunogen. Anti GST antibody was raised in sheep against the GST protein (S902A, 1 st bleed). Anti GFP (green fluorescent protein) antibody (S268B) was raised in sheep against recombinant GFP protein and affinity-purified against the antigen. Secondary antibodies coupled to horseradish peroxidase were obtained from Thermo Scientific and fluorescent secondary antibodies employed for the Odyssey Infrared System were from Rockland.

Immunological procedures

Cell lysates (20-30 Hg) and immunoprecipitates were subjected to SDS-PAGE electrophoresis using 10% gels and transferred onto nitrocellulose membranes. Membranes were blocked for 20 minutes at room temperature in 10% (w/v) dried non-fat milk powder in TBS-T (Tris buffered saline with 1% Tween-20). All primary antibodies used for immunoblotting were used at 1 Hg/ml in 5% milk in TBS-T. Secondary antibodies used were either horseradish peroxidase-conjugated or fluorescent antibodies. For immunoprecipitations, TTBK2 antibody was covalently coupled to protein G-Sepharose beads at a ratio of 1 Hg antibody/Hl of beads using a dimethyl pimelimidate (DMP) procedure, or GFP-antibody coupled beads were used. The indicated amount of cell lysates were immunoprecipitated using 5 Hl bead volume of coupled antibody for 1 hour, washed twice with lysis buffer containing 0.5 M NaCl and twice with Buffer A. Immunoprecipitates were either subjected to protein kinase activity as described below or to immunoblot analysis.

TTBK2 immunoprecipitation kinase assays

Peptide kinase assays were set up in a total volume of 50 Hl in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MgCl2 and 0.1 mM -32 P-ATP (~500-1000 cpm/pmol) in the presence of the indicated concentration of peptide substrate. The assays were performed for 20 minutes at 30 °C. When purified proteins were used, reactions were terminated by applying 40 Hl of the reaction mixture on to P81 phosphocellulose paper. In the case of immunoprecipitated TTBK2, reactions were terminated by adding 10 Hl of 0.2M EDTA and gentle mixing. After a short spin, 50 Hl of the Fluorescence microscopy HEK293 Flp-in T-REx stable cell lines harbouring GFP-tagged wild-type and mutant forms of TTBK2 were generated using standard protocols. Cells were grown on coverslips and induced with 0.1 Fg/ml doxycycline for 16 hours. Cells were fixed for 10 minutes with 4% paraformaldehyde at room temperature and washed twice for 5 minutes. After permeabilisation in phosphate-buffered saline (PBS) containing 1% Triton-X (v/v) for 10 minutes at room temperature, cells were incubated for 10 minutes with donkey serum (Sigma D9663) and then for 1 hour with 0.5 Fg/ml of anti-TTBK2 antibody. Cells were washed 3 times with PBS and incubated 30 minutes with the secondary antibody (Alexa Fluor 594) diluted 1:500 in PBS. After three further washes with PBS, cells were incubated with DAPI (4',6-diamino-2phenylindole) for 10 minutes and mounted onto slides using Hydromount (National Diagnostics). Images were collected on a Zeiss LSM 700 confocal microscope using an Plan-Apochromat x100 objective.

Quantitation of GFP-TTBK2 localisation

GFP expressing cell lines were fixed and stained with DAPI to identify their nuclei. Fields of cells selected on the basis of the DAPI staining were chosen at random and single optical sections through the nucleus and cytoplasm in the DAPI and GFP channels were collected sequentially on the Zeiss LSM 700 confocal microscope using the alpha Plan-Apochromat 100x objective. These images were quantified using the image-processing software Volocity (Perkin-Elmer). The nuclei were identified using the DAPI channel and the average GFP nuclear intensity measured for each cell. The whole cells including the nuclei were identified from the GFP channel and the area of the previously identified nuclei removed from these areas. The average intensity in the remaining cytoplasm was then measured for each cell. The nuclear intensity was then expressed as a ratio of the cytoplasmic intensity in order to avoid variability in protein expression by the cells. For each cell lines between 260 and 460 cells were counted over 10 fields of cells.

Animals and the generation of TTBK2 knock-in mouse

Animal studies were approved by the University of Dundee Ethics Committee and performed under a UK Home Office project license. Animals were obtained from Harlan Ltd (UK) and Taconic Artemis pharmaceuticals and maintained under specific pathogen-free conditions. The knock-in mice were generated by Taconic Artemis on an inbred C57BL/6J background as described in Figure 5. Genotyping was performed by PCR on DNA extracted from ears or embryonic membranes. Primers 1 (5'-CATTTGTTGGCATTATTTCAAAGG-3') and 2 (5'-AGTAGTAGTAGTAGTAGTAACATGG-3') were used to detect the wild-type and knock-in alleles. The PCR program consisted of 2 min at 95°C, 15 sec at 95°C, 30 sec at 60°C, and 10 sec at 72°C for 35 cycles.

Embryos and mouse embryonic fibroblasts culture

To generate embryos, TTBK2 heterozygous mice were crossed and embryonic development was estimated considering the day of vaginal plug observation as 0.5 dpc. Pregnant females were sacrificed and uterine horns were collected in PBS. The embryos were dissected from the decidua in M2 media (Sigma M7167) at room temperature and fixed in 4 % PFA for 2 hours and washed and stored in PBS for phenotypic analysis. Mouse embryonic fibroblasts were generated from embryos at 9.5 to 10.5 dpc. Embryos were removed from M2 media and placed in trypsin for 10 minutes at 37°C. From this stage, embryos were cultured in advanced DMEM/F12 media (Invitrogen 12634) supplemented with 10% FBS, 1X MEM non-essential amino acids, 2 mM L-glutamine and 0.1 mM 2-mercaptoethanol.

Results

Analysis of substrate specificity of TTBK2 by positional scanning peptide library approach

We initially utilised the positional scanning peptide library approach [15, 16] to probe the substrate specificity preferences of TTBK2. This assay employs 198 biotinylated peptide libraries that each contain a 1:1 mixture of serine and threonine at the central position and one additional position fixed to one of the 20 amino acids, as well as phosphothreonine, or phosphotyrosine. Phosphothreonine and phosphotyrosine were included to allow substrate specificity analysis of kinases such as CK1 to which TTBK2 is related that have preferences for priming phosphorylation sites. All other positions contain an equimolar degenerate mixture of natural amino acids (except serine, threonine, and cysteine). We screened the 198 libraries using -32 P-ATP and recombinant TTBK2(1-450) or kinase inactive TTBK2[D163A](residues 1-450) expressed in HEK293 cells. Biotinylated peptides were then captured on a streptavidin-coated membrane. The relative amino acid preference at each position was determined by quantifying 32 P-radioactivity incorporation following phosphoimaging. The key striking preference observed from this analysis was for a phosphotyrosine residue at the +2 position downstream from the site of phosphorylation (Fig 1B). For experiments undertaken with the kinase-inactive TTBK2[D163A], vastly lower overall levels of phosphorylation were observed with no preference for a +2 phosphotyrosine.

Elaboration of TTBKtide as an optimal peptide substrate

As TTBK2 is a member of the CK1 family we decided to verify whether TTBK2 would phosphorylate a model peptide substrate termed CK1tide that is frequently employed to assess CK1 isoform activity [START_REF] Marin | Design and synthesis of two new peptide substrates for the specific and sensitive monitoring of casein kinases-1 and -2[END_REF]. CK1 phosphorylates CK1tide (RRKDLHDDEEDEAMS*ITA) at the Ser residue marked with an asterisk [START_REF] Marin | Design and synthesis of two new peptide substrates for the specific and sensitive monitoring of casein kinases-1 and -2[END_REF], and we confirmed that TTBK2[1-450] also phosphorylates CK1tide specifically at this Ser residue (Fig S1). We next investigated the effects of introducing a phosphotyrosine residue at every position from the -5 to the +4 residue. This confirmed the findings of the positional scanning peptide library approach, revealing that introducing a phosphotyrosine residue at the +2 position, but not at any other position, strongly promoted peptide phosphorylation, principally by decreasing the Km approximately 8-fold (Fig 1C &D). The CK1tide peptide with a phosphotyrosine residue at the + 2 position was named TTBKtide (RRKDLHDDEEDEAMSIYpA). Introducing a phosphothreonine rather than a phosphotyrosine at the +2 position failed to enhance peptide phosphorylation (Fig 1D). Substituting the phosphorylated Ser residue in TTBKtide for a Thr did not effect phosphorylation by TTBK2, but substitution with a Tyr residue abolished phosphorylation (Fig 1D ).

Molecular basis for phosphate-priming

CK1 isoforms possess a well-known requirement for priming phosphorylation [START_REF] Flotow | Phosphate groups as substrate determinants for casein kinase I action[END_REF][START_REF] Flotow | Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase[END_REF][20]. The crystal structure of yeast CK1 [START_REF] Xu | Crystal structure of casein kinase-1, a phosphate-directed protein kinase[END_REF] and human CK1 [START_REF] Longenecker | Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition[END_REF] reveals the presence of a sulfate-binding groove on the small lobe of the kinase domain, predicted to function as the phosphate-priming site (Fig 2A). Four highly conserved basic Lys/Arg residues on CK1 isoforms form ionic interactions with the sulfate residue (Fig 2A). Three of these residues are conserved in TTBK1 and TTBK2 (Lys50, Lys143 and Arg181). The fourth residue is an Ala in TTBK2 (Ala184) as well as in TTBK1 (Fig 2B). To verify whether these residues on TTBK2 could be involved in enhancing phosphorylation of +2 phosphotyrosine containing substrates, we studied the effect that mutation of Lys50, Lys143, Arg181 and Ala184 had on the phosphorylation of TTBKtide. Mutation of Lys50 or Lys143 to either Glu or Ala inactivated/destabilised TTBK2 (SFig 2A) and equivalent mutations in CK1 had similar effects (SFig 2B). In contrast, mutation of Arg181 or Ala184 did not affect stability (Fig 2C ) or the intrinsic TTBK2 kinase activity as judged by the ability of these mutants to phosphorylate a peptide lacking the +2 phosphotyrosine residue (Fig 2D). However, the ability of TTBK2[Arg181Ala or Glu] or TTBK2[Ala184Gly or Glu] mutants to phosphorylate tyrosine-phosphorylated TTBKtide was markedly impaired (Km values 100-150 M compared to 18 M for wild-type TTBK2) (Fig 2D). This data suggests that TTBK2 possesses a phosphate-priming groove similar to CK1, except that it is involved in recognising +2 phosphotyrosine residues rather than N-terminal phosphoserine/phosphothreonine.

Disease causing TTBK2 mutations markedly increase protein expression and inhibit kinase activity

We next investigated how disease-causing truncation mutations affect TTBK2 protein expression as well as kinase activity. We generated HEK293 cells that stably express GFP-tagged versions of full-length TTBK2, family-1 and family-2 disease variants of TTBK2 (see Fig 1) as well as a TTBK2[1-450] truncation mutant. We observed that both family-1 and family-2 mutations and residue 450-truncation mutants were expressed at higher levels than full-length wild-type or kinase-inactive GFP-TTBK2[D163A] (Fig 3A). Similarly, we also observed increased expression of the truncation mutants of TTBK2 possessing FLAG (SFig 3A) or GST (SFig 3B) tags.

Monitoring the rate of degradation following pulse labelling of cells with 35 Smethionine revealed that both wild-type and disease mutant family-1 TTBK2 displayed a similar ~24 h half-life in HEK293 cells, indicating that there was not a significant difference in the rate at which these proteins are degraded in cells (SFig 4).

We next immunoprecipitated GFP-TTBK2 variants and quantified their intrinsic activity employing the TTBKtide peptide substrate. We calculated specific activity of wild-type and mutant forms of TTBK2 by normalising for differences in protein expression using quantitative LICOR immunoblot analysis. This revealed that fulllength wild-type TTBK2 possessed approxamately10-fold higher specific activity than the family-1 and family-2 disease mutants or the TTBK2(1-450) mutant (Fig 3A). The kinase-inactive full-length TTBK2[D163A] or truncated TTBK2[D163A](1-450) displayed no detectable activity, establishing that activity of TTBK2, rather than that of a contaminating kinase is being measured (Fig 3A). Similar analysis of the specific activity of wild-type and mutants of TTBK2 variants with FLAG (SFig 3A) or GST (SFig 3B) epitope tags confirmed that SCA11 truncating mutations markedly suppressed kinase activity.

Disease causing TTBK2 mutations promote nuclear localisation

We analysed the cellular localisation of stably expressed variants of GFP-TTBK2 in HEK293 cells. This revealed that full-length wild-type TTBK2 (Fig 4

Generation of TTBK2 (family-1 mutation) knock-in mice

Knock-in mice which precisely mimic the one-base insertion of adenosine in exon-13 at nucleotide 1329, observed in SCA11 family-1 [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF], were generated as described in Figure 5. We observed that heterozygous TTBK2 fmly1/+ mice were viable and fertile. The oldest TTBK2 fmly1/+ mice we have thus far analysed are ~1 year of age and display no overt phenotype. Anatomical dissection of 1-year-old mice brains also revealed no discernable abnormality. Breeding of heterozygous TTBK2 fmly1/+ mice resulted in no homozygous TTBK2 fmly1/fmly1 mice being born (Fig 6A ), indicating that the homozygous mutation resulted in embryonic lethality. We therefore dissected embryos at different stages of development and found that at E10-TTBK2 fmly1/fmly1 embryos were detected at the expected Mendelian frequency but displayed major abnormalities compared to littermate TTBK2 fmly1/+ or TTBK2 +/+ embryos. By stage E11 no homozygous TTBK2 fmly1/fmly1 embryos were observed, suggesting that these embryos perished before this stage. Homozygous E10-TTBK2 fmly1/fmly1 embryos were developmentally delayed, lacking prominent sub-divisions of the developing brain and were smaller than wild-type littermates (Figure 6B). These embryos failed to complete embryonic turning movements or undergo normal caudal extension of the body; in several cases the rudimentary caudal body, which contained some somites (future vertebrae, muscle and dermis) and spinal cord, remained folded back on more rostral tissues (Figure 6B). This distortion and under-development of the main body axis (Figure 6B) are the likely causes of embryonic lethality.

Expression and activity of mutant TTBK2 in tissues and fibroblasts derived from knock-in mice

We next analysed endogenous TTBK2 expression and activity in various mouse tissues. This revealed significantly higher levels of TTBK2 protein and activity in brain and testes than other tissues investigated (Fig 7A). In wild-type TTBK2 +/+ brain and testes, TTBK2 migrated on an SDS polyacrylamide gel at the expected ~150 kDa size. As predicted, in tissues of the heterozygous TTBK2 fmly1/+ mice the level of fulllength TTBK2 is reduced by ~50% and a truncated form of TTBK2 migrating at ~50 kDa that is not seen in the wild-type mice is observed (Fig 7B ). We also analysed endogenous TTBK2 catalytic activity after immunoprecipitation from brain and testes extracts and found that TTBK2 activity was reduced ~40 to 50% in tissues derived from heterozygous TTBK2 fmly1/+ compared to wild-type littermate animals, consistent with the truncating mutation suppressing TTBK2 catalytic activity (Fig 7B ).

We also analysed TTBK2 activity in mouse embryonic fibroblasts derived from wildtype TTBK2 +/+ , heterozygous TTBK2 fmly1/+ and homozygous TTBK2 fmly1/fmly littermate stage E10 embryos. TTBK2 activity derived from homozygous TTBK2 fmly1/fmly1 cells was ~90% lower than activity observed in wild-type TTBK2 +/+ fibroblasts (Fig 7C). The heterozygous TTBK2 fmly1/+ displayed intermediate TTBK2 activity.

Discussion

We have undertaken the first characterisation of TTBK2 catalytic activity and substrate specificity, and we find that it possesses a marked preference for a phosphotyrosine residue at the + 2 position from the site of phosphorylation (Fig 1). Sequence comparisons indicate that TTBK2 possesses a phosphate-priming binding site in its catalytic domain equivalent to that in related CK1 isoforms. We find that mutating two key conserved residues within this putative phosphate-binding groove (Arg181 or Ala184) did not affect the ability of TTBK2 to phosphorylate a nontyrosine phosphorylated peptide, but inhibited phosphorylation of a tyrosine phosphorylated peptide (Fig 2). The residues making up this putative phosphatebinding pocket are conserved on TTBK1 suggesting this isoform may also possess a preference for +2 residue phosphotyrosine priming. The key difference in the putative phosphate-binding groove between CK1 and TTBK1/TTBK2 is the presence of a non-basic Ala residue in TTBK1 and TTBK2 rather than a basic Lys in all CK1 isoforms. It would be interesting to explore whether this contributes towards the preference of TTBK2 for phosphotyrosine rather than phosphoserine/phosphothreonine priming in CK1 isoforms.

One of the most studied consensus phosphorylation site for CK1 isoforms is S/Tp-X-X-S/T, where S/Tp refers to a phospho-serine or phospho-threonine, X refers to any amino acid, and the underlined residues refer to the target site [START_REF] Flotow | Phosphate groups as substrate determinants for casein kinase I action[END_REF][START_REF] Flotow | Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase[END_REF][20]. In the case of CK1, the priming phosphorylation site is at the -3 position rather than at the + 2 position for TTBK2. If both TTBK2 and CK1 use a similar phosphate-binding site within their catalytic domain, there would need to be marked differences in the mechanism and orientation at which primed phosphorylated substrates interact with CK1 and TTBK2. In future work it would be interesting to co-crystallise the catalytic domain of TTBK2 with a +2 tyrosine phosphorylated peptide to define the molecular mechanism by which TTBK2 interacts with such substrates and establish whether this putative phosphate binding groove we have analysed is involved. To our knowledge the structure of CK1 bound to an S/Tp-X-X-S/T motif containing peptide has not been reported, but would be of interest to compare with that of TTBK2. It would also be important to mine phosphorylation site databases for proteins phosphorylated on Sp/Tp-X-Yp motifs and determine whether any of these proteins might comprise physiological substrates for TTBK2.

Our analysis has enabled us to elaborate a reasonable peptide substrate for assessing TTBK2 activity (TTBKtide-RRKDLHDDEEDEAMSIYpA) that is phosphorylated with a Km 18 M and a Vmax of 76 U/mg. We have used this assay to investigate how SCA11 truncating mutations impact on TTBK2 activity and demonstrated that two familial SCA11 mutations analysed, induce ~10-fold reduction in TTBK2 protein kinase activity. Truncation of TTBK2 at residue 450 in close proximity to where the familial TTBK2 truncating mutations occurs, similarly suppressed TTBK2 activity. The observation that TTBK2 immunoprecipitated from TTBK2 fmly1/+ mice possessed ~40% less activity ( The truncated TTBK2 mutants were expressed in HEK293 cells at significantly higher levels than the full-length wild-type enzyme (Fig 3 and Supplementary Fig 3). However, in the testes and brain of TTBK2 fmly1/+ mice, more similar levels of expression of the endogenous truncated and full-length forms of TTBK2 were observed (Fig 6). Although the relative expression of full-length and truncated mutants of TTBK2 in patients with SCA11 mutations has not yet been investigated, TTBK2 mRNA levels from lymphoblasts of affected individuals were significantly lower compared to lymphoblast mRNA from unaffected individuals [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. This suggests that the premature stop codon in the TTBK2 mRNA, leads to nonsense-mediated decay of mutated mRNA. It is still likely that some of the truncated SCA11 protein is expressed in SCA11 patients as the mutant transcript was still detected [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. Treating lymphoblasts from SCA11 affected individuals with cycloheximide an inhibitor of nonsense-mediated decay, increased TTBK2 mRNA to levels that are similar in the normal transcript [START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. Taking this data into consideration it is therefore possible that the truncated TTBK2 protein is indeed expressed at much higher levels than the fulllength protein, but due to nonsense-mediated decay, less of the truncated protein is produced. This could account for why in the TTBK2 fmly1/+ mice, similar levels of wild type and truncated mutant protein are observed. In future work it would be important to analyse the relative levels of wild type and mutant TTBK2 mRNA in TTBK2 fmly1/+ mice. We also detected enhanced nuclear localisation of the truncated forms of TTBK2, indicating that inappropriate localisation within the nucleus could potentially contribute to the mechanism by which mutations in TTBK2 lead to SCA11. Potentially, the truncated TTBK2 mutants result in the inappropriate phosphorylation of nuclear proteins.

The developmental delay and small size of homozygous TTBK2 fmly1/fmly1 embryos are consistent with a role for TTBK2 activity in the regulation of fundamental cellular processes, such as cell proliferation. The failure of embryonic turning and under development of the body axis might also lead to a poor placental connection and hence embryonic lethality. TTKB1 expression is prominently localised in cortical and hippocampal neurons [START_REF] Sato | Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation[END_REF]. Further insight into the TTKB2 phenotype and its role in spinocerebellar ataxia would be gained by establishing its expression pattern, particularly with respect to its localisation in the cerebellum. The TTBK2 fmly1/+ mice as well as TTBK2 fmly1/fmly1 MEFs will be useful in validation of potential TTBK2 substrates that are identified.

In conclusion, our study provides some initial insights into substrate specificity of TTBK2 and how SCA11 causing mutations impact on kinase activity and localisation. In future studies it would be important to define the substrates that TTBK2 phosphorylates and investigate whether reduced phosphorylation of these targets contributes to the development of SCA11. In particular, it would be interesting to determine whether physiological TTBK2 substrates were primed with a +2 phosphotyrosine and how regulation of tyrosine phosphorylation was coupled to the control of these TTBK2 targets. Identifying the key targets of TTBK2 could provide vital new insights into the molecular mechanism underpinning the development of spinocerebellar ataxia and result in new ideas as to how this debilitating disease might be better treated in the future. TTBK2 was immunoprecipitated from 30 Fg of cell extract and subjected to kinase activity assay (upper panel) followed by immunoblot analysis (lower panel). TTBK2 kinase activity was quantified employing 30 M TTBKtide and specific activity was calculated by correcting the amount of phosphate incorporation for protein levels in the immunoprecipitate using quantitative immunoblot with the Odyssey system and is presented as cpm/absorbance units (cpm/LICOR Arbitrary Units). The data are the average for duplicate experiments that were repeated four separate times with similar results. Dotted lines indicates that blots were on separate gels. The grey rectangles represent TTBK2 exons. The grey and black triangles represent FRT and Lox P sites, respectively. The positions of the TTBK2 primers (P1 and P2) used for genotyping are represented as short black lines with arrowheads. (B) Genomic DNA purified from the targeted ES cells of the indicated genotypes was digested with either ScaI or AvrI and subjected to Southern analysis with the corresponding DNA probes (positions shown). In the case of the 5' probe, the wildtype allele produces a 9.8kb fragment while the conditional knockin allele generates a 7.3kb fragment. Similarly, the 3' probe detects a fragment of 23.8 kb from the wildtype allele and a 15.kb fragment from the conditional knockin allele.(C) Genomic DNA was PCR amplified with TTBK2 primers P1, P2. The wild-type allele (detected using P1 and P2) generates a 220 bp product while the knockin allele generates a 381 bp product.

Figure 6. Embryonic lethality and knock-in embryo description

(A) TTBK2 fmly1/+ mice were mated and the progeny genotyped as described in the Methods. The number of mice obtained is indicated for each genotype. (B) Wild-type and homozygous TTBK2 fmly1/fmly1 embryos at E10 were detected at the expected Mendelian frequency. Mutant embryos are smaller and developmentally delayed, lacking prominent sub-divisions of the brain (arrowheads on wild-type embryo). Incomplete embryonic turning movements may result in failure to extend the body axis. 27 separate E10 TTBK2 fmly1/fmly1 embryos have been analysed and similar phenotypes were observed.

Figure 7. Study of TTBK2 in wild-type and TTBK2 fmly1/+ knock-in mice (A) The indicated tissue extracts were generated from wild-type mice. Extracts were immunoblotted for TTBK2 (lower panel) or TTBK2 was immunoprecipitated and subjected to TTBK2 kinase assay employing the TTBKtide peptide substrate. The data are the average for duplicate experiments that were repeated four separate times with similar results. (B) Brain and testes lysates were generated from TTBK2 +/+ and TTBK2 fmly1/+ mice and subjected to immunoblot or TTBK2 kinase assay analysis, as in (A). (C) Mouse embryonic fibroblasts (MEFs) were generated from TTBK2 +/+ , TTBK2 fmly1/+ and TTBK2 fmly1/ fmly1 E10 embryos as described in the Methods. TTBK2 activity was assessed following immunoprecipitation as in (A). Due to the low levels of TTBK2 protein expressed in MEFs and high antibody background in immunoprecipitates, we were unable to detect expression of TTBK2 by immunoblot analysis. Licenced copy. Copying is not permitted, with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110276 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110276 A c c e p t e d M a n u s c r i p t reaction was spotted onto P81 phosphocellulose paper. After extensive washing in 50 mM phosphoric acid, reaction products were quantified by Cerenkov counting. Km and Vmax parameters were calculated using the Michaelis-Menten model of nonlinear regression with the GraphPad Prism V5 program.

Biochemical

  Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110276 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110276 A c c e p t e d M a n u s c r i p t

  , panel 1) or full-length, kinase-inactive TTBK2[D163A] (Fig 4, panel 2) was expressed at higher levels in the cytosol than the nucleus. However, TTBK2(family-1 mutation) (Fig 4, panel 3) or truncated TTBK2(1-450) (Fig 4, panel 4&5 ) were expressed at similar levels in both the cytosol and the nucleus. Quantitation of the ratio's of nuclear versus cytosolic TTBK2, establishes that ~2-fold higher proportion of TTBK2 (family-1 mutation) and truncated TTBK2(1-450) is localised in the nucleus compared to full length TTBK2 (Fig 4, panel 7).

  Fig 7B) and TTBK2 immunoprecipitated from TTBK2 fmly1/fmly1 MEFs (Fig 7C) had ~10-fold lower activity, also supports the notion that SCA11 mutations drastically reduce endogenous TTBK2 activity. Thus these findings indicate that loss of TTBK2 activity could underpin development of SCA11 in patients. It would be important to study in more detail the mechanism by which the SCA11truncating mutations lead to suppression of TTBK2 protein kinase activity. The SCA11 truncating mutations leave the kinase catalytic domain intact removing a Cterminal non-catalytic region possessing no obvious functional domains (Fig 1A). The removal of the C-terminal non-catalytic region of TTBK2 might delete a motif required for kinase activation, activity or proper folding of the catalytic domain. Depending upon the mechanism by which SCA11 mutations suppress TTBK2 catalytic activity, it may be worth undertaking a drug screen to determine if it is Biochemical Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110276 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110276 A c c e p t e d M a n u s c r i p t possible to identify compounds that could lead to the re-activation of truncated TTBK2 mutants. Such compounds could have potential for treating patients with SCA11.

Biochemical

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Figure Legends Figure 1 .

 1 Figure LegendsFigure 1. Domain structure and substrate specificity of TTBK2 (A) Schematic representation of the domain structure of TTBK2 showing the location of the three reported SCA11 causing mutations. Numbering of residues corresponds to human TTBK2. (B) Recombinant HEK293 purified GST-TTBK2 (1-450) WT and catalytically inactive GST-TTBK2 (1-450) D163A were used to screen a positional scanning peptide library consisting of 189 biotinylated peptide libraries in individual kinase assays. Reaction products were bound to streptavidin-coated membrane and, after washing, phosphorylation was visualised by phospho-imaging. (C) Peptides with various positions of the phosphotyrosine residue from +4 to -5 relative to the phosphorylated serine residue were synthesised and analysed for their ability to phosphorylate GST-TTBK2 (1-450) WT purified from HEK293 cells. Km and Vmax values were derived by non-linear regression analysis as described in the Methods section. (D) Three TTBKtide variants with serine, threonine and tyrosine at the phospho-acceptor position and a TTBKtide variant with a phosphothreonine at the +2 position were synthesised and the kinetics of their phosphorylation by GST-TTBK2[1-450] was analysed. Km and Vmax values were derived by non-linear regression analysis. NP denotes that the peptide was phosphorylated too poorly to undertake accurate kinetic analysis.

Figure 2 .

 2 Figure 2. Molecular basis for phosphate priming (A) High resolution structure of CK1 delta protein showing the sulphate binding site on the C-lobe of the kinase domain predicted to function as the phosphate priming region [22]. Lysine 38 (site A), Lysine 130 (site B), Arginine 168 (site C) and Lysine 171 (site D) form ionic interactions with the sulphate residue and are shown in orange. (B) Sequence alignment of the indicated species of TTBK1, TTBK2 and CK1 family enzymes showing the sequence conservation of the sulphate-binding residues. (C) Coomassie-stained gel of the GST-TTBK2[1-450] truncated form of the indicated wild-type (WT) and mutant proteins expressed and purified from HEK293 cells. (D) The indicated GST-TTBK2[1-450] truncated forms expressed in (C) were tested for their ability to phosphorylate TTBKtide (RRKDLHDDEEDEAMSIY P A) and a variant of TTBKtide (RRKDLHDDEEDEAMSIYA) where the tyrosine at the +2 position is not phosphorylated. Km and Vmax values were derived by non-linear regression analysis.

Figure 3 :

 3 Figure 3: Truncated form of TTBK2 are less active but express at a higher levels than the full-length kinase HEK293 cells were transiently transfected with the wild-type (WT) and indicated mutants of Flag-tagged (A & B) or GST-tagged (C & D) TTBK2. (A & C) Cells were lysed and subjected to immunoblotting with the anti-TTBK2 antibody and the other indicated antibodies. (B & D)TTBK2 was immunoprecipitated from 30 Fg of cell extract and subjected to kinase activity assay (upper panel) followed by immunoblot analysis (lower panel). TTBK2 kinase activity was quantified employing 30 M TTBKtide and specific activity was calculated by correcting the amount of phosphate incorporation for protein levels in the immunoprecipitate using quantitative immunoblot with the Odyssey system and is presented as cpm/absorbance units (cpm/LICOR Arbitrary Units). The data are the average for duplicate experiments that were repeated four separate times with similar results. Dotted lines indicates that blots were on separate gels.

Biochemical

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Figure 4 .

 4 Figure 4. SCA11 disease mutations promote TTBK2 nuclear localisationHEK293 cells stably expressing the indicated forms of GFP-TTBK2 were treated with 1 g/ml tetracycline for 16 hours to induce the expression of TTBK2 (panels 1 to 6). Cells were fixed with 4% paraformaldehyde and stained with antibody recognising TTBK2 and with DAPI for nuclear staining. Fluorescent imaging was performed on a confocal microscope. Similar results were obtained in four independent experiments. Quantitation of nuclear versus cytoplasmic levels of TTBK2 (panel 7) was undertaken as described in the Materials and methods. For each cell lines between 260 and 460 cells were counted over 10 fields of cells.

Figure 5 .

 5 Figure 5. Targeting strategy used to generate TTBK2 knock-in mutations (A) Diagram describing the knock-in construct, the endogenous allele containing exon 13 and the targeted allele with the puromycin cassette removed by Flp recombinase.The grey rectangles represent TTBK2 exons. The grey and black triangles represent FRT and Lox P sites, respectively. The positions of the TTBK2 primers (P1 and P2) used for genotyping are represented as short black lines with arrowheads. (B) Genomic DNA purified from the targeted ES cells of the indicated genotypes was digested with either ScaI or AvrI and subjected to Southern analysis with the corresponding DNA probes (positions shown). In the case of the 5' probe, the wildtype allele produces a 9.8kb fragment while the conditional knockin allele generates a 7.3kb fragment. Similarly, the 3' probe detects a fragment of 23.8 kb from the wildtype allele and a 15.kb fragment from the conditional knockin allele.(C) Genomic DNA was PCR amplified with TTBK2 primers P1, P2. The wild-type allele (detected using P1 and P2) generates a 220 bp product while the knockin allele generates a 381 bp product.

Biochemical

  FIGURE 1

FIGURE 4

 4 FIGURE 4

Biochemical

  Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110276 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110276 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  FIGURE 5

  FIGURE 6

  Biochemical Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110276 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110276

  Biochemical Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110276 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110276

Probe 5' Digest:ScaI Probe 3' Digest:AvrII +/+ +/+ +/fmly1 +/fmly1 +/fmly1 +/fmly1

			12		Lox P Frt	F rt	13	Lox P	14
	Targeting vector	Tk					Puro
			Avr II ScaI	ScaI					ScaI	ScaI	Avr II ScaI ScaI
		1	3	1 2			13	14		15	16
	Endogenous allele
				5' Probe				3' Probe
					9.8 kb
					15 kb
			Avr II ScaI	ScaI			ScaI				ScaI	ScaI	ScaI ScaI	Avr II
	(after homologous Targeted allele	1	3	1 2	Lox P Frt	Puro	F rt 13 Lox P	14	15	16
	recombination)
					7.3 kb
					23.8 kb
	Constitutive KI allele	1		3	1 2	Lox P Frt	13 Lox P	14	15	16
	affter Flp
	recombination
		9.8 kb					23.8 kb
		7.3 kb					15 kb

+/

+ +/fmly1 fmly1/ fmly1 SOUTHERN BLOTS PCR

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

We thank Gail Fraser for genotyping of the mice, the DNA Sequencing Service (School of Life Sciences, University of Dundee, Scotland) and the protein production and antibody purification teams [Division of Signal Transduction Therapy (DSTT), University of Dundee] coordinated by Hilary McLauchlan and James Hastie. NE is funded by an MRC-UK Studentship. EF is a PhD student supported by the Wellcome Trust and KGS is funded by an MRC programme grant G0600234. We thank the Medical Research Council, the European Regional Development Fund grant for an Innovation Pipeline for Translational Science (LUPS/ERDF/2008/2/1/0429) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KgaA and Pfizer) for financial support.

Author

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


K Baczynska
email: k.a.baczynska@gmail.com

J Billowes

P Campbell

F C Charlwood

B Cheal

T Eronen

D H Forest

A Jokinen

T Kessler

I D Moore

M Rüffer

G Tungate

J Äystö

Nuclear spin determination of 100m Y by collinear laser spectroscopy of optically pumped ions

Keywords: numbers: 21.10.Ft, 21.10.Hw, 21.10.Ky, 42.62.Fi

published or not. The documents may come

Nuclear spin determination of 100mY by collinear laser spectroscopy of optically pumped ions

Region of the sudden onset of deformation around Z = 40

Nuclear structure close to the Z = 40 subshell has been the subject of extensive interest for many years. Studies of this region have included recent precise mass determinations [1,2], γ-spectroscopy [3,4] and optical measurements. The optically measured Kr [5], Rb [6], Sr [7,8,9], Y [10], Zr [11,12], Nb [13] and Mo [14] isotope chains provide considerable experimental information about ground and isomeric state properties. In the cases of Rb and Sr, the optical measurements revealed two regions of strong ground state deformation, with static quadrupole deformation parameters β 2 ≈ 0.4, for N ≈ 38 and N ≈ 60. The Zr, Y and Nb isotopes were subsequently measured using collinear laser spectroscopy and all three isotope chains also show a sharp onset of strong deformation at N = 60. Figure 1 illustrates the change in meansquare charge radius as a function of the neutron number and shows the similarities of these isotope chains. The Kr isotope chain measurements show that the evolution of the mean-square charge radius is similar to that in higher Z chains, except at the neutron number N = 60. The lack of a sudden onset of deformation at N = 60 sets the lower limit in Z for this phenomenon. The corresponding upper limit in Z for this shape change has recently been determined from measurements of the Mo isotope chain. Figure 1 shows that the charge radius in this system increases smoothly from the N = 50 shell closure across N = 60 with no sudden increase.

The sudden shape change from soft, weakly oblate to more rigid, strongly prolate shapes observed for the isotopes with 42 > Z > 36 has also been studied theoretically. Shell model calculations attribute the onset of deformation to the isoscalar component of the n-p interaction between nucleons in the spin-orbit partner orbitals (1g 9/2 ) π and (1g 7/2 ) ν [15]. However, mean-field models explain the deformation in terms of the occupation of low K components of the (1h 11/2 ) ν orbital [16]. Recent calculations using the projected shell model suggest that both of these mechanisms play a rôle in the sudden onset of strong deformation at N = 60 [17].

Yttrium study

The main interest of the odd-Z yttrium chain (Z = 39) was based on the investigation of the nuclear shapes and deformations along the isotope chains in the region of Z = 40. The yttrium study was especially important for the region between the neutron shell closure and onset of strong deformation in order to address the lack of information on quadrupole moments and deformations for strontium [8] and zirconium isotopes [11].

Isotope shift and hyperfine structure measurements provide complementary information about the nuclear deformation. There are two main contributions to the mean-square charge radius r 2 : the nuclear size, and mean-square deformation. Any variation from a spherical shape contributes to the mean-square charge radius and to the lowest order in deformation, r 2 is given by: where r 2 s is the mean-square charge radius of an equivalent volume spherical nucleus, which can be calculated from the liquid drop model, or more reliably from the finiterange droplet model of Myers and Schmidt [18], and β 2 2 is the mean-square quadrupole deformation parameter which includes both static and dynamic contributions:

r 2 = r 2 s 1 + 5 4π β 2 2 (1)
β 2 2 = β 2 2 + β 2 2 -β 2 2 = β 2 static + β 2 dynamic (2)
The deformation, β 2 , may be extracted from the hyperfine structure via the electric quadrupole interaction term, which provides the spectroscopic electric quadrupole moment, Q s . This can be related to β 2 via the projection from the intrinsic frame [START_REF] Kopfermann | Nuclear Moments[END_REF]:

Q s = Q 0 I (2I -1) (I + 1) (2I + 3) (3)
and the relation

Q 0 ≈ 5Z r 2 s √ 5π β 2 (1 + 0.36 β 2 ) ( 4 )
It is evident from (3) that Q s can only be measured for isotopes with nuclear spins I ≥ 1. The lack of quadrupole moment information for the Sr and Zr isotope chains is due to many of their odd-A isotopes having ground state spins of I = 1/2.

In order to define the nuclear behaviour in this region the isotopes 86-90,92-102 Y have been measured using collinear laser spectroscopy at the IGISOL facility at the University of Jyväskylä, Finland. The strong transition from the Y + ground state, 5s 2 1 S 0 → 4d5p 1 P 1 at 363.31 nm, was used in two experiments reported in reference [10]. The isotope shift and hyperfine structure analysis of these data provided the magnetic dipole, µ, and spectroscopic electric quadrupole, Q s , moments and changes in meansquare charge radii, δ r 2 . These showed that below the N = 50 shell closure, the isomers at least are oblate with significant dynamic deformation, which is demonstrated by the large difference between the measured r 2 and that calculated from (1) using only the contribution from the static deformation, as shown in figure 2.

As the neutron number increases from N = 50 the nuclear deformation exhibits an increasingly oblate and increasingly soft nature [10]. At N = 60 there is a sudden change to more rigid, strongly deformed prolate shapes.

Yttrium isotopes seem to follow the Rb, Sr and Zr trends. The large shift observed at N = 59 between the 98 Y ground and isomeric states is a consequence of the nuclear shape changing from oblate to strongly prolate. Unfortunately, for this ionic ground state transition there is a maximum of only three hyperfine structure components, which made it impossible to simultaneously determine µ, Q s , δ r 2 A,A and the nuclear spin for 97m2,98m,100,102 Y. The situation in 100 Y is complicated further as the observed hyperfine structure could arise from either the τ 1/2 = 735 ms ground state with I = 1 or 2, or from the τ 1/2 = 0.94 s isomeric state at 145 keV with I = 3, 4 or 5 [START_REF] Singh | [END_REF]. The measured spectrum was fitted for the five different spin possibilities, I = 1 -5, giving various spin-dependent values for the nuclear parameters µ, Q s and δ r 2 89,100 . Unfortunately only one value, with nuclear spin I = 1, can be rejected because the analysis gives an unphysically large static deformation parameter β 2 > 0.7 [10]. The predicted r 2 , using a static contribution only for spins I = 3, 4 and 5, are included in figure 2. Therefore, a definitive spin assignment is required to define the nuclear properties. This work describes how an unambiguous nuclear spin assignment was achieved for the observed state in 100 Y.

Preparation to experiment and efficiency tests

In the previous experiment [10], the transition from 1 S 0 to 1 P 1 in Y + was measured. This kind of transition is preferable for laser spectroscopy when the nuclear spin is known because it significantly reduces the number of hyperfine structure components allowing the whole structure to be measured in a short time. However, to complete the nuclear spin investigations another strong transition needs to be measured with an atomic spin J > 1 to complement existing data. During exploratory efficiency tests, a stable 89 Y + ion beam of up to 1 picoampere, produced by a discharge source at the IGISOL [21], was brought onto resonance with ∼ 1 mW of laser light. Results showed that the best measured photon to ion efficiency for the transition from the low lying metastable state at 1045.083 cm -1 was at least two times lower than the efficiency for the transition previously measured from the ground state. Optical pumping [13] was applied to enhance the population of the 1045.083 cm -1 state, improving the spectroscopic efficiency. The 3 D 2 state population was enhanced by illuminating the ion beam in the cooler/buncher [22] with ∼ 5 mW of 363.31 nm laser light. This was produced by frequency doubling the output of a Ti:Sapphire laser which was pumped by a pulsed Nd:YAG laser working at 10 kHz. The effect of the optical pumping and the level scheme for the pumping are shown in figure 3. The optical pumping enhanced the efficiency sufficiently to make the transition from the 3 D 2 state just possible for on-line measurements.

The on-line experiment

Radioactive yttrium isotopes from mass A = 96 to A = 100 were produced at the IGISOL, JYFL by proton induced fission of natural uranium at 30 MeV. The reaction products were thermalized and partially neutralized to singly charged ions in fast flowing helium gas, electrostatically extracted and mass separated. A helium gas pressure of 250 mbar was found to give the best production yields of the radioactive isotopes. In order to keep the plasma in the reaction part of the chamber, and slow down the fast fission fragments, a thin separation foil made from 89 Y was used [21]. Ion rates ranged from 2,100 ions/s for A = 100 to 2,800 ions/s for A = 98, the most strongly produced. The 89 Y + beam produced from recoils from the thin 89 Y foil had an ion rate of 86,000 ions/s. A dye mixture of Rhodamine 6G and Rhodamine 640 in a Spectra Physics 380 dye laser with an intra cavity β-BBO frequency doubling crystal were used to generate 0.8 mW of 321.67 nm UV light. The laser was locked to the iodine absorption line at 15526.1851 cm -1 [START_REF] Gerstenkorn | Atlas du Spectre D'Absorption de la Molecule d'Iode[END_REF]. For the optical pumping step less than 5 mW of laser power was produced at 363.31 nm wavelength. Spectra were taken for stable 89 Y and radioactive isotopes 96,98,99 Y to provide calibration of the mass and field factors of the isotope shift [START_REF] King | Isotope Shifts in Atomic Spectra[END_REF] and the hyperfine A and B coefficients for the 321.67 nm transition.

The spectra of the yttrium isotopes measured using optical pumping are presented in figure 4. Complete structures were measured for 89,96,98,99 Y. Due to low yield and the distribution of spectral strength over many hyperfine structure components, it was necessary, but sufficient, to select only the two strongest components of the 100 Y structure. The mass and field factors for the 321.67 nm transition, along with the hyperfine A and B coefficients measured for 89 Y and 99 Y in this work, were used together with the data from [10] to estimate the location of these strongest hyperfine components of 100 Y for possible spin values in the range I = 1 to 5.

Data analysis

The hyperfine splitting of an atomic or ionic energy level is given by [START_REF] Kopfermann | Nuclear Moments[END_REF]:

E hfs = A 2 K + B 4 3 2 K(K + 1) -2I(I + 1)J(J + 1) I(2I -1)J(2J -1) . ( 5
)
where K=F (F +1)-J(J+1)-I(I+1), F is the total angular momentum (where F=I+J) and A and B are the magnetic dipole and electric quadrupole hyperfine structure coefficients, respectively. These parameters are related to the nuclear magnetic dipole moment, µ, and spectroscopic quadrupole moment, Q s , via:

A = µB el IJ , B = eQ s ∂ 2 V el ∂z 2 . ( 6
)
As the magnetic field, B el , and the gradient of the electric field, ∂ 2 V el /∂z 2 , produced by the atomic electrons at the nucleus are independent of the isotope, the magnetic moment, µ x , and the spectroscopic quadrupole moment, Q x s , for an isotope x can be determined from:

A x = µ x I x I y µ y A y , B x = Q x s Q y s B y (7)
where the hyperfine parameters A y and B y , moments µ y and Q y s , and the nuclear spin, I y , are known for a reference isotope y.

The change in mean-square charge radius between two isotopes, δ r 2 A,A , is related to the measured isotope shift, δν A,A , via [START_REF] King | Isotope Shifts in Atomic Spectra[END_REF]:

δν A,A = M A -A AA + F δ r 2 A,A (8)
where M and F are the transition dependent, but isotope independent, mass and field factors respectively. The nuclear spin determination is based on the predictions of the hyperfine structure constants and isotope shifts for the possible spin values. As the hyperfine structure is dominated by the magnetic dipole interaction, it was essential to find very accurately the ratio of hyperfine A values for the upper and lower states involved in the transition. Spectra of 89 Y measured during the off-line tests were fitted using a chi-squared minimization technique to extract the centroid of the structure and the hyperfine A parameters for the lower 3 D 2 and upper 3 P 1 states. A weighted average over all the individual measurements gave the final values of A( 3 D 2 )=-220.17 [START_REF] Gerstenkorn | Atlas du Spectre D'Absorption de la Molecule d'Iode[END_REF] MHz and A( 3 P 1 )=-97.98(39) MHz, with their ratio A( 3 P 1 )/A( 3 D 2 )=+0.445 (2). From the data in table 1, it can also be seen that the hyperfine A factors for 99 Y have the same ratio, +0.445(3), which indicates that there is no significant hyperfine anomaly in this transition.

Spectra for 96,98,99 Y measured during the on-line experiment were also fitted using a chi-squared minimization routine to obtain the centroids and the hyperfine structure parameters. To eliminate any time dependent frequency drifts, the reference isotope 89 Y was regularly measured between measurements of radioactive isotopes. Table 1 displays the isotope shifts relative to the reference 89 Y determined from the fitted centroids and the hyperfine structure parameters A and B.

Spin determination of 100m Y

The isotope shifts given in table 1 were combined with those measured previously in the 363.31 nm transition [10] via a King Plot [START_REF] King | Isotope Shifts in Atomic Spectra[END_REF], as shown in figure 5. Using the field, F 363 = -3181(57) MHz fm -2 , and mass, M 363 = +1789(40) GHz, factors determined for the 363.21 nm ground state transition [10], the King Plot allowed the field and mass factors of the 321.67 nm transition measured in this work to be evaluated as F 321 = -1343(25) MHz fm -2 and M 321 = +107(17) GHz respectively. The isotope shifts and nuclear moments evaluated in reference [10] for spin values I = 1 to 5 were used with the calibration data obtained in this work to predict the hyperfine structure and centroid of the produced 100 Y state relative to the centroid of the 89 Y structure for the 3 D 2 to 3 P 1 transition for each possible spin. The δ r 2 , µ and Q s values from reference [10], along with the predicted isotope shifts relative to the 89 Y centroid and hyperfine structure coefficients for the 3 D 2 level are given in table 2 for each possible spin considered. The hyperfine parameters for the 3 P 1 level were estimated using the values calculated for the 3 D 2 level, the ratio of A factors determined from 89 Y and the ratio of B coefficients from 99 Y.

Figure 6 illustrates the predicted spectra for the nuclear spins I = 1 to 5 together Table 2. The spin-dependent values of δ r 2 89,100 and nuclear moments from reference [10] along with the predicted isotope shift and hyperfine structure constants for all possible spin assignments.

Spin δ r 2 89,100 δν 89,100 with the two experimentally measured peaks for 100 Y. It can be seen that the two measured peaks are only matched with the predicted spectrum for the nuclear spin I = 4. The structure observed in this work, and in the work of reference [10], therefore arises from the τ 1/2 = 0.94 s isomeric state in 100 Y (which is either spin 3, 4 or 5 [START_REF] Singh | [END_REF]) rather than from the lower spin ground state. The uncertainty in the predicted centroid positions is only ∼ 0.2%, which is too small to significantly affect the predicted peak positions. The magnetic dipole and electric quadrupole moments are known with a precision of 1% and 11% respectively. These give, together with the statistical errors from the fitting routine, a precision of the A and B values of 1% and 25% respectively. As the hyperfine structure is dominated by the magnetic dipole interaction, the 25% uncertainty on the B coefficient has a negligible effect on the predicted spectra.

µ A( 3 D 2 ) Q s B( 3 D 2 ) I (fm 2 ) (MHz) (µ N ) (MHz) (b) (MHz) 1

Discussion and Interpretation

The unambiguous spin assignment of 100m Y allows the nuclear deformation to be determined from the data of reference [10], where the full hyperfine spectrum was measured. As presented in figure 2, 100m Y follows the sudden shape change at N = 60 observed in neighbouring isotopes. For spin I = 4, β 2 = +0.35(4) was taken from the data of reference [10] and it shows that the nucleus has a prolate shape. The difference between the deformation obtained from the isotope shift data and that obtained from the hyperfine structure data, as seen in figure 2, is quite large which indicates a sizable dynamic contribution to the deformation.

The behaviour of 100m Y reported here is notably different to that observed in the ground states of its neighbouring even-N isotopes, 99,101 Y, which display static and rigid deformations. The nature of the deformation in 100m Y does however closely resemble that observed in the deformed isomeric state of 98 Y [10]. In reference [10] it was possible to show, irrespective of the spin assignment in 98m Y, that this system had a large meansquare charge radius and yet a relatively small (but prolate) quadrupole deformation. The N = 59 isomer was reported to be the softest configuration in yttrium observed to date and it was believed that after the shape change, more rigidly deformed shapes were being observed with increasing neutron number. For the odd-N isomeric system 100m Y this is clearly not the case. Moreover if the 102 Y structure reported in reference [10] arises from the isomeric configuration then this nucleus must also be assigned a soft structure (although a spin determination will be required before a definitive assignment can be made). Unlike the zirconium isotopes beyond N = 59, the known yttrium systems now show an alternating character in their deformation between rigid shapes observed in the even-N ground states to the soft deformations displayed by the odd-N isomers.

The assignment of I = 4 for 100m Y also gives information on the probable twoquasiparticle configuration of this isomer. Studies of the β --decay from this state, and the lowest-lying single-particle Nilsson orbitals in neighbouring nuclei, led Lhersonneau et al to conclude that the most likely two-quasiparticle configurations were either {π The two remaining possible configurations can be distinguished by considering the magnetic moment. The moment for a two-quasiparticle state can be estimated using the additivity theorem [27]:

µ = I I + 1 (g R + g Kπ K π + g Kν K ν ) ( 9 )
where K π and K ν are the projections of the uncoupled proton and neutron spins along the nuclear symmetry axis respectively, and I = |K π ± K ν |. The g-factors g Kν = -0.48(3) and g Rν = +0.26(4) for the ν[411]3/2 orbital were deduced from laser spectroscopic [11,12] and integrated perturbed angular correlation [28], measurements of 101 Zr, while g Kπ = +1.61(4) and g Rπ = 0.44 (10) can be extracted for the π[422]5/2 Nilsson state using the magnetic moment, µ = +3.18(2) µ N , intrinsic quadrupole moment, Q 0 = +4.34(48) b [10] and (g K -g R )/Q 0 = 0.27(3) [26] measured for 99 Y. The g-factors for the π[303]5/2 state can be estimated as g Kπ g Rπ = Z/A = 0.39 because (g K -g R )/Q 0 ∼ 0 for this orbital [26]. Using equation 9, these gfactors give estimates of µ = +2.92 µ N and µ = +0.46 µ N for the K π = 4 + and K π = 4 -configurations respectively. Clearly, only the {π[422]5/2 ⊗ ν[411]3/2} 4 + twoquasiparticle configuration gives a moment that is consistent with the measured value of +2.75(1) µ N .

Conclusion

In order to extend the investigation of the nuclear shape and deformation of the yttrium isotope chain above the sudden shape change at N = 60, the nuclear spin of 100m Y has been measured by collinear laser spectroscopy with optical pumping at the IGISOL facility at the University of Jyväskylä, Finland.

The unambiguous spin assignment I = 4 provides critical information about the magnetic dipole and electric quadrupole moments, mean-square charge radii difference, δ r 2 89,100m , the nuclear deformation and the two-quasiparticle configuration. It is observed that 100m Y follows the sudden shape change to a strongly prolate shape, seen in the neighbouring isotope chains: Sr, Rb, Zr and Nb. However, unlike the 99,101 Y isotopes, this state in 100 Y also has a strong dynamic component to its deformation. This discrepancy illustrates the richness of nuclear behaviour in this region, and provides compelling reasons for extending the nuclear spin investigations for the remaining 100g,102g,m Y isotopes.

Figure 3 .

 3 Figure 3. Spectra showing the three hyperfine components for the 321.67 nm transition in 89 Y + with and without optical pumping. Insert shows the pumping scheme used.

Figure 4 .

 4 Figure 4. Resonance fluorescence spectra for 89,96,98,99 Y and two peaks from 100 Y for the ionic transition at 321.67 nm using optical pumping.

-Figure 5 .

 5 Figure 5.King Plot of the modified isotope shifts of the 96,98,99 Y isotopes relative to the reference isotope 89 Y obtained from the 321.67 nm transition plotted against the modified δν 89,96 , δν 89,98 and δν 89,99 from the 363.31 nm transition.

Figure 6 .

 6 Figure 6. The predicted spectra of 100 Y for the nuclear spin I=1,2,3,4,5 with two measured peaks from 100 Y.

  [422]5/2 ⊗ ν[411]3/2} 4 + or {π[422]5/2 ⊗ ν[532]5/2} 5 -[25]. Also, the configuration {π[303]5/2 ⊗ ν[411]3/2} 4 -must be considered, as this corresponds to the parallelcoupled partner of the {π[303]5/2 ⊗ ν[411]3/2} 1 -configuration tentatively assigned to the ground state of 100 Y [26]. Of these candidates, only the K π = 5 -possibility is eliminated by the spin assignment of I = 4.

  The yttrium mean-square charge radii for the ground and isomeric states. Experimentally determined mean-square charge radii are indicated by filled symbols.

		20					I=2
		19.75		Ground state		I=3
	<r 2 > (fm 2 )	18.75 19 19.25 19.5	|β 2 |= 0 . 4 |β 2 |= 0 . 5	Isomeric state Unidentified state	I=5 I=4	I=5 I=4	|β 2 |= 0 .0 |β 2 |= 0 . 1 |β 2 |= 0 .2 I=3
		18.25 18.5	|β 2 |= 0 . 3
		18
		47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17.75
				Neutron Number, N
			Figure 2.

Open symbols indicate mean-square charge radii calculated from (1) using only the contribution from the static deformation. Isodeformation contours are included as dashed lines.

Table 1 .

 1 Isotope shifts, δν 89,A , relative to 89 Y, and the hyperfine A and B parameters for the lower and upper states extracted from the analysis of the on-line data. Quoted errors are the statistical error arising from uncertainties in the fitting of the spectra.

	Mass I π	δν 89,A	A( 3 D 2 )	A( 3 P 1 )	B( 3 D 2 )	B( 3 P 1 )
	A			( M H z )	( M H z )	( M H z )	( M H z )	( M H z )
	89 96	1 2 0 --1068(10) --0 -220.17(23) -97.98(39) ---	--
	98	0 --1414(4)	-	-	-	-
	99	5 2	+	-2550(10) +1023(4)	+455(2)	+200(38) -145(10)

Acknowledgments

This work has been supported by the UK STFC, the EU 6th Framework programme "Integrating Infrastructure Initiative-Transnational Access", Contract No. 506065 (EURONS) and by the Academy of Finland Centre of Excellence Programme 2006-2011 (Nuclear and Accelerator Based Physics Programme at JYFL).