Adrian Alder

Iris Holdermann

Peter Beyer

Salim Al-Babili
email: salim.albabili@biologie.uni-freiburg.de

Carotenoid oxygenases involved in plant branching catalyze a highly specific, conserved apocarotenoid cleavage reaction

Keywords: Apical Dominance, Apocarotenoids, β-Apo-13-Carotenone, Carotenoid Cleavage, Plant Branching Inhibitor, Plant Development CCD, carotenoid cleavage dioxygenase, max, more axillary growth, rms, ramosus, HPLC, high performance liquid chromatography, LC-MS, liquid chromatography-mass spectrometry

come

Carotenoid oxygenases involved in plant branching catalyze a highly specific, conserved apocarotenoid cleavage reaction

INTRODUCTION

Apart from their established roles as photoprotective and light-harvesting pigments in photosynthesis, carotenoids fulfill more ubiquitous functions as precursors for an ever-increasing number of physiologically important compounds, termed apocarotenoids. Examples of such carotenoid derivatives are represented by the opsin chromophore retinal, the morphogen retinoic acid, the phytohormone abscisic acid (ABA) and the fungal pheromone trisporic acid. In general, apocarotenoids are synthesized through oxidative cleavage of their precursors mediated by carotenoid cleaving (di)oxygenases (CCD; the mono-or dioxygenase mechanism is still under debate) [for reviews, see [START_REF] Moise | Related enzymes solve evolutionarily recurrent problems in the metabolism of carotenoids[END_REF][START_REF] Bouvier | Oxidative tailoring of carotenoids: a prospect towards novel functions in plants[END_REF][START_REF] Auldridge | Plant carotenoid cleavage oxygenases and their apocarotenoid products[END_REF][START_REF] Kloer | Structural and biological aspects of carotenoid cleavage[END_REF].

The research on carotenoid oxygenases was paved by the identification of VP14 (viviparous14) from maize. VP14 is a 9-cis-epoxy-carotenoid dioxygenase catalyzing the formation of the ABA-precursor xanthoxin [START_REF] Schwartz | Specific oxidative cleavage of carotenoids by VP14 of Maize[END_REF], which represents the rate-limiting step in ABA-biosynthesis [START_REF] Qin | Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance[END_REF][START_REF] Iuchi | Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoids dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis[END_REF]. Subsequently, the occurrence of homologs in all taxa was discovered in silico, which allowed to elucidate the biosynthesis of several carotenoid-derived compounds, such as retinal in animals [START_REF] Von Lintig | Filling the gap in vitamin A research[END_REF][START_REF] Redmond | Expression, and substrate specificity of a mammalian β-carotene 15,15´-dioxygenase[END_REF] and fungi [START_REF] Prado-Cabrero | Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi[END_REF], the pigments bixin [START_REF] Bouvier | Biosynthesis of the food and cosmetic plant pigment bixin (annatto)[END_REF], saffron [START_REF] Bouvier | Oxidative remodeling of chromoplast carotenoids: Identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis[END_REF] and neurosporaxanthin [START_REF] Prado-Cabrero | Identification and biochemical characterization of a novel carotenoid oxygenase: elucidation of the cleavage step in the Fusarium carotenoid pathway[END_REF]. In addition, carotenoid oxygenases mediating the formation of volatile compounds, such as β-ionone, were identified from several plants [START_REF] Schwartz | Characterization of a novel carotenoid cleavage dioxygenase from plants[END_REF][START_REF] Simkin | The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone[END_REF]. Moreover, the characterization of homologous enzymes unveiled novel reaction mechanisms, such as the specific cleavage of apocarotenoids instead of bicyclic carotenoids and their conversion into retinal in cyanobacteria [START_REF] Ruch | Retinal biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF][START_REF] Scherzinger | Retinal is formed from apo-carotenoids in Nostoc sp. PCC7120: in vitro characterization of an apo-carotenoid oxygenase[END_REF][START_REF] Marasco | Identification of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120 with different cleavage activities[END_REF].

Apart from ABA, additional carotenoid-derived signals occur in plants, awaiting their molecular identification [START_REF] Mouchel | Novel phytohormones involved in long-range signaling[END_REF]. It was shown, for instance, that the Arabidopsis bypass1 mutant, affected in several developmental processes, can be partially rescued by inhibitors of carotenoid biosynthesis [START_REF] Van Norman | Dissecting the biosynthetic pathway for the bypass1 root-derived signal[END_REF].

Moreover, apical dominance mutants of several plant species caused by lesions in two divergent carotenoid oxygenase genes indicated the involvement of an apocarotenoid signal required for maintaining normal plant architecture [START_REF] Leyser | The fall and rise of apical dominance[END_REF][START_REF] Dun | Apical dominance and shoot branching. Divergent opinions or divergent mechanisms?[END_REF][START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF][START_REF] Sorefan | MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea[END_REF].

Apical dominance is mediated by apically derived auxin, which is transported basipetally. However, several lines of evidence excluded a direct mode of action by auxin [START_REF] Mcsteen | Shoot branching[END_REF], rather it is thought to exert its function through second messengers, like cytokinin [START_REF] Leyser | Regulation of shoot branching by auxin[END_REF][START_REF] Turnbull | Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance[END_REF][START_REF] Nordstrom | Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin-cytokinin-regulated development[END_REF]. However, the analysis of the more axillary growth (max1-4) mutants from Arabidopsis [START_REF] Turnbull | Micrografting techniques for testing long-distance signalling in Arabidopsis[END_REF], the ramosus (rms1, 2, 5) mutants from pea [START_REF] Beveridge | Branching in pea (action of genes rms3 and rms4)[END_REF][START_REF] Beveridge | Long-distance signalling and a mutational analysis of branching in pea[END_REF][START_REF] Morris | Mutational analysis of branching in pea. Evidence that RMS1 and RMS5 regulate the same novel signal[END_REF], and the decreased apical dominance 1 (dad1) mutant from petunia [START_REF] Napoli | Highly branched phenotype of the petunia dad1-1 mutant is reversed by grafting[END_REF], all impaired in apical dominance, provided new insights. Grafting studies showed that wild-type rootstocks were able to rescue the branching phenotype of mutant shoots and suggested the involvement of an entirely novel, upwardly mobile signal acting as a relay between the auxin message and the response of the axillary buds [START_REF] Leyser | The fall and rise of apical dominance[END_REF][START_REF] Dun | Apical dominance and shoot branching. Divergent opinions or divergent mechanisms?[END_REF]. It was shown that the synthesis of this signal required the activities of the genes MAX1, MAX3, MAX4 and RMS1, RMS5 in A. thaliana and P. sativum, respectively [START_REF] Leyser | The fall and rise of apical dominance[END_REF][START_REF] Johnson | Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals[END_REF].

The link between apocarotenoids and plant branching was uncovered through the identification of MAX4 from Arabidopsis, RMS1 from Pisum, and DAD1 from Petunia [START_REF] Sorefan | MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea[END_REF][START_REF] Snowden | The decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE 8 gene affects branch production and plays a role in leaf senescence, root growth and flower development[END_REF]. These enzymes constitute a subgroup of the plant carotenoid oxygenase family (carotenoid cleavage dioxygenase 8, CCD8), while MAX3 [START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF] and RMS5 [START_REF] Johnson | Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals[END_REF] represent members of the CCD7 subfamily. It has been shown that AtCCD7 (MAX3) mediated the cleavage of several carotenoids at the 9-10 and/or 9´-10´ double bond [START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF][START_REF] Schwartz | The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching[END_REF] and that the co-expression of AtCCD7 and AtCCD8 (MAX4) in β-caroteneaccumulating E. coli cells led to the formation of a C 18 -ketone, β-apo-13-carotenone [START_REF] Schwartz | The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching[END_REF]. These data led to the hypothesis that the synthesis of the branching inhibitory signal is initiated by two sequential cleavage reactions, where AtCCD7 converts a carotenoid into an apocarotenoid, which represents the substrate for AtCCD8. However, this presumed apocarotenoid specificity of AtCCD8 contradicts recent observations indicating that this enzyme can cleave carotenoids [START_REF] Auldridge | Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family[END_REF]. The enzymatic activities of the pea enzymes PsCCD8 (RMS1) and PsCCD7 (RMS5) have not been described so far.

Monocots and dicots share similarities with respect to branching [START_REF] Mcsteen | Shoot branching[END_REF]. During the vegetative growth of rice, grain-bearing branches, called tillers, are formed on the unelongated basal internodes and develop their own adventitious roots. The tillering of rice involves the formation of axillary buds and their subsequent outgrowth [START_REF] Doust | Grass architecture: genetic and environmental control of branching[END_REF]. The number of tillers is an important agronomic breeding trait for grain production and many rice tillering mutants have been reported. For instance, the monoculm1 mutant does not develop any tillers due to a lesion in the MONOCULM1 (MOC1) gene. MOC1 is expressed in the axillary buds and encodes a nuclear protein, which initiates the formation of axillary buds [START_REF] Li | Control of tillering in rice[END_REF]. In contrast, the rice mutant fc1 (Os teosinte branched 1, Os tb1) exhibits a high-tillering phenotype combined with dwarfism. The gene OsTB1 encodes a putative transcription factor, which acts as a negative regulator of tillering by inhibiting the outgrowth of axillary buds [START_REF] Takeda | The OsTB1 gene negatively regulates lateral branching in rice[END_REF]. Recently, a closely related Arabidopsis gene, BRANCHED1 (BRC1), has been identified and shown to arrest the development of axillary buds [START_REF] Aguilar-Martínez | Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds[END_REF]. Adding to these similarities, the characterization of the High Tillering Dwarf 1 (HTD1) and DWARF10 (D10) rice mutants led to the discovery of the putative carotenoid oxygenase genes OsCCD7 [START_REF] Zou | The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds[END_REF] and OsCCD8b [START_REF] Arite | DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice[END_REF]. It was supposed that these are orthologs of the dicot enzymes AtCCD7 /AtCCD8 or PsCCD7/PsCCD8, mediating the synthesis of a carotenoid-derived branching inhibitor.

In the current work, we investigated the biosynthesis of the branching inhibitor in vitro, placing emphasis on the second proposed cleavage reaction. Using heterologously expressed enzymes, we show that the rice OsCCD8b, the Arabidopsis AtCCD8 and the pea PsCCD8 are highly specific apocarotenoid cleavage enzymes mediating a common reaction leading to the production of β-apo-13carotenone (C 18 ), which may be a precursor of the branching inhibitor in monocot and dicot plants.

EXPERIMENTAL PROCEDURES

Cloning of cDNAs and generation of expression plasmids

A synthetic cDNA encoding OsCCD8b (Accession No. NM_191187) deduced from the corresponding gene OSJNBa0014K08 was obtained from Epoch Biolabs, Inc.; TX, USA and cloned into pBluescript-SK to yield pBSK-OsCCD8b. For the cloning of PsCCD8, five microgram of total RNA, isolated from two weeks-old auxin treated pea seedlings, was used for cDNA synthesis using SuperScript™ RnaseH -reverse transcriptase (Invitrogen, Paisley, UK) according to the instructions of the manufacturer. Two microliter of the cDNA was then used for amplification with the following primers: RMS1-I: 5´-atggctttcatagcctcaccaactc-3´ and RMS1-II: 5´-ttactgttttggaacccagcatcc-3´. The PCR reaction was performed using 500 nM of each primer, 150 µM dNTPs and 2.5 U OptiTaq Polymerase (Roboklon, Berlin, Germany) in the buffer provided, as follows: 2 min initial denaturation at 94 °C, 32 cycles (20 s 95 °C, 30 s 63 °C, 105 s 72 °C) and 5 min final polymerization at 72 °C. For the cloning of AtCCD8, total cDNA was synthesized from RNA isolated from A. thaliana two weeksold seedlings as above. Five microliter cDNA containing oligo-dT, 100 ng of the 5´-specific primer AtCCD8-I: 5´-atggcttctttgatcacaacc-3´ and 100 µM dNTPs were used in a PCR reaction performed with 5 U Taq-polymerase (Eppendorf, Germany) in the buffer provided, as follows: 2 min initial denaturation at 94 °C, 32 cycles (30 s 94 °C, 30 s 50 °C, 2 min 72 °C) and 10 min final polymerization at 72 °C. The obtained PsCCD8 and AtCCD8 PCR fragments were purified using GFX TM PCR DNA and Gel Band Purification Kit (Amersham Biosciences, NJ, USA), and cloned into the pCR2.1®-TOPO® to yield pCR-PsCCD8 and pCR-AtCCD8, respectively.

For in vitro assays, the three cDNAs were amplified from the obtained plasmids using the primers Os 8-I: 5´-atgtctcccgctatgctgcaggcgt-3´and Os 8-II: 5´-ttacttgctgttccttttcctgggca-3´ for OsCCD8b, AtCCD8-I and AtCCD8-II: 5´-tcatccaacagctttctccaacttc-3´ for AtCCD8, and RMS1-I, -II for PsCCD8.

The PCR reactions were performed with OptiTaq Polymerase (Roboklon, Berlin, Germany) according to the instructions of the manufacturer. The OsCCD8b, AtCCD8 and PsCCD8 fragments were then purified as described above and cloned into pBAD/THIO-TOPO® TA (Invitrogen, Paisley, UK) to yield pThio-Os8b, pThio-At8 and pThio-Ps8, respectively. The identity of the obtained plasmids was verified by sequencing.

Protein expression and purification

For protein purification, the OsCCD8b-cDNA was excised from pBSK-OsCCD8b with the restriction enzymes KpnI and EcoRI and ligated into pET32a (Novagen, Darmstadt, Germany) to yield pET32a-Os8b. This plasmid was then transformed into BL21(DE3) E. coli cells. 4 ml of overnight cultures grown in LB-medium were inoculated into 50 ml 2 * YT-medium. The cultures were induced at an OD 600 (0.5) with 0.5 mM isopropyl β-D-thiogalactopyranoside and grown for 24 h at 18 °C. Cells were then harvested and the thioredoxin-OsCCD8b fusion protein was purified using TALON-resin (Clontech, CA, USA) according to the instructions of the manufacturer.

For crude assays, the plasmids pThio-At8, pThio-Ps8 and pThio-Os8b were transformed into BL21(DE3) E. coli cells harboring the plasmid pGro7 (Takara Bio Inc.; Mobitec, Göttingen, Germany), which encodes the groES-groEL-chaperone system under the control of an arabinoseinducible promoter. 2.5 ml of overnight cultures of transformed cells were then inoculated into 50 ml of 2*YT-medium, grown at 28 °C to an OD 600 of 0.5 and induced with arabinose (0.2%, w/v) for 4h.

Cells were harvested and resuspended in 1 ml of the following buffer: 50 mM NaH 2 PO4, 300 mM NaCl, 1 mg/ml lysozyme, 1 mM dithiothreitol and Triton X-100 (0.1%, v/v); pH 8.0. After incubation for 30 min on ice, cells were sonicated and centrifuged at 12000 g at 4 °C for 30 min. The isolated supernatant was then used for in vitro assays.

Enzymatic assays

Substrates were purified using thin-layer silica gel plates (Merck, Darmstadt, Germany). Plates were developed in light petroleum/diethylether/acetone (40:10:10, v/v/v). Substrates were scraped off in dim daylight and eluted with acetone. Synthetic apocarotenals were kindly provided by BASF (Ludwigshafen, Germany). β-carotene and lycopene were obtained from Roth (Karlsruhe, Germany).

Zeaxanthin was isolated from Synechocystis sp. PCC6803; neoxanthin, and lutein from spinach.

Rosafluene-dialdehyde was prepared enzymatically using the AtCCD1 from A. thaliana and β-apo-8´carotenal according to [START_REF] Schmidt | The carotenase AtCCD1 from Arabidopsis thaliana is a dioxygenase[END_REF]. β-apo-10´-carotenol was prepared by reducing the corresponding aldehyde with NaBH 4 in an EtOH solution. β-apo-10´-carotenoic acid was obtained enzymatically from the corresponding aldehyde using a NAD-dependent dehydrogenase from Neurospora crassa [START_REF] Estrada | The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway[END_REF].

In vitro assays were performed in a total volume of 200 µl. 50 µl of substrates (160 µM) in EtOH were mixed with 50 µl of ethanolic Triton X-100 (0.4%, v/v; Sigma, Deisenhofen, Germany), dried using a vacuum centrifuge and then resuspended in 50 µl water. The prepared substrates were then mixed with 100 µl of 2 X incubation buffer containing 2 mM TCEP, 0.4 mM FeSO 4 and 2 mg/ml catalase (Sigma, Deisenhofen, Germany) in 100 µl of 200 mM Hepes-NaOH, pH 8. Purified OsCCD8b was then added to a final concentration of 400 ng/µl. Crude assays were performed using 50 µl of the soluble fractions of overexpressing cells. The assays were incubated for 2 h at 28 °C. Extraction was done by adding two volumes of each acetone and light petroleum/diethylether (1:4, v/v). After centrifugation, the epiphase was collected.

For in vivo assays, carotenoid-accumulating strains were generated by transforming XL-1 Blue E. coli cells with the plasmids pLYC, p-β and pZEA, pACYC (Fermentas GmbH; St. Leon-Rot, Germany) derivatives encoding the Erwinia herbicola genes required for lycopene, β-carotene and zeaxanthin biosynthesis, respectively. The obtained strains were then transformed with pThio-At8, pThio-Ps8, pThio-Os8b and the control plasmid pThio. Overnight cultures of transformed cells were inoculated into 50 ml of LB-medium, grown at 28 °C to an OD 600 of 0.5 and induced with arabinose (0.2%, w/v) for 16 h. Cells were then harvested, extracted with chloroform/MeOH (2:1, v/v) and dried extracts were subjected to HPLC analyses.

Analytical methods

Substrates were quantified spectrophotometrically at their individual λ max using extinction coefficients calculated from E1% as given by Barua and Olson [START_REF] Barua | β-Carotene is converted primarily to retinoids in rats in vivo[END_REF]. The concentrations of the rosafluenedialdehyde, β-apo-10´-carotenol and -carotenoic acid were estimated based on the concentrations of the precursor apocarotenals. Protein concentration was determined using the BioRad protein assay kit (BioRad, Hercules, CA). For HPLC, a Waters system (Eschborn, Germany) equipped with a photodiode array detector (model 996) was used. The separation was performed using a YMC-Pack C 30 -reversed phase column (250 x 4.6 mm i.d., 5 µm; YMC Europe, Schermbeck, Germany) with the solvent systems B: MeOH/ tert-butylmethyl ether/water (120:4:40, v/v/v) and A: MeOH/ tertbutylmethyl ether (500:500, v/v). The column was developed at a flow-rate of 1 ml/min with a gradient from 100% B to 43% B within 45 min, then to 0% B within 1 min, maintaining the final conditions for another 14 or 29 min at a flow-rate of 2 ml/min. For the analyses of assays containing rosafluene-dialdehyde, the separation was performed using the solvent systems B: MeOH/water (35:65, v/v) and A: MeOH/tert-butylmethyl ether/water (500:500:100, v/v/v). The column was developed for 10 min with solvent B at a flow rate of 1.4 ml/min, followed by a linear gradient from 100% B to 50% B within 10 min at a flow rate of 1.4 ml/min, then to 100% A and a flow rate of 2 ml/min within 1 min, maintaining these final conditions for another 4 min. LC-MS analyses of HPLC-purified products were performed using a Thermo Finnigan LTQ mass spectrometer coupled to a Surveyor HPLC system consisting of a Surveyor Pump Plus, Surveyor PDA Plus and Surveyor Autosampler Plus (Thermo Electron, Waltham, MA, USA). Separations were carried out using a Thermo Hypersil GOLD C 18 -reversed phase column (150 x 4.6 mm i.d., 3 µm) with the solvent system A: MeOH/water/t-butylmethyl ether (50/45/5, v/v/v) and B: MeOH/water/tbutylmethyl ether (27/3/70, v/v/v) with the water containing 0.1 g/l ammonium acetate. The column was developed isocratically at a flow rate of 450 µl min -1 with 90% A and 10 % B for 5 min followed by a linear gradient to 5% A and 95% B in 10 min. The final conditions were maintained for 5 min at a flow-rate of 900 µl min -1 . The identification of β-apo-13-carotenone was carried out using APCI in positive mode. Nitrogen was used as sheath and auxiliary gas, which were set to 20 and 5 arbitrary units, respectively. The vaporizer temperature was 225 °C and capillary temperature 175 °C. The

RESULTS

In vivo and in vitro assays with recombinant OsCCD8b

To investigate the carotenoid cleavage activity of OsCCD8b, the corresponding cDNA, encoded in the plasmid pThio-Os8b, was expressed in β-carotene-, zeaxanthin-and lycopene-accumulating E. coli strains, a frequently used in vivo system to determine the enzymatic activity of carotenoid synthesizing and cleaving enzymes [START_REF] Cunningham | Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene[END_REF][START_REF] Von Lintig | Filling the gap in vitamin A research[END_REF]. Subsequent HPLC analyses revealed a pronounced reduction in the carotenoid levels in lycopene and zeaxanthin strains. However, no cleavage products were detected (Supplemental Fig. 1). Due to the limited variety of substrates that can be offered in vivo, we further explored the activity of the enzyme in vitro using supernatants of OsCCD8b-expressing BL21(DE3) E. coli cells. Based on the assumption that OsCCD8b may cleave apocarotenoids rather than their carotenoid precursors, we applied synthetic apocarotenals as substrates. Among the apocarotenoids presented in Fig. 1, we could only detect a weak cleavage activity with β-apo-10´-carotenal, indicated by the appearance of small amounts of a novel product (data not shown). To improve the enzymatic activity, the thioredoxin-OsCCD8b fusion was expressed in BL21(DE3) E. coli cells harboring the vector pGro7, which encodes the chaperones groES-groEL. This resulted in a striking improvement of the enzymatic activity, as indicated by the strong bleaching (not shown) of the β-apo-10´-carotenal assays. HPLC analyses revealed the conversion of β-apo-10´-carotenal into a more polar compound (Fig. 2A). Based on the UV-Vis-spectrum and the elution pattern, we assumed that the product detected was β-apo-13-carotenone. To prove identity, the product formed was purified and analyzed using LC-MS. As shown in Fig. 3B, the OsCCD8b product exhibited the expected [M+H] + molecular ion of 259.27 and a MS 2 spectrum identical to the authentic reference compound (Fig. 3A). This suggests that OsCCD8b cleaves β-apo-10´-carotenal (C 27 ) at the C13-C14-double bond yielding the ketone β-apo-13-carotenone (C 18 ). This activity must also lead to the formation of a C 9 -dialdehyde (Fig. 4), which was not detected with the extraction and separation protocol used.

Apocarotenals are frequently reduced to alcohols or oxidized to form acids in vivo. To investigate the impact of these variants on the cleavage activity of OsCCD8b, we produced the corresponding alcohol using NaBH 4 and acid with an aldehyde dehydrogenase from Neurospora crassa [START_REF] Estrada | The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway[END_REF]. The HPLC analyses showed that the incubation of β-apo-10´-carotenol resulted in the formation of β-apo-13carotenone (C 18 ) as above (Fig. 2B), while β-apo-10´-carotenoic acid was not converted (Supplemental Fig. 2C).

In contrast to β-apo-10´-carotenal/ol, there was no conversion with any of the other apocarotenals differing in chain lengths (Supplemental Fig. 3), i.e., β-apo-12´-(C 25 ), β-apo-8´-carotenal (C 30 ) and rosafluene-diladehyde (C 14 ). Other C 27 -compounds with different end-groups, the acyclic apo-10´lycopenal and the ring-hydroxylated, (3R)-3-OH-β-apo-10´-carotenal, were not cleaved either (Supplemental Fig. 2). To further explore the substrate specificity, we applied several C [START_REF] Takeda | The OsTB1 gene negatively regulates lateral branching in rice[END_REF] activity was detected in the subsequent HPLC analyses (Supplemental Fig. 4). It has been reported that the expression of AtCCD7 in β-carotene-accumulating E. coli cells led to the formation of β-ionone [START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF]. This implies the formation of β-apo-10´-carotenal and/or rosafluene-dialdehyde as further products. In consideration of the orthology of AtCCD7 and OsCCD7, and accounting for the possibility that the latter may also produce rosafluene-diladehyde, we incubated OsCCD8b with this compound. However, we did not detect any significant conversion (Supplemental Fig. 3C).

In vitro and in vivo assays with recombinant AtCCD8 and PsCCD8

The rice enzyme OsCCD8b shows about 74% similarity to the dicot enzymes AtCCD8 (MAX4) and PsCCD8 (RMS1). To compare the substrate specificities of AtCCD8 and PsCCD8 with the rice enzyme, the corresponding cDNAs were cloned into the pBAD/Thio expression-system. Based on the experience with OsCCD8b, the two cDNAs were co-expressed with the chaperones groES-groEL in BL21(DE3) E. coli cells. In vitro assays were then performed with the synthetic substrates depicted in Fig. 1 using the supernatants of expressing cells. Subsequent HPLC analyses (Fig. 2) revealed a clearcut conversion of β-apo-10´-carotenal and β-apo-10´-carotenol into β-apo-13-carotenone (C 18 ). The identity of the produced ketone was verified using LC-MS analyses (as above; not shown). In contrast, none of the other synthetic substrates was cleaved, as determined by HPLC (Supplemental Fig. 2,3).

Similarly, we did not detect any cleavage activity with rosafluene-diladehyde (Supplemental Fig. 3), β-apo-10´-carotenoic acid (Supplemental Fig. 2) and with the C 40 -carotenoids lycopene, β-carotene, zeaxanthin, lutein and neoxanthin (Supplemental Fig. 4). In line with these in vitro results, the expression of the two cDNAs, embedded in the corresponding pThio-plasmids, in E. coli cells accumulating lycopene, β-carotene and zeaxanthin did not result in any HPLC-detectable products, albeit a reduction in the carotenoid contents occurred (Supplemental Fig. 1, see Discussion).

In vitro assays with purified OsCCD8

The in vitro results presented in this study were all obtained with supernatants of CCD8s-expressing E. coli cells. To confirm these results, we performed in vitro assays using purified thioredoxin-OsCCD8 fusion (Supplemental Fig. 5). The substrate specificity was identical, however, the overall activity was significantly lower (data not shown).

DISCUSSION

Tillering is an important agronomic trait for grain production in rice and other monocot crops; of axillary buds [START_REF] Zou | The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds[END_REF][START_REF] Arite | DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice[END_REF]. This indicated the presence of a novel, unidentified signalling molecule, which, by orthology and analogy with the situation in Arabidopsis, may be a carotenoid derivative.

In A. thaliana, lateral branching is supposed to be under the control of an apocarotenoid derivative acting in concert with auxin. This derivative is initially synthesized by the successive actions of the carotenoid oxygenases AtCCD7 (MAX3) and AtCCD8 (MAX4) [START_REF] Leyser | The fall and rise of apical dominance[END_REF][START_REF] Schwartz | The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching[END_REF], further modified by MAX1 [START_REF] Booker | MAX1 encodes a cytochrome P450 family member that acts downstream of MAX3/4 to produce a carotenoid-derived branch-inhibiting hormone[END_REF], a class III cytochrome P450, and perceived by MAX2 [START_REF] Stirnberg | MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching[END_REF], a F-box leucine-rich repeat protein.

The carotenoid origin of this signal molecule was deduced from the enzymatic activities of AtCCD7 and AtCCD8. However, conflicting data have been reported on the corresponding substrate specificities. It was shown that AtCCD7 exhibited wide substrate specificity in carotenoidaccumulating E. coli cells, converting the linear C 40 carotenoid ζ-carotene and the cyclic β-carotene and potentially also lycopene, δ-carotene and zeaxanthin [START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF]. Regional specificity was determined at the C9´-C10´ double bond; no information could be given whether cleavage also occurred at the C9-C10 position. This resulted in the formation of the respective C 13 -compounds and implied the formation of a C 27 -aldehyde and/or a C 14 -dialdehyde (rosafluene-dialdehyde). Another report confirmed lycopene and β-carotene cleavage but excluded zeaxanthin as a substrate. However, the second cleavage product was identified as a C 27 -apocarotenoid, and hence asymmetric cleavage was proposed, confirmed by in vitro assays showing the conversion of β-carotene into β-apo-10´-carotenal (C 27 ) [START_REF] Schwartz | The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching[END_REF]. The second enzyme involved, AtCCD8, was not investigated in vitro so far. However, it showed wide substrate specificity in E. coli as well [START_REF] Auldridge | Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family[END_REF], where β-carotene-, lycopene-and zeaxanthinaccumulating cells reduced their carotenoid content upon its expression. This contradicts results

showing that this enzyme did not produce apocarotenoids when expressed alone in carotenoidaccumulating E. coli cells. Only upon co-expression with AtCCD7, the C 18 ketone β-apo-13carotenone was detected as a new compound together with the AtCCD7 products. By sequence homology and the similarities of the phenotypes obtained, it was assumed that PsCCD7/PsCCD8 (RMS5/RMS1) from pea and OsCCD7/OsCCD8b from rice are functionally equivalent to AtCCD7/AtCCD8 from Arabidopsis.

Given the conflicting data, it is unclear whether firstly, AtCCD8 can cleave carotenoids, and secondly whether it can convert apocarotenoids other than β-apo-10´-carotenal (C 27 ). The cleavage of several substrates could result in many different products, and thus may imply the occurrence of a family of branching inhibitors in A. thaliana, instead of only one. Third, the question arises whether the CCD8 family members PsCCD8, OsCCD8b and AtCCD8 catalyze the identical reaction to produce one precursor of the branching inhibitor that is identical across plant species. To clarify these points, we have undertaken the outlined approach with CCD8 enzymes, considering plants, for which respective mutants have been characterized, i.e., rice as a monocot and the dicots pea and Arabidopsis.

To address these questions, we first expressed the three cDNAs in carotenoid-accumulating E. coli cells. However, we could not detect any cleavage product, albeit decreases in the substrate contents [START_REF] Auldridge | Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family[END_REF]. To explain these contradictory data, we checked the cleavage of several carotenoids, including the ones accumulated in the E. coli system, in vitro, using enzyme preparations readily active on β-apo-10´-carotenal/ol. As shown in Supplemental data, Fig. 4, we could neither observe significant reductions in the substrate levels, nor detect any cleavage product. Hence, we concluded that the decreased carotenoid contents in the E. coli cells are the result of interference with carotenoid biosynthesis through pleiotropic effects caused by the overexpression. In fact, we occasionally observe bleaching of such cells due to the formation of inclusion bodies even when the expressed oxygenase gene is inactivated by mutation.

OsCCD8b was insoluble in E. coli; however, the problem could be solved satisfactorily by expressing a thioredoxin-fusion together with the groES-groEL chaperone system. All carotenoids tested were not converted, pointing to apocarotenoids as the authentic substrates. The use of synthetic apocarotenoids showed that this was in fact the case. However, in marked contrast to other apocarotenoid-cleaving oxygenases investigated so far (see below), the enzyme showed a very narrow specificity, being active exclusively with β-apo-10´-carotenal/ol. Only these substrates were converted into the C 18 -compound β-apo-13-carotenone. The elucidation of the substrate specificity of AtCCD8 and PsCCD8 revealed the identical selectivity in vitro. Consequently, we did not detect any conversion of the C 14 -compound rosafluene-dialdehyde, which may arise through the cleavage of carotenoids at the C9-C10 and C9´-C10´ double bonds, catalyzed by AtCCD7 [START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF].

The high selectivity reported here seems unique. Several carotenoid oxygenases, such as CCD1 from A. thaliana [START_REF] Schwartz | Characterization of a novel carotenoid cleavage dioxygenase from plants[END_REF][START_REF] Schmidt | The carotenase AtCCD1 from Arabidopsis thaliana is a dioxygenase[END_REF] and the enzymes CarX [START_REF] Prado-Cabrero | Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi[END_REF] and CarT [START_REF] Prado-Cabrero | Identification and biochemical characterization of a novel carotenoid oxygenase: elucidation of the cleavage step in the Fusarium carotenoid pathway[END_REF] from Fusarium, were shown to convert C 40 -carotenoids and apocarotenoids. In contrast, the cyanobacterial enzymes SynACO [START_REF] Ruch | Retinal biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF] and NosACO [START_REF] Scherzinger | Retinal is formed from apo-carotenoids in Nostoc sp. PCC7120: in vitro characterization of an apo-carotenoid oxygenase[END_REF] cleaved only apocarotenoids, but of varying chain lengths and functional groups.

However, these inhomogenous apocarotenoid substrates were all targeted at the same site, the C15-C15´double bond, to produce the C 20 -compounds retinal and its derivatives. The structure of SynACO provides some clues on the parameters determining substrate specificities and, in case of relaxed specificities, on the maintenance of high regional specificity of cleavage [50]. SynACO contains a hydrophobic substrate binding tunnel leading to the Fe 2+ -4-His arrangement in the reaction center. The geometry of this tunnel accounts for the wide substrate specificity where the β-ionone ring is arrested at the entrance while its depth allows to accommodate C 25 -up to C 35 -apocarotenoids. The cleavage site is then determined by the constant distance between the β-ionone ring and the reaction center, leading to cleavage at the C15´-C15 double bond in all cases. The high substrate and regional specificity of the CCD8 family members investigated here may be the result of a similar structure in which the distance between the β-ionone ring and the reaction center governs the C13-C14 cleavage site. However, it can be speculated that the substrate tunnels of these enzymes are less deep and therefore restrictive with respect to chain length. In addition, the geometry of the tunnel-entrance could exclude hydroxylated βionone rings by an unknown mechanism. The absence of a more branching phenotype in the Arabidopsis mutant lut2, which lacks α-carotene and its derivatives [START_REF] Pogson | Arabidopsis carotenoid mutants demonstrate that lutein is not essential for photosynthesis in higher plants[END_REF], suggests that the branching inhibitor is synthesized either from acyclic or from β-cyclic carotenoids. Similarly, the normal branching of mutants impaired in violaxanthin and neoxanthin synthesis [START_REF] Rock | The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis[END_REF] indicates that these epoxy-carotenoids play no role as carotenoid precursors.

Hence, it can be concluded that the branching inhibitor is derived from β-zeacarotene, γ-carotene, βcarotene or β-cryptoxanthin, since the cleavage of these carotenoids by CCD7 at the C9´-C10´ double bond results in the formation of the CCD8 substrate β-apo-10´-carotenal (Fig. 4). The high substrate specificity of the CCD8 enzymes implies an equivalently high product specificity of the CCD7 family members. In support of this, it was shown that the max3-Arabidopsis phenotype, caused by a lesion in AtCCD7, can be rescued by introducing the OsCCD7 cDNA under the control of the constitutive 35S promoter [START_REF] Zou | The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds[END_REF]. It is therefore surprising to note the wide substrate specificity of AtCCD7 and its ability to cleave several carotenoids in vivo [START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF][START_REF] Schwartz | The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching[END_REF]. This may indicate that the biological function of CCD7s is not restricted to the formation of the branching inhibitor.

Taken together, the concordance of the reactions mediated by the CCD8s investigated here suggests an ancient pathway for the synthesis of the branching inhibitor, which arose prior to the monocot-dicot divide. Moreover, it can be assumed that β-apo-13-carotenone represents the common apocarotenoid precursor of the branching inhibitor. Adding on the evolutionary perspectives, β-apo-13-carotenone is supposed to be the precursor of the well-known fungal pheromone trisporic acid [START_REF] Schachtschabel | Biological activity of trisporoids and trisporoid analogues in Mucor mucedo (-)[END_REF]. During the preparation of this manuscript, a report appeared describing a novel function of β-apo-13-carotenone, designated as d'orenone, in blocking the growth of root hairs by interfering with PIN2-mediated auxin transport [START_REF] Schlicht | D'orenone blocks polarized tip-growth of root hairs by interfering with the PIN2-mediated auxin transport network in the root Apex[END_REF], thus supporting the biological relevance of our findings. The purified product of OsCCD8b (compound 1, Fig. 1) showed the expected [M+H] + molecular ion of 259.27. The MS 2 spectrum was identical with the authentic reference (A) including the 217, 201, 185, 175, 159, 119 and 85 [M+H] + fragments. These data suggest the OsCCD8b-product to be β-apo-13-carotenone; its structure is given in A.

Figure 4 Proposed pathway for the synthesis of the plant branching inhibitor

The first step, catalyzed by CCD7s, must lead to β-apo-10´-carotenal, the CCD8-substrate determined in this work. In the second, very specific step, the CCD8s (OsCCD8b, AtCCD8 and PsCCD8) mediate the cleavage of only β-apo-10´-carotenal/ol at the C13-C14 double bond leading to the C 18 -ketone βapo-13-carotenone and a C 9 -product. Further modifications of one of the two products, likely β-apo-13-carotenone, then lead to the branching inhibitor(s) active in monocots and dicots.

7 Biochemical

 7 source current was set to 5 µA and the capillary voltage was 49 V. Journal Immediate Publication. Published on 18 Jul 2008 as manuscript BJ20080568 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080568 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  -carotenoids, including lycopene, β-carotene, zeaxanthin, lutein, and neoxanthin in vitro. However, no cleavage 8 Biochemical Journal Immediate Publication. Published on 18 Jul 2008 as manuscript BJ20080568 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080568 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  however, the molecular factors that control this branching process have just begun to emerge. Recent mutant analyses of high-tillering rice mutants suggest the involvement of the two carotenoid oxygenases OsCCD7 and OsCCD8b in determining the number of tillers, by inhibiting the outgrowth 9 Biochemical Journal Immediate Publication. Published on 18 Jul 2008 as manuscript BJ20080568 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080568 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  were observed. Our results are consistent with a former in vivo study on AtCCD8 in which no 10 Biochemical Journal Immediate Publication. Published on 18 Jul 2008 as manuscript BJ20080568 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080568 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society cleavage products were detected in decolorized E. coli cultures

11 Biochemical

 11 Journal Immediate Publication. Published on 18 Jul 2008 as manuscript BJ20080568 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080568 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 18 Jul 2008 as manuscript BJ20080568 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080568 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Structure of the apocarotenoid substrates used for in vitro assays with OsCCD8b, AtCCD8 and PsCCD8 I: rosafluene-dialdehyde (C 14 ), II: β-apo-12´-carotenal (C 25 ), III: β-apo-10´-carotenal (C 27 ), IV: β-apo-10´-carotenol (C 27 ), V: β-apo-10´-carotenoic acid (C 27 ), VI: (3R)-3-OH-β-apo-10´-carotenal (C 27 ), VII: apo-10´-lycopenal (C 27 ) and VIII: β-apo-8´-carotenal (C 30 ). The three enzymes converted only compound III and its corresponding alcohol (IV).

Figure 2 HPLC

 2 Figure 2 HPLC analysis of in vitro incubations with β-apo-10´-carotenal /ol A: The incubations (2 h) of β-apo-10´-carotenal (2) with the enzymes OsCCD8b, PsCCD8 and AtCCD8 led to the formation of product (1) which resembled β-apo-13-carotenone in its UV/Vis spectrum and elution characteristics. B: The substrate β-apo-10´-carotenol (3) yielded the same product (1). The UV/Vis spectra of the substrates and the product are shown in the insets. For structures, see Figs. 1 and 4.

Figure 3

 3 Figure 3 LC-MS analysis of the authentic β-apo-13-carotenone standard (A) and the purified OsCCD8b product (B)

Figure 4 Biochemical

 4 Figure 4

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Graduiertenkolleg 1305, "Signalsysteme in pflanzlichen Modellorganismen", and by the HarvestPlus programme (www.harvestplus.org). We are indebted to Dr. Hansgeorg Ernst, BASF, for providing the apocarotenoids. We thank Dr. Jorge Mayer and Daniel Scherzinger for valuable discussions.


Elisa M Nevalainen

Aneta Skwarek-Maruszewska

Attila Braun

Markus Moser

Dr Pekka Lappalainen
email: pekka.lappalainen@helsinki.fi

Two biochemically distinct and tissue-specific twinfilin isoforms are generated from mouse twinfilin-2 gene by alternative promoter usage

Keywords: Twinfilin, actin dynamics, cytoskeleton, isoform Biochemical Journal Immediate

Twinfilin is an evolutionarily conserved regulator of actin dynamics composed of two actin-depolymerizing factor homology (ADF-H) domains. Twinfilin binds actin monomers and heterodimeric capping protein with high affinity. Previous studies demonstrated that mammals express two twinfilin isoforms, twinfilin-1 and twinfilin-2, of which at least twinfilin-1 also regulates cytoskeletal dynamics by capping actin filament barbed ends. Here, we show that alternative promoter usage of the mouse twinfilin-2 gene generates two isoforms, which differ from each other only at their very N-terminal region. Of these isoforms, twinfilin-2a is predominantly expressed in non-muscle tissues, whereas expression of twinfilin-2b is restricted to heart and skeletal muscle. Both proteins bind actin monomers and capping protein, as well as efficiently cap actin filament barbed ends. However, the N-terminal ADF-H domain of twinfilin-2b interacts with ADP-Gactin with five-fold higher affinity than with ATP-G-actin, whereas the corresponding domain of twinfilin-2a binds ADP-G-actin and ATP-G-actin with equal affinities. Together, these data show that, like twinfilin-1, mouse twinfilin-2 is a filament barbed end capping protein, and that two tissue-specific and biochemically distinct isoforms are generated from the twinfilin-2 gene through alternative promoter usage.

INTRODUCTION

The actin cytoskeleton plays an essential role in numerous cell biological processes, including motility, cell division, polarized growth, cytokinesis, endocytosis, and signal transduction. The structure and dynamics of the actin cytoskeleton are both spatially and temporally regulated by a large number of actin-binding proteins, which interact with filamentous and/or monomeric actin [reviewed in 1 and 2].

Twinfilin is an evolutionarily conserved multifunctional actin-binding protein, which is involved in the regulation of motile and morphological processes in organisms from yeasts to mammals [START_REF] Goode | Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomersequestering protein[END_REF][START_REF] Wahlström | Twinfilin is required for actin-dependent developmental processes in Drosophila[END_REF][START_REF] Pelkmans | Genome-wide analysis of human kinases in clathrin-and caveolae/raft-mediated endocytosis[END_REF][START_REF] Helfer | Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: implications on motility[END_REF][START_REF] Iwasa | Spatial and temporal relationships between actin-filament nucleation, capping, and disassembly[END_REF][START_REF] Yamada | Identification of twinfilin-2 as a factor involved in neurite outgrowth by RNAi-based screen[END_REF]. Twinfilin forms a stable, high affinity complex with ADPactin monomers, decreases the rate of nucleotide exchange on actin monomers and prevents their assembly to filament ends [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF][START_REF] Palmgren | Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin[END_REF]. Interaction between twinfilin and Gactin can be down-regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at least in vitro [START_REF] Palmgren | Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin[END_REF]. Twinfilin also interacts with the heterodimeric capping protein, and at least in budding yeast this interaction is necessary for twinfilin's correct sub-cellular localization to the cortical actin cytoskeleton [START_REF] Palmgren | Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin[END_REF][START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF][START_REF] Falck | Biological role and structural mechanism of twinfilin-capping protein interaction[END_REF]. In addition, recent studies revealed that mouse twinfilin-1 caps filament barbed ends with a preferential affinity to ADP-bound filament ends. This activity appears to be essential for the role of twinfilin-1 in motile processes [START_REF] Helfer | Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: implications on motility[END_REF]. Furthermore, yeast twinfilin was shown to induce filament severing at low pH and this activity is inhibited by binding to heterodimeric capping protein [START_REF] Moseley | Twinfilin is an actin-filament-severing protein and promotes rapid turnover of actin structures in vivo[END_REF].

Twinfilins are composed of two actin-depolymerizing factor homology (ADF-H) domains separated by a short linker and followed by a 35-residue C-terminal tail region [START_REF] Palmgren | Twinfilin, a molecular mailman for actin monomers[END_REF]. The two ADF-H domains are ~20% homologous to ADF/cofilins and to each other [START_REF] Lappalainen | The ADF homology (ADF-H) domain: a highly exploited actin-binding module[END_REF]. Studies on mouse twinfilin-1 revealed that the high-affinity actin monomer-binding site is located in the C-terminal ADF-H domain, whereas the presence of both ADF-H domains is required for filament barbed end capping [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF][START_REF] Paavilainen | Structural basis and evolutionary origin of actin filament capping by twinfilin[END_REF].

The capping protein binding site is located in the C-terminal tail region in yeast twinfilin and in mouse twinfilin-1 [START_REF] Falck | Biological role and structural mechanism of twinfilin-capping protein interaction[END_REF].

Yeasts, worms, and flies have only one twinfilin isoform. In mammals, however, two twinfilin genes (twinfilin-1 and twinfilin-2) exist. Twinfilin-1 and twinfilin-2 proteins 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.

share ~75 % identity. Both mouse twinfilin isoforms bind actin monomers and capping protein with high affinity, but show different tissue distributions. Twinfilin-1 is the major isoform in the developing embryo and in most adult mouse non-muscle tissues, whereas twinfilin-2 expression is most abundant in heart, skeletal muscle, and spleen [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF].

Here we show that the mouse twinfilin-2 gene encodes for two different twinfilin-2 isoforms (designated twinfilin-2a and twinfilin-2b) that are generated by alternative promoter usage. The two twinfilin-2 variants differ only in the very N-terminal region and are identical to each other from twinfilin-2a residue 9 and twinfilin-2b residue 7 onwards. Our results show that twinfilin-2a and twinfilin-2b display distinct tissuespecific expression patterns and differ from each other in respect to interaction with actin monomers. Together, these data suggest that alternative promoter usage of the twinfilin-2 gene generates two biochemically distinct twinfilin isoforms that fulfil specific needs for regulation of actin dynamics in specialized mammalian cell-types.

MATERIALS AND METHODS

Plasmid construction

Plasmids for twinfilin-1 and twinfilin-2a expression (pPL144 and pPL182, respectively) as a His-tagged fusion protein in Escherichia coli have been described earlier [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF][START_REF] Vartiainen | Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics[END_REF]. The DNA fragments corresponding to full-length mouse twinfilin-2b as well as to twinfilin-2a N-terminal domain (residues 1-142), twinfilin-2b N-terminal domain (residues 1-140) and twinfilin-2 C-terminal domain (residues 169-316 in twinfilin-2a and 167-314 in twinfilin-2b) were amplified by PCR from pPL182 plasmid. The oligonucleotides introduced NcoI and HindIII sites at the 5' and 3' ends of the PCR products, respectively. The PCR fragments were digested with NcoI and HindIII and ligated into the pHAT2 [START_REF] Peränen | T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli[END_REF] vector to create plasmids pPL481, pPL482, pPL483 and pPL484. The plasmid for expressing mouse 1 2 capping protein in E. coli has been described previously [START_REF] Palmgren | Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin[END_REF]. Generation of the plasmid for expressing myctagged twinfilin-1 in mammalian cells (pPL79) was described earlier [START_REF] Vartiainen | Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics[END_REF]. For generation of the constructs for expressing myc-tagged twinfilin-2a and twinfilin-2b, the corresponding cDNA fragments were amplified by PCR. The oligonucleotides

Protein purification

The expression and purification of full-length mouse twinfilin-1, twinfilin-2a, and twinfilin-2b and the individual domains of twinfilin-2a and twinfilin-2b as his-tagged proteins were carried out as previously described [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF]. Mouse 1 2-capping protein was purified as previously described [START_REF] Palmgren | Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin[END_REF]. Actin was purified from rabbit skeletal muscles [START_REF] Pardee | Purification of muscle actin[END_REF] and labelled by NBD-Cl [START_REF] Detmers | 7-Chloro-4nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization[END_REF][START_REF] Weeds | Interactions of pig plasma gelsolin with G-actin[END_REF] and pyrenyl as described [START_REF] Egile | Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actinbased motility[END_REF][START_REF] Cooper | Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization[END_REF].

Recombinant human gelsolin was obtained from Cytoskeleton Inc. Spectrin-actin seeds were isolated from human erythrocytes as described [START_REF] Casella | Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments[END_REF].

RNA extraction

Total RNA was isolated from various mouse tissues by TRIzol (Invitrogen) according to the manufacturer's protocol. Briefly, tissue samples were homogenized in a 15 ml tube with Polytron (Glen Mills) in 1.0 ml of TRIzol per 100 mg of tissue.

Homogenates were incubated 10 min at room temperature and centrifuged at 12,000×g for 15 min at 4°C to pellet insoluble material and high-molecular-weight DNA.

Chloroform was added, tubes were shaken for 15 seconds, incubated 3 min at room temperature and centrifuged at 12,000×g for 15 min at 4°C. After phase separation, RNA was precipitated with isopropanol, washed with 75% ethanol and resuspended in an appropriate volume of DEPC-treated water. RNA purity and quantity was ascertained from optical density at 260 and 280 nm. The samples were stored at 80°C until use.

Real-time quantitative PCR

Three micrograms of total RNA was reverse-transcribed at 42°C for 1 h in 20 l solution containing 200 units of SuperScript II Reverse Transcriptase (Invitrogen) and 500 ng of Oligo(dT). The resulting cDNA was either immediately used for nonquantitative RT-PCR or quantitative PCR (Q-PCR) or stored at 20°C until use. Q-PCR was applied to determine the expression levels of each isoform in different Q-PCR was performed with the following temperature conditions: 95°C for 5 min and 40 cycles at 95°C for 10 s, 58°C for 15 s and 72°C for 15 s. Primers designed for specific detection of different twinfilin isoforms and GAPDH are listed in Table 1. All primer pairs resulted in a ~100 bp product. The amplification of the twinfilin-1 cDNA was performed using primers ES110 and ES121, twinfilin-2a with primers ES122 and ES124, twinfilin-2b with primers ES124 and ES125. GAPDH served as housekeeping gene for the adjustment of relative expression data. The amplification of the GAPDH cDNA was performed using primers ES126 and ES127. To verify the identity of the various PCR products, RT-PCR was performed and the DNA fragments were separated on 2% agarose gels, extracted by NucleoSpinExtract II Band Extraction Kit (Macherey Nagel) and subjected to sequencing.

Actin monomer binding assay

Actin monomer binding experiments were performed by monitoring the change in the fluorescence of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled G-actin at different twinfilin concentrations as described earlier [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF]. ADP-actin was prepared by incubating NBD-actin with hexokinase-agarose beads (Sigma) and glucose for 2 h at 4 °C [START_REF] Pollard | Rate constants for the reactions of ATP-and ADP-actin with the ends of actin filaments[END_REF]. The final concentration of actin in these assays was 0.2 M, and the twinfilin concentrations ranged from 0.05 to 14 M. Experiments were carried out at room temperature in F-buffer (2 mM Tris-HCl (pH 8.0), 0.1 mM CaCl 2 , 0.1 mM DTT, 0.2 mM ADP or ATP, 1 mg/ml bovine serum albumin, 2 mM MgCl 2 , 0.1 M KCl). The reactions were measured using a BioLogic MOS-250 fluorescence spectrophotometer and the data analyzed as described previously [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF].

Actin polymerization assays

The effect of twinfilin on actin filament assembly kinetics was monitored by the increase in pyrenyl-actin fluorescence in the presence of spectrin-or gelsolin-capped filaments as described earlier [START_REF] Helfer | Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: implications on motility[END_REF]. Experiments were carried out with 2.5 M actin (10% pyrene labeled) and twinfilin concentrations ranging from 0.1 to 12 M. The excitation and emission wavelengths were 365 and 407 nm, respectively. 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.

Polymerization was monitored at room temperature with a BioLogic MOS-250 fluorescence spectrophotometer at 10 second intervals. The initial rate of filament growth was normalized to the value of 1 measured in the absence of twinfilin. The data were analyzed as described previously using the program SigmaPlot 9.0 [START_REF] Helfer | Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: implications on motility[END_REF].

Native gel electrophoresis assay

Interaction of the twinfilin isoforms with capping protein was assayed with 6% native polyacrylamide gels. Reactions containing 2 M wild-type twinfilin-1, twinfilin-2a or twinfilin-2b or a mutant twinfilin-1 lacking the capping protein binding capacity and 4 M 1 2 capping protein in 10 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM DTT were incubated for 60 min at room temperature. Fifteen l of the reactions were mixed with 5 l of loading buffer (125 mM Tris (pH 8.8), 250 mM NaCl, 2.5 mM DTT, 50% glycerol) and loaded onto a 6% native polyacrylamide gel [START_REF] Falck | Biological role and structural mechanism of twinfilin-capping protein interaction[END_REF]. The gel was run at 100 V for 180 min using native running buffer (25 mM Tris, 194 mM glycine pH 8.5, 0.5 mM DTT), and the proteins were detected by Coomassie Blue staining.

Immunofluorescence microscopy

Isolation, culturing, transfection and immunofluorescence microscopy of neonatal rat cardiomyocytes was carried out as described previously [START_REF] Chereau | Leiomodin is an actin filament nucleator in muscle cells[END_REF]. The cells were fixed 24 hours after transfection. Endogenous twinfilins were visualized with anti-twinfilin antibody recognizing twinfilin-1 and twinfilin-2 (1:80 dilution), myc-tagged fusion proteins with mouse anti-myc antibody (1:500), and secondary antibodies conjugated

RESULTS

Identification of two twinfilin-2 variants

A previous study identified two twinfilin isoforms (twinfilin-1 and twinfilin-2) from mammals [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF]. To reveal the biological roles of these proteins, we generated twinfilin-2 knockout mice. In the knockout construct, the cassette was inserted into the first coding exon of the twinfilin-2 gene. During our analysis of the knockout mice, we noticed that while twinfilin-2 protein and mRNA were absent from all non-muscle tissues examined, twinfilin-2 mRNA and protein were still present in striated muscle and heart (Nevalainen et al., unpublished). Subsequent EST database searches revealed two potential mouse twinfilin-2 variants.

In contrast to the previously identified twinfilin-2 variant [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF] (which will be referred to as twinfilin-2a), twinfilin-2b uses an alternative translation initiation codon, which is located 18 nucleotides upstream from the 3' splice acceptor site of the twinfilin-2a intron 1 (Fig. 1A). A TATA box located ~20 residues upstream of the translation initiation codon of twinfilin-2b as well as a conserved putative promoter region 5' to the TATA box is also present (data not shown). This gives rise to a twinfilin-2b mRNA that contains an alternative first exon (referred to as exon 2b in Fig. 1A) encoding for a twinfilin-2b protein of 347 residues as compared to twinfilin-2a consisting of 349 residues (Fig. 1B). Alignment of the amino acid sequences of mouse twinfilin-2a and twinfilin-2b shows that they are identical to each other with the exception of the 6/8 amino terminal residues.

Tissue-specific expression patterns of twinfilin-2a and twinfilin-2b

To investigate whether twinfilin-2b mRNA is expressed in vivo and to analyse its expression pattern in comparison to twinfilin-1 and twinfilin-2a, RNA from mouse heart, lung, kidney, spleen, liver, skeletal muscle, and brain was extracted. RNAs were reverse transcribed and the expression of each isoform from these tissues was first examined by a non-quantitative RT-PCR using isoform specific primer pairs (Table 1).

Twinfilin-2a was expressed in all tissues examined and twinfilin-1 in all tissues except skeletal muscle. However, based on this experiment, twinfilin-2b expression was A c c e p t e d M a n u s c r i p t restricted to heart and skeletal muscles (Fig. 2A). The PCR products were also isolated from an agarose gel and sequenced to verify their identity (data not shown).

The relative amounts of each isoforms were subsequently analysed by Q-PCR (Fig 2B). Q-PCR analysis revealed that twinfilin-2a was expressed in all tissues examined and represents the most abundant isoform in spleen. In contrast, twinfilin-2b expression is restricted to skeletal muscle and heart. Interestingly, twinfilin-2b is the dominant isoform in these tissues at mRNA levels when compared to twinfilin-1 and twinfilin-2a expression. As reported previously [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF], twinfilin-1 mRNA was widely expressed in non-muscle tissues, but was absent from skeletal muscle (Fig 2B).

Actin monomer binding properties of twinfilin-2a and twinfilin-2b

To compare the biochemical properties of the twinfilin isoforms, we produced recombinant mouse twinfilin-2a and twinfilin-2b as well as their individual ADF-H domains as His-tagged proteins. All proteins were soluble and monomeric according to their elution position from a gel-filtration column (data not shown). We first examined actin monomer binding of all three twinfilin isoforms under physiological ionic conditions by using a fluorometric assay with NBD-labelled actin monomers. As reported previously for twinfilin-1 and twinfilin-2a [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF][START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF], also twinfilin-2b binds ADP-G-actin with a significantly (>8-fold) higher affinity than ATP-G-actin (Fig. 3A-C). As previously shown for twinfilin-1 [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF], the C-terminal ADF-H domain, common to both twinfilin-2 isoforms, binds ADP-G-actin with very high affinity (Fig. 3F).

However, the actin monomer binding properties of the N-terminal ADF-H domains of twinfilin-2a and twinfilin-2b differed significantly from each other. Whereas the Nterminal ADF-H domains of twinfilin-2a (Fig. 3D) and twinfilin-1 [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF] bind ADP-Gactin with a relatively low affinity (K D ~500 -700 nM), the corresponding domain of twinfilin-2b bound ADP-G-actin with significantly higher affinity (Fig. 3E). It is also important to note that in contrast to all ADF-H domains examined so far [START_REF] Ojala | The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers[END_REF][START_REF] Maciver | Actophorin preferentially binds monomeric ADP-actin over ATP-bound actin: consequences for cell locomotion[END_REF][START_REF] Carlier | Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility[END_REF][START_REF] Blanchoin | Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin[END_REF],

the N-terminal ADF-H domain of twinfilin-2a does not show preferential binding to ADP-actin, but instead binds ADP-and ATP-G-actin with equal affinities (Fig. 3D).

All mouse twinfilins cap actin filament barbed ends

Recent data showed that, when using mammalian actin, mouse twinfilin-1 caps actin filament barbed ends, whereas budding yeast and Drosophila melanogaster twinfilins do not display barbed end capping activity [START_REF] Helfer | Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: implications on motility[END_REF]. Therefore, we examined whether mouse twinfilin-2a and twinfilin-2b also display filament barbed end capping or if this activity is specific for mammalian twinfilin-1. Barbed and pointed end capping activity was assayed by examining the effects of purified twinfilin-2a and twinfilin-2b on the growth of spectrin-or gelsolin-capped actin filaments, respectively. We used mouse twinfilin-1, which inhibits pointed-end growth by sequestration and prevents barbed-end assembly by both sequestering actin monomers and capping filament ends, as a reference in the experiments.

Like twinfilin-1, twinfilin-2a and twinfilin-2b inhibited filament barbed end growth in a range of concentrations substoichiometric to G-actin (Fig. 4A-C). This is not possible by mere sequestering, but is consistent with capping of the barbed ends in addition to sequestration of actin monomers. The pointed end growth was inhibited by sequestering actin monomers only. K F values for binding of twinfilin-1, twinfilin-2a and twinfilin-2b to actin filament barbed ends were 0.14 µM, 0.19 µM, and 0.26 µM, respectively and K T values for ATP-G-actin sequestration were 0.76 µM, 0.91 µM, and 0.81 µM, respectively. These data show that also twinfilin-2a and twinfilin-2b are actin filament barbed end capping proteins.

Twinfilin-2a and twinfilin-2b bind heterodimeric capping protein

In addition to actin, twinfilin-1 and twinfilin-2a were shown to bind mouse 1 2 capping protein in vitro [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF]. The interaction with heterodimeric capping protein is essential for the correct subcellular localization of twinfilin in budding yeast [START_REF] Palmgren | Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin[END_REF], but the possible biological role of this interaction in mammalian cells is presently not known. Yeast twinfilin does not affect the filament barbed end capping activity of yeast capping protein [START_REF] Falck | Biological role and structural mechanism of twinfilin-capping protein interaction[END_REF]. However, because mammalian twinfilin-1 and twinfilin-2a also display filament barbed end capping activity [6 and To examine whether also twinfilin-2b binds capping protein, we performed a native gel electrophoresis assay with purified full-length mouse twinfilins and mouse 1 2capping protein. The capping protein binding site resides in the C-terminal tail of yeast twinfilin and mouse twinfilin-1, and thus a mutant twinfilin-1 construct lacking the Cterminal tail was used as a negative control [START_REF] Falck | Biological role and structural mechanism of twinfilin-capping protein interaction[END_REF]. Purified capping protein runs as a single band below the migration positions of all full-length twinfilin isoforms on a native polyacrylamide gel (Fig. 5; lane 1). When mixed with each other in a 1:2 molar ratio, a complex of twinfilin and capping protein forms. On a native gel, this complex migrates above the positions of uncomplexed twinfilin and capping protein (Fig. 5; upper band on lane 3), while the band corresponding to the migration position of uncomplexed twinfilin disappears. As expected, the C-terminally deleted twinfilin-1 protein did not display interaction with capping protein (Fig. 5; lanes 4 and 5).

However, both twinfilin-2 isoforms interacted with capping protein similar to twinfilin-1 (Fig. 5; lanes 6-9).

Subcellular localizations of twinfilins in rat cardiomyocytes

In mammalian non-muscle cells, twinfilin-1 and twinfilin-2a show predominantly punctuate cytoplasmic localization that at least in the case of twinfilin-1 partially overlaps with endosomes. In addition, twinfilin-1 localizes to cell-cell contacts and to lamellipodial actin network in cells expressing dominant active forms of Cdc42 and Rac GTPases [START_REF] Helfer | Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: implications on motility[END_REF][START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF]. To examine the subcellular localizations of twinfilins in muscle cells, we first performed immunofluorescence microscopy on cultured neonatal rat cardiomyocytes by using an antibody that recognizes all three twinfilin isoforms. These cells display regular, contractile myofibrils and thus provide a good model for studying the subcellular localizations of twinfilins in heart cells, where all three isoforms are expressed (see Fig. 2). Immunofluorescence microscopy revealed that twinfilins display predominantly punctuate cytoplasmic staining also in cardiomyocytes, although in some cells endogenous twinfilins also concentrated to myofibrils (Fig. 6A).

A c c e p t e d M a n u s c r i p t

To compare the subcellular localizations of each twinfilin isoform in cardiomyocytes, we expressed N-terminally myc-tagged twinfilin-1, twinfilin-2a and twinfilin-2b in these cells. Similarly to endogenous twinfilin, also the myc-tagged twinfilins displayed mainly punctuate cytoplasmic localizations. Twinfilin-1 and twinfilin-2b were also enriched along myofibrils in a subset of cells, whereas in the case of twinfilin-2a significant enrichment to myofibrils was not observed (Fig. 6B). Instead, twinfilin-2a was often concentrated to regions between myofibrils (Fig. 6B and data not shown).

DISCUSSION

Twinfilin is an evolutionarily conserved protein that regulates actin dynamics in organisms from yeasts to mammals. Mammals have two twinfilin genes that were previously shown to encode biochemically very similar proteins with slightly different expression patterns [START_REF] Vartiainen | Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated[END_REF]. Here we demonstrate that a third mammalian twinfilin isoform is produced through alternative promoter usage of the twinfilin-2 gene.

Interestingly, the two twinfilin-2 variants display distinct tissue-specific expression patterns, suggesting that promoter usage of twinfilin-2 is tightly and tissue-specifically regulated. Whereas the expression of twinfilin-2b is restricted to striated muscle, where it is also the predominant twinfilin isoform, twinfilin-2a is mainly co-expressed with twinfilin-1 in most non-muscle tissues. In cultured cardiomyocytes, twinfilins show mainly punctuates cytoplasmic localization, although that twinfilin-1 and twinfilin-2b also concentrate to myofibrils. Similar as described here for twinfilin-2, previous studies showed that the two ADF/cofilin family actin-binding proteins of Caenorhabditis elegans, UNC-60A and UNC-60B, are generated from the unc-60 gene. However, the UNC60A and UNC60B isoforms are generated through alternative splicing instead of alternative promoter usage as shown here for twinfilin-2 [START_REF] Mckim | The Caenorhabditis elegans unc-60 gene encodes proteins homologous to a family of actin binding proteins[END_REF]. UNC-60A is expressed in various tissues and required for early embryogenesis, whereas UNC-60B is specifically expressed in body wall muscle and is essential for myofibril assembly [START_REF] Ono | UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle[END_REF][START_REF] Ono | Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments[END_REF]. Muscle specific isoforms of central actin-regulating proteins are common in mammals. However, they are usually encoded by a separate gene from their corresponding non-muscle specific isoforms, mainly as a result of gene duplications during evolution. These include for 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.

example muscle-specific ADF/cofilin, cyclase-associated-protein, and -actinins [START_REF] Ono | Characterization of a novel cofilin isoform that is predominantly expressed in mammalian skeletal muscle[END_REF][START_REF] Vartiainen | The three mouse actin-depolymerizing factor/cofilins evolved to fulfil cell-type-specific requirements for actin dynamics[END_REF][START_REF] Bertling | Cyclase-associated-protein 1 promotes cofilin-induced actin dynamics in mammalian nonmuscle cells[END_REF][START_REF] Beggs | Cloning and characterization of two human skeletal muscle alpha-actinin genes located on chromosomes 1 and 11[END_REF][START_REF] Honda | Actinin-4, a novel actin-bundling protein associated with cell motility and cancer invasion[END_REF].

In our database searches, we found twinfilin-2b homologues from rat and chicken, suggesting that the alternative promoter usage that produces the two variants of twinfilin-2 is evolutionarily conserved in vertebrates. Furthermore, alternative splicing of chicken twinfilin-2 gene from another position was also recently reported [START_REF] Tang | Characterising alternate splicing and tissue specific expression in the chicken from ESTs[END_REF].

Interestingly, we did not find the twinfilin-2b variant from human ESTs, suggesting that this specific alternative promoter usage event may have been lost during the evolution of primates.

Our study demonstrates that all three mammalian twinfilin isoforms bind capping protein and cap filament barbed ends in a similar manner. Thus these data provide evidence that actin filament barbed end capping activity is not restricted to twinfilin-1 only, but may be a general feature of all mammalian twinfilins. In addition, all three twinfilins bind actin monomers, but display differences in these interactions. The isolated N-terminal ADF-H domain of twinfilin-2b binds ADP-G-actin with approximately 2,5-fold higher affinity compared to the corresponding domain from twinfilin-2a. However, the N-terminal domain of twinfilin-2a displays ~2-fold higher affinity to ATP-G-actin than the corresponding twinfilin-2b domain. Consequently, the full-length twinfilin-2a binds ATP-G-actin with higher and ADP-G-actin with lower affinity than twinfilin-2b. These differences in G-actin interactions can be explained by the fact that the N-terminal region, which differs between twinfilin-2a and twinfilin-2b, was shown to play a central role in G-actin binding of twinfilin-1 [START_REF] Paavilainen | Structural conservation between the actin monomerbinding sites of twinfilin and actin-depolymerizing factor (ADF)/cofilin[END_REF].

In conclusion, we have identified two biochemically distinct variants of twinfilin-2 protein, and show that they are expressed in differentially. Future cell biological and knockout mouse studies are required to elucidate the biological roles of these two twinfilin isoforms and to reveal the possible redundant roles of twinfilin-2a and twinfilin-1 in non-muscle tissues. Furthermore, it will be important to examine the role of twinfilin-2b in skeletal and heart muscle cells. 1. GAPDH amplification was used as a control. Twinfilin-2b is the most abundant isoform in heart and skeletal muscles, whereas twinfilin-2a and twinfilin-1 are expressed widely in non-muscle tissues. Twinfilin-2a is the predominant isoform in spleen and twinfilin-1 in lung, kidney, liver, and brain. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608 A c c e p t e d M a n u s c r i p t introduced EcoRV and HindIII sites at the 5' and 3' ends of the PCR products, respectively. The PCR fragments were digested with EcoRV and HindIII and ligated into the myc-tagged pEGFP-N1 based vector (Clonetech) to create plasmids pPL80 and pPL554, respectively.

Biochemical

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  tissues. Q-PCR reactions were carried out on a LightCycler 480 System (Roche) by using the SYBR Green I Master kit (Roche). Reactions had a final volume of 20 l and contained 4 pmol of each primer and 0.4 l of cDNA obtained by reverse transcription.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608

  to fluorescein or rhodamine (Molecular Probes, Invitrogen). Actin filaments were visualized with alexa 488 or rhodamine-conjugated phalloidin diluted 1:300 (Molecular Probes, Invitrogen) Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608A 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.

  Fig. 4 of this study], their Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608 A c c e p t e d M a n u s c r i p t possible effects on the barbed end capping activity of heterodimeric capping protein can not be reliably examined.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080608 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080608

Biochemical

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FIGURE LEGENDS FIGURE 1 .

 LEGENDS1 FIGURE LEGENDS

FIGURE 2 .

 2 FIGURE 2. Expression profiles of the three mouse twinfilin isoforms A) A non-quantitative RT-PCR analysis of the expression of different twinfilin and GAPDH mRNAs in selected mouse tissues. B) Quantitative PCR (Q-PCR) analysis from mRNAs isolated from indicated mouse tissues. The PCR primers used are listed in Table1. GAPDH amplification was used as a control. Twinfilin-2b is the most

FIGURE 3 .

 3 FIGURE 3. Interactions of mouse twinfilins and the isolated ADF-H domains of twinfilin-2 with actin monomers. Increase in the fluorescence of NBD-labelled ATP-G-actin (open circles) or ADP-G-actin (closed circles) was measured at different concentrations of mouse twinfilin isoforms and the isolated domains of twinfilin-2a and -2b. The experiment was carried out with 0.2 M actin under physiological ionic conditions. Symbols indicate data and the solid lines are fitted binding curves for a complex with 1:1 stoichiometry. A) Full-length twinfilin-1, B) full-length twinfilin-2a, C) full-length twinfilin-2b, D) N-terminal ADF-H domain of twinfilin-2a, E) Nterminal ADF-H domain of twinfilin-2b, and F) C-terminal ADF-H domain of twinfilin-2a/b.

FIGURE 4 .

 4 FIGURE 4. All mouse twinfilin isoforms cap actin filament barbed ends. In a pyrene-actin polymerization assay, barbed-end growth (filled circles) and pointed-end

FIGURE 5 .

 5 FIGURE 5. All mouse twinfilin isoforms interact with heterodimeric capping protein. Binding of twinfilin isoforms to capping protein was studied by a native gel electrophoresis assay. Mouse full-length twinfilins and a mutant twinfilin-1 lacking the capping protein binding site were loaded on a gel either separately or as a mixture with mouse 1 2-capping protein. The concentration of twinfilins was 2 M and the concentration of capping protein was 4 M. Lane 1 shows the mobility of capping protein alone, lanes 2, 4, 6 and 8 show the mobilities of wild-type twinfilin-1, mutant twinfilin-1, twinfilin-2a and twinfilin-2b, respectively. When mixed with capping protein, the twinfilin-1, twinfilin-2a and twinfilin-2b disappear from their original migration position and a new band corresponding to the twinfilin/capping protein complex appears above the original migration positions of the proteins (lanes 3, 7 and 9). In contrast, the electrophoretic mobility of the mutant twinfilin-1 lacking the capping protein binding site (see ref. 12) is not affected by the presence of capping protein (lane 5).

FIGURE 6 .TABLE 1 .

 61 FIGURE 6. Localization of the twinfilin isoforms in heart muscle cells. A)Subcellular localization of endogenous twinfilins in cultured neonatal rat cardiomyocytes. An antibody recognizing all three twinfilin isoforms showed punctuate cytoplasmic localization and also revealed enrichment of twinfilins to myofibrils in a subset of cells. B) In cells expressing myc-tagged twinfilins, twinfilin-1 and twinfilin-2b displayed mainly punctuate cytoplasmic localization, but were also enriched in myofibrils (arrowheads in the insets). Twinfilin-2b displayed punctuate

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© 2008 The Authors Journal compilation © 2008 Biochemical Society

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ACKNOWLEDGMENTS

We thank Mikko Frilander, Reinhard Fässler, Ville Paavilainen and Martina Serlachius for critical reading of the manuscript. We thank Maria Vartiainen for reagents. This study was supported by grants from Sigrid Juselius Foundation and Finnish Cancer Organizations to P.L.