Elsa Wiame

Donatienne Tyteca

Nathalie Pierrot

François Collard

Mustapha Amyere

Gaëtane Noel

Jonathan Desmedt

Marie-Cécile Nassogne

Miikka Vikkula

Jean-Noël Octave

Marie-Françoise Vincent

Pierre J Courtoy

Eugen Boltshauser

Emile Van Schaftingen
email: emile.vanschaftingen@uclouvain.be

Emile Van Schaftingen

Molecular Identification of Aspartate N-acetyltransferase and its Mutation in Hypoacetylaspartia

Keywords: N-acetylaspartate, N-acetyltransferase, hypoacetylaspartia, membrane proteins, endoplasmic reticulum 2

The brain-specific compound N-acetylaspartate (NAA) occurs almost exclusively in neurons, where its concentration reaches 20 mM. Its abundance is determined in patients by magnetic resonance spectroscopy to assess neuronal density and health. The molecular identity of the N-acetyltransferase that catalyses NAA synthesis has remained unknown, because this enzyme is membrane-bound and difficult to purify. Database searches indicated that, among the putative N-acetyltransferases (i.e., proteins homologous to known Nacetyltransferases, but with uncharacterized catalytic activity) encoded by the human and mouse genomes, two, NAT8L and NAT14, were almost exclusively expressed in brain. Transfection studies in HEK293T cells indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its K M for aspartate and its sensitivity to detergents are similar to those described for brain aspartate Nacetyltransferase. Confocal microscopy analysis of CHO cells and neurons expressing recombinant NAT8L indicates that it is associated with the endoplasmic reticulum, but not with mitochondria. Mutation search in the NAT8L gene in the only patient known to be deficient in NAA disclosed the presence of a homozygous 19-bp deletion resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme opens perspectives for the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

INTRODUCTION

N-acetylaspartate (NAA), a brain-specific molecule discovered more than 50 years ago, is the second most abundant metabolite in brain after glutamate. It is essentially present in neurons, where its concentration reaches 20 mM. Its abundance can be determined by MRS (magnetic resonance spectroscopy) to monitor neuronal density and health in neurological disorders, including Alzheimer's disease, multiple sclerosis and AIDS encephalopathy [START_REF] Moffett | N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology[END_REF]. NAA is formed from aspartate and acetyl-CoA by aspartate N-acetyltransferase (Asp-NAT), a membrane-bound enzyme [START_REF] Goldstein | Biosynthesis of N-acetyl-L-aspartic acid[END_REF][START_REF] Knizley | The enzymatic synthesis of N-acetyl-L-aspartic acid by a waterinsoluble preparation of a cat brain acetone powder[END_REF][START_REF] Goldstein | The enzymatic synthesis of N-acetyl-L-aspartic acid by subcellular preparations of rat brain[END_REF], which is unstable in the presence of detergents [START_REF] Madhavarao | NAA synthesis and functional roles[END_REF] and could therefore not be purified. Its subcellular localization has been variously reported as mitochondrial [START_REF] Patel | Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport[END_REF][START_REF] Ariyannur | N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes[END_REF] or microsomal [START_REF] Goldstein | The enzymatic synthesis of N-acetyl-L-aspartic acid by subcellular preparations of rat brain[END_REF][START_REF] Lu | N-Acetylaspartate synthase is bimodally expressed in microsomes and mitochondria of brain[END_REF]. NAA is not degraded in neurons, but it is transferred by an undefined mechanism to oligodendrocytes, the myelin-forming cells, where it is hydrolyzed by aspartoacylase (aminoacylase II). The released acetate serves as a precursor for the synthesis of fatty acids, required for the formation of myelin lipids [START_REF] Chakraborty | Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelinassociated aspartoacylase[END_REF]. NAA serves also as a precursor for N-acetylaspartylglutamate [START_REF] Tyson | Labeling of N-acetylaspartate and Nacetylaspartylglutamate in rat neocortex, hippocampus and cerebellum from [1-13 C]glucose[END_REF][START_REF] Arun | Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators[END_REF], the most abundant dipeptide present in brain and possibly a neurotransmitter [START_REF] Wroblewska | N-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytes[END_REF]. Other proposed roles for NAA are osmoregulation or disposal of amino groups resulting from glutamate transamination [START_REF] Moffett | N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology[END_REF]. Except that NAA does not appear to be a neurotransmitter [START_REF] Moffett | Preface: a brief review of N-acetylaspartate[END_REF], no agreement exists on the relative importance of these roles.

NAA metabolism is disrupted in two inborn errors of metabolism. Deficiency of aspartoacylase, caused by mutations in the ASPA gene, gives rise to a leukodystrophy known as Canavan disease (MIM 271900), which is characterized by onset in early infancy, dystonia, severe mental defect, blindness, megalencephaly, and death by 18 months on the average. [START_REF] Matalon | Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease[END_REF]. NAA concentration is raised in brain, CSF, and urine. The 'opposite' disorder, i.e. the absence of brain NAA, has been described in only one patient, with truncal ataxia, marked developmental delay, seizures and secondary microcephaly [START_REF] Martin | Absence of Nacetylaspartate in the human brain: impact on neurospectroscopy?[END_REF][START_REF] Boltshauser | Follow-up of a child with hypoacetylaspartia[END_REF][START_REF] Burlina | Hypoacetylaspartia: clinical and biochemical follow-up of a patient[END_REF]. This disorder, designated 'hypoacetylaspartia', is presumably due to a deficiency of Asp-NAT, but this has not been demonstrated.

A better definition of the role of NAA and the identification of the defect underlying hypoacetylaspartia would greatly benefit from the molecular identification of Asp-NAT. This identification was the purpose of the present work.

EXPERIMENTAL Preparation of expression vectors

Human and mouse total brain cDNA were synthesized with random primers and 1.5-2.5 µg total RNA with the M-MuLV reverse transcriptase from Fermentas (St Leon-Rot, Germany) according to the manufacturer's instructions. FirstChoice human brain total RNA was from Applied Biosystems and mouse total brain RNA was prepared as described in [START_REF] Maliekal | Molecular identification of mammalian phosphopentomutase and glucose-1,6bisphosphate synthase, two members of the alpha-D-phosphohexomutase family[END_REF].

The open reading frames of mouse NAT14 (UCSC refseq gene NM_201355) and mouse NAT8L (UCSC refseq gene NM_001001985) were PCR-amplified using 2.5 U of PWO polymerase (Roche Applied Science) in the presence of 1.5 M betaine (Sigma-Aldrich) with mouse total brain cDNA as a template. For the non-tagged constructions, a 5'-primer (for primer sequences, see Table S1 in the Supplementary data) containing the putative ATG preceded by a perfect Kozak sequence and a KpnI site, and a 3'-primer containing the putative stop codon flanked by a XbaI site were used. For the C-tagged constructs, the 5'-primers were the same, while the putative stop codon in the 3'-primers was mutated and flanked by a XbaI site. For the N-tagged constructs, the 5'-primers were devoid of Kozak sequence and the 3'primers were the same as for the wild-type amplifications. The PCR amplifications started with an initial denaturation step at 96°C for 3 min, followed by 35 cycles for 30 sec at 96°C, 1 min at 60°C and 1 min at 72°C, and ended with a final extension step at 72°C for 5 min. All PCRs reported in this paper were performed with this 5-step program except for site-directed mutagenesis (see below). PCR products of the expected size were obtained, subcloned into pBlueScript plasmid and checked by sequencing. The KpnI/XbaI digested products were prepared from E. coli XL1blue strain (C-tagged constructs) or from E. coli JM110 strain (wild-type and N-tagged constructs) and inserted in pCMV5, pEF6MycHisA and pEF6HisB expression vectors for the wild-type, C-tagged and N-tagged constructs, respectively. For human NAT8L, amplifications were performed with 2.5 U of Pfu polymerase (Fermentas) in the presence of 1 M betaine, with 2 µl of human total brain cDNA as a template. The three types of construct were prepared as for the mouse sequences and checked by sequencing.

Site-directed mutagenesis

Fifty ng of the pBlueScript vector containing the human NAT8L sequence designed to be expressed with a polyHis tag at its C-terminus were used as a template, in a 50 µl-PCR reaction containing 25 pmol of each primer (Table S1), 0.2 mM dNTP, 1 M betaine, and 2.5 U Pfu polymerase in 1 X Pfu buffer. All these primers were phosphorylated at their 5' end. This mixture was subjected to thermal cycling at 96°C for 30 sec for denaturation, followed by 30 sec at 55°C for annealing, and 5 min at 72°C for extension. A total of 20 cycles was used and the procedure was ended by 7 min at 72°C. For both mutants, 3 identical reactions were done and pooled at the purification step in 35 µl of 10 mM TrisHCl, pH 8.5. The purified PCR product (30 µl) was incubated overnight at 16°C with 5 U of T4 DNA ligase (Fermentas) in the presence of 5 % PEG 4000 in 50 µl of 1 X ligase buffer. The ligation product was then digested for 2 hours at 37°C with 20 U of DpnI in order to eliminate the wild-type template. Fifteen µl of this mixture were used to transform 100 µl of E. coli XL1blue bacteria. The complete NAT8L coding region of all plasmids was sequenced to verify the introduced deletion and the absence of random mutations. The KpnI/XbaI fragment was then subcloned in pEF6MycHisA vector and both mutants were expressed as polyHis/myc-tagged fusion proteins.

Transfections of HEK 293T cells and western blotting

HEK 293T cells were cultured and transfected essentially as described by Rzem et al. [START_REF] Rzem | A gene encoding a putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is mutated in L-2-hydroxyglutaric aciduria[END_REF] using the jetPEI procedure. After 48 h at 37°C, the cells were washed once with 5 ml of cold phosphate-buffered saline (PBS). They were scraped in 0.8 ml of 20 mM Hepes (pH 7.1) containing 5 µg/ml leupeptin and antipain, frozen in liquid nitrogen, thawed and lysed by vortexing. The extracts were incubated with 100 U/ml DNase I (from Sigma-Aldrich; prepared in 5 mM MgSO 4 ) for 1 hour on ice before use. Protein concentration was determined with the Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], using gamma-globulin as a standard. Western blots were performed as described [START_REF] Rzem | A gene encoding a putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is mutated in L-2-hydroxyglutaric aciduria[END_REF] with a monoclonal pentaHis antibody (Qiagen) diluted 1:2000 in PBS containing 1 % bovine serum albumin. After washing, membranes were probed for 1 hour at room temperature with peroxidase-conjugated anti-mouse antibody. Detection was performed using the ECL Western blotting analysis system from GE Healthcare according to the manufacturer's protocol.

N-acetylaspartate measurements by GC/MS

Quantification of NAA in cell media was performed by isotope dilution gas chromatographymass spectrometry as described by Kelley and Stamas [START_REF] Kelley | Quantification of N-acetyl-L-aspartic acid in urine by isotope dilution gas chromatography-mass spectrometry[END_REF] with N-[d 3 ]-acetylaspartate from VU Medical Center, Metabolic Laboratory (Dr H.J. ten Brink, Amsterdam, The Netherlands). Samples (0.2 ml) were treated with hydroxylamine hydrochloride in the presence of N-[d 3 ]acetylaspartate as internal standard, and extracted with ethyl acetate followed by diethyl ether. After evaporation of the combined extracts under a stream of nitrogen, the residue was resuspended in acetonitrile, and derivatization was achieved with N,Obis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA/TMCS). Analysis was performed by gas-chromatography (Hewlett-Packard 6890 series GC system equipped with a 30 m x 0.25 mm fused silica capillary column CP-SIL 8CB, Varian) with helium as carrier gas, coupled with a Hewlett-Packard 5973 Mass selective detector under electron-impact fragmentation. Mass spectrometric data were collected in the selected ion mode at m/z = 274 for native NAA, and m/z = 277 for synthetic isotopic NAA. Calibration curves were constructed by mixing 4.5 g of the isotopic NAA with standard solution containing 0 -6 g of NAA, followed by drying under nitrogen and direct derivatization with the silylating agent.

Assay of aspartate N-acetyltransferase

Asp-NAT was assayed radiochemically in a mixture (200 µl final volume) comprising, unless otherwise stated 10 mM potassium phosphate, 20 mM Hepes, pH 7.1, 1 mM MgCl 2 , 50 µM Laspartate, 10 5 cpm [U- 14 C]L-aspartate (GE Healthcare), 200 µM acetyl-CoA (Sigma-Aldrich), and 10 µl of a cell extract containing 5-10 mg protein/ml. After 20-40 min at 30°C, the reaction was arrested by a 5-min incubation at 80°C and 1 ml of 5 mM Hepes, pH 7.1 was added. The sample was centrifuged and the supernatant was applied on a 1-ml Dowex AG1X8 column (Cl -form, 100-200 mesh, from Acros Organics) prepared in a Pasteur capillary pipette. The latter was washed successively with 2 ml 5 mM Hepes, pH 7.1, 5 ml 150 mM NaCl in the same buffer to elute unreacted aspartate, and 5 ml 300 mM NaCl to elute NAA. Radioactivity was counted in the presence of Ultima Gold TM (Perkin Elmer) in a liquid scintillation counter. The kinetic studies shown in Figure 3 and Figures S2-4 were performed using washed pellets, obtained by centrifuging extracts for 20 min at 16 000 x g, resuspending them in the initial volume of extraction buffer, recentrifugation and resuspension in the original volume of extraction buffer.

Mutation search

Genomic DNA analysis of the child with hypoacetylaspartia [START_REF] Boltshauser | Follow-up of a child with hypoacetylaspartia[END_REF] was performed with informed consent of the legal parents. The 3 exons of the NAT8L gene were PCR-amplified using 0.5 µM of forward and reverse primers (Table S1) with 75 ng of genomic DNA as a template. These reactions (50 µl) were performed using Biotools DNA polymerase (Kordia Life Sciences, Leiden, the Netherlands) in its provided buffer, 0.2 mM dNTP, and 1 M betaine. PCR products were purified and sequenced directly on both strands by use of a CEQ2000 sequencer (Beckman) or by the sequencing service of Macrogen (Seoul, South Korea). For the screening of the 118 healthy controls, the region surrounding the deletion was amplified from genomic DNA with a specific couple of primers (Table S1). PCR conditions were the same except that a concentration of 1.5 M betaine was used. The PCR products were directly loaded on a 2 % agarose gel containing 0.5 µg/ml ethidium bromide to distinguish wild-type (223 bp) and mutant (204 bp) alleles.

Neuronal cultures and infection

Primary cultures of cortical neurons were prepared from 17-18 days old Wistar rat embryos as described previously [START_REF] Macq | The long-term adenoviral expression of the human amyloid precursor protein shows different secretase activities in rat cortical neurons and astrocytes[END_REF]. Brains were dissected, transferred to HBSS buffer medium (1 mM sodium pyruvate, 10 mM Hepes without Ca 2+ and Mg 2+ ), dissociated in the same medium containing 1.26 mM Ca 2+ and Mg 2+ (0.50 mM MgCl 2 and 0.41 mM MgSO 4 ) and centrifuged for 10 min at 200 g (room temperature). Cells were plated in culture dishes pre-treated with 10 µg/ml poly-L-lysine in PBS and cultured for 7 days in NEUROBASAL medium supplemented with 2% (v/v) B-27 medium and 0.5 mM L-glutamine prior to infection with recombinant lentivirus. The NAT8L-pEF6MycHisA plasmid was utilized for construction of recombinant lentivirus, as previously described [START_REF] Salmon | Production and titration of lentiviral vectors[END_REF]. After 7 days of culture, neurons were incubated in the presence of the lentivirus for 3 days before analysis.

Immunofluorescence in transfected CHO cells and infected primary neurons

CHO cells were propagated in DMEM/F-12 supplemented with 10 % fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were seeded at 50,000 cells/cm 2 in 24-well plates on glass coverslips coated with 25 µg/µl fibronectin (in PBS) and grown for one day ( 80 % confluency). Transfection with the NAT8L-pEF6MycHisA plasmid or the PDIb-HA construct [START_REF] Raykhel | A molecular specificity code for the three mammalian KDEL receptors[END_REF] was carried out at 0.5 µg total DNA and 1 µl lipofectamine/cm 2 at 37°C for 5 h followed by reincubation overnight in DMEM/F-12/10% FBS without antibiotics. After 24 h, cells were processed for immunofluorescence as described by Mettlen et al. [START_REF] Mettlen | Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells[END_REF].

For immunofluorescence experiments, neurons were seeded at 10 5 cells/cm 2 on glass coverslips, fixed with paraformaldehyde 4% (v/v) for 15 min at room temperature and permeabilized thereafter with 1% Triton X100 (v/v) in PBS during 15 min. Non-specific immunostaining was prevented by 1 h incubation in a PBS solution containing 3% non-fat dry milk at room temperature.

For primary antibodies, we used mouse IgG 2a anti-Myc-Tag (9B11) monoclonal antibody (1/500; Cell Signalling), mouse IgG 1 anti-His monoclonal antibody (1/50; Qiagen), rabbit affinity-purified specific antibodies against NAT8L (1/100), rabbit anti-HA antibodies (1/300), rat monoclonal antibody recognizing KDEL-bearing proteins (1/300; Abcam), either alone or combined with mouse IgG 2b monoclonal antibody to PDI (1/50; Abcam), rabbit MAP-2 primary antibody (B9; serum used at 1/1000 dilution) and Alexa-labelled secondary antibodies were from Molecular Probes/Invitrogen (each used at 5 µg/ml). For live mitochondria labelling, cells were incubated with MitoTracker-red (CMXRos; Molecular Probes; 250 nM in DMEM/F-12) at 37°C for 30 min followed by chase in MitoTracker-free medium for 30 min, before fixation-permeabilization. All preparations were mounted in Mowiol in the dark overnight and examined with a LSM 510 META confocal microscope (Zeiss, Jena, Germany) using a Plan-Apochromat 63X/1.4 oil DIC objective.

Specific antibodies against NAT8L

Antibodies were raised against two peptides of mouse NAT8L (C)NTAFRGLRQHPRTQLL (P44) and (C)MSVDSRFRGKGIAKALG (P45) previously shown to be suitable for immunization [START_REF] Niwa | A novel molecule "shati" is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference[END_REF]. These peptides were conjugated to keyhole limpet hemocyanin and injected together to rabbits 4 times at 2 weeks intervals. Serum was taken from the rabbits one month after the final injection of these peptides. IgG were isolated from the sera by affinity chromatography on protein-G (HITRAP protein G HP, GE Healthcare). IgG were bound to the column in PBS buffer and eluted with 50 mM glycine-HCl, pH 2.7, containing 150 mM NaCl. Collected fractions were immediately neutralized with 1 M Tris-HCl, pH 8. Antibodies specific to the P44 and P45 NAT8L peptides were then purified by affinity chromatography on columns coupled with the P44 or P45 peptide. Peptides were coupled through their aminoterminal cysteine to iodoacetamide immobilized on a crosslinked agarose support (Sulfolink, Thermo scientific PIERCE). IgG fractions were loaded on the peptide-coupled column in PBS. Antibodies bound to the column were eluted with 50 mM glycine-HCl, pH 2.7, containing 150 mM NaCl, and immediately neutralized with 1 M Tris-HCl, pH 8.

RESULTS

Database searches

The characteristics of Asp-NAT are probably shared by only a limited number of mammalian gene products. Indeed, (1) this enzyme most likely belongs to a family of Nacetyltransferases; (2) it is mainly, if not exclusively, expressed in brain; (3) it is membranebound; (4) it should have close homologues only in species that synthesize NAA. We decided therefore to search mammalian genomes for genes encoding proteins with these characteristics.

A search in the human and mouse genomes with N-acetyltransferase (NAT) as a query yielded about 30 genes encoding N-acetyltransferases. These comprised a few well-identified sequences (e.g. N-acetyltransferases acting on arylamines, glucosamine-6-phosphate, glutamate, and spermine/spermidine) and a majority of putative N-acetyltransferases (i.e. hypothetical proteins showing sequence similarity to well characterized N-acetyltransferases) with undefined specificity. mRNA expression data of the Symatlas database [START_REF] Su | A gene atlas of the mouse and human protein-encoding transcriptomes[END_REF] indicated that two of them were mainly present in brain: NAT14 and NAT8L. NAT14, also called Klp1 (K562 cell-derived leucine-zipper-like protein 1) is thought to be a transcription factor regulating the expression of coproporphyrinogen oxidase by binding to a promoter regulatory element [START_REF] Takahashi | Cloning of a coproporphyrinogen oxidase promoter regulatory element binding protein[END_REF]. NAT8L, also designated Shati, is overexpressed in brain after administration of metamphetamine to mice [START_REF] Niwa | A novel molecule "shati" is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference[END_REF]. Both proteins showed at least one putative transmembrane domain. Close homologues (> 50 % sequence identity) of NAT8L and NAT14 were only found in vertebrates. Taken together, these findings indicated that both NAT8L and NAT14 were good candidates for Asp-NAT.

Gene structure, tissue distribution, and protein sequence analysis of NAT8L

NAT8L is encoded by 3-exon genes on mouse chromosome 5 and human chromosome 4 (band 4p16.3). Previous PCR data obtained with mouse NAT8L (Shati) indicated that the mRNA encoding this protein is expressed not only in brain, but also in liver and kidneys [START_REF] Niwa | A novel molecule "shati" is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference[END_REF]. We therefore performed quantitative PCR amplification (as described in [START_REF] Maliekal | Molecular identification of mammalian phosphopentomutase and glucose-1,6bisphosphate synthase, two members of the alpha-D-phosphohexomutase family[END_REF]) on mouse tissue cDNA with couples of primers situated in exons 1 and 2, or in exons 2 and 3. Our findings indicated that NAT8L was amplified from brain cDNA and to a lesser extent from thymus or spleen cDNA. The abundance of the mRNA was calculated to be about ten-fold lower in these tissues than in brain. No specific amplification was observed with liver, kidney, lung, skeletal muscle, testis, and heart cDNA (results not shown).

The NAT8L cDNA was also amplified from human brain cDNA using primers situated in exons 1 and 3. Sequencing of the amplified products confirmed the deduced protein sequence registered under NCBI accession number EAW82541. No amplification could be observed with primers corresponding to a cDNA sequence encoding a protein (Swissprot Q8N9F0) that differs from human and mouse NAT8L in its first 90 residues. This cDNA contains in fact an exon from the upstream human gene (LOC401115 in human) fused to exons 2 and 3 of the NAT8L gene.

The NAT8L sequences of various species are aligned in Figure 1. As previously noted [START_REF] Niwa | A novel molecule "shati" is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference[END_REF], the C-terminal region is homologous to N-acetyltransferases. NAT8Ls also comprise a conserved N-terminal sequence of 15 residues, a proline-and alanine-rich region of variable length (residues 40-73 in the human sequence), and a highly hydrophobic region of about 30 amino acids that just precedes the N-acetyltransferase domain.

Targeting prediction programmes (TargetP [START_REF] Emanuelsson | Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[END_REF]; PSort2 [START_REF] Nakai | PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization[END_REF]) did not suggest that NAT8L had a mitochondrial propeptide or a signal peptide. It should be noted that exon 1 of human, mouse and rat NAT8L genes contains an in-frame stop codon that precedes the initiator ATG codon. This indicates that the protein sequence truly starts with the methionine shown as Met1 in the alignment. NAT14 is not described in detail as we show in the next section that it does not correspond to Asp-NAT.

Identification of aspartate N-acetyltransferase as NAT8L

Both mouse NAT8L and NAT14 were overexpressed in HEK293T cells, as native proteins or as fusion proteins with a polyHis tag at the C-or the N-terminus. GC-MS analysis [START_REF] Kelley | Quantification of N-acetyl-L-aspartic acid in urine by isotope dilution gas chromatography-mass spectrometry[END_REF] of media collected after 48 hours indicated the presence of a product co-eluting with authentic NAA in medium from cells transfected with all NAT8L constructs (see Figure S1 in the Supplementary data), whereas this compound was below the detection limit (5 µM) in control cells (Figure S1A), or those transfected with NAT14 constructs. The fragmentation pattern of this product (insert of Figure S1B) was identical with that of authentic NAA, with major fragments of m/z ratios 73, 147, 184, 245, 274 and 376 [START_REF] Kelley | Quantification of N-acetyl-L-aspartic acid in urine by isotope dilution gas chromatography-mass spectrometry[END_REF]. Quantification of NAA by isotope dilution with the trideuterated compound (Figure 2B) indicated that the concentration of NAA in the medium amounted to 200-250 µM in cells transfected with the different NAT8L constructs, corresponding to an amount of 300 nmol/mg cell protein. Analysis of cell extracts indicated that this compound was also present intracellularly at concentrations of about 60 nmol/mg protein (results not shown).

Asp-NAT activity was assayed in cell extracts as the acetyl-CoA-dependent conversion of [ 14 C]L-aspartate to a more acidic product (Figure 2C). In full agreement with the results mentioned above, this activity was observed in extracts of cells transfected with the three types of NAT8L constructs, but not in other extracts. Slightly lower activities were observed with the N-tagged and the C-tagged forms as compared to untagged NAT8L (59 % and 75 %, respectively). Western blot analysis with an anti-polyHis tag antibody (Figure 2A) revealed that all His-tagged versions of NAT8L and NAT14 were expressed. This indicated that the absence of Asp-NAT activity in cells transfected with NAT14 was not due to lack of expression of this protein.

Kinetic properties of NAT8L

The Asp-NAT activity was further characterized in extracts of cells expressing wild-type mouse NAT8L. The enzymatic activity was almost completely (> 90%) recovered in the pellet after a 20-min centrifugation of frozen-thawed extracts at 16.000 x g (not shown), indicating that it is membrane-bound. The kinetic studies were therefore performed on washed pellets, to eliminate soluble, potentially interfering enzymes. Detergents showed a biphasic effect on the activity (Figure 3A), increasing it at low concentration, presumably because they allowed access of the catalytic site to substrate, and being inhibitory at higher concentrations. Double-reciprocal plots of the saturation curve for aspartate was linear both in the absence and in the presence of 0.5 mM CHAPS, which increased V max by 30 % without affecting the K M ( 90 µM) for aspartate (Figure S2). With acetyl-CoA as varying substrate, the double-reciprocal plots were non-linear in the absence of detergent, consistent with part of the enzyme being poorly accessible to acetyl-CoA, but well in the presence of CHAPS (Figure 3B). The K M values amounted to 9 and 13 µM in the presence of 0.5 and 1 mM CHAPS, respectively.

[ 14 C]Glutamate was also used by the enzyme, but with an about 50-fold lower affinity (K M 5 mM) and a similar V max compared to aspartate (not shown), in agreement with previous results [START_REF] Truckenmiller | N-Acetylation of L-aspartate in the nervous system: differential distribution of a specific enzyme[END_REF][START_REF] Madhavarao | Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain[END_REF]. Accordingly, the incorporation of [ 14 C]-aspartate into NAA was inhibited by glutamate competitively with respect to aspartate with a K i of 6 mM (Figures 3C andS3A). No inhibition was observed with other amino acids, indicating that the enzyme is specific. The enzyme was also inhibited by its reaction product NAA. This inhibition was essentially competitive with respect to aspartate (K i = 0.56 mM; Figures 3D andS3B) and uncompetitive versus acetyl-CoA (Figure S4).

Subcellular localization of NAT8L

Because of the controversy on the subcellular localization of Asp-NAT, it was of interest to check the localization of NAT8L in transfected cells. This was done by confocal microscopy analysis of CHO cells transfected with myc-tagged (at the C-terminus), his-tagged (at the Nterminus) or untagged NAT8L, and of neurons in primary culture infected with a lentiviral vector allowing the expression of myc-tagged NAT8L. Immunolabelling of myc-tagged NAT8L expressed in CHO cells or in primary neurons delineated the nuclear envelope (arrows in Figures 4 and5) and produced a reticular pattern, strongly suggestive of the endoplasmic reticulum. This was confirmed by the substantial (though not complete) colocalization of myc-tagged NAT8L with KDEL-bearing proteins of the endoplasmic reticulum (arrowheads in Figures 4 and5). Analysis of the colour intensity profiles in the cytoplasm showed that the peaks of NAT8L-labelling most often coincided with the peaks of ER marker labelling (Figures 4D and5C; see also Figure S5C). Large but not full co-localization is in keeping with the well-known heterogeneity among endoplasmic reticulum domains [START_REF] Villa | Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, Bip[END_REF][START_REF] Krijnse-Locker | The organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons[END_REF]. In contrast, the myc-tagged NAT8L signal was fully resolved from the Golgi complex, recognized by co-transfection with the PDIb-HA construct [START_REF] Raykhel | A molecular specificity code for the three mammalian KDEL receptors[END_REF] (Figure 4B), and from mitochondria labelled in vivo by MitoTracker red (Figures 4C,4E), as confirmed by analysis of the intensity profiles (Figure 5B). Targeting to the nuclear envelope and the endoplasmic reticulum was independent of epitope tagging, since NAT8L fused to a myc-tag at its Cterminus or to a polyHis-tag at its N-terminus showed a similar localization pattern to native NAT8L recognized with affinity-purified antibodies (Figure S5A).

Mutation of NAT8L in a patient with hypoacetylaspartia

To check whether NAT8L was mutated in the patient deficient in NAA, we amplified the three exons of the NAT8L gene and screened them for mutations. A 19-bp deletion in the homozygous form was observed in the first exon (Figure 6A). This mutation leads to a change in the reading frame after amino acid 69 and it should therefore lead to no production of functional protein. To rule out the possibility that this mutation would still allow the production of an active protein by codon-slipping or by reinitiation at a later AUG codon, we prepared an expression vector for this mutated form (provided with a C-terminal his-tag) and transfected it in HEK293T cells. Extracts prepared from these cells did not contain any detectable Asp-NAT activity or material reacting with the anti-polyHis tag antibody, whereas extracts of cells transfected with the non-mutated human cDNA were enzymatically active and contained an immunoreactive band of the appropriate size (Figure 6B andC).

Several human NAT8L protein entries (e.g. BAC04426.1) comprise only the last 134 AA of NAT8L. The sequence of this (putative) shorter protein is unaffected by the mutation and it was therefore important to verify if it displayed Asp-NAT activity. Transfection of a vector allowing the expression of this shorter form resulted in the expression of a polypeptide of the expected size ( 18 kDa) but to no detectable enzymatic activity (Figure 6B andC). Taken together, these findings indicated that the mutation found in the patient corresponds indeed to a null mutation.

The patient is an adopted child and no material was therefore available to check the presence of the mutation in the biological parents. The mutation found in the patient could be detected by PCR amplification of genomic DNA and gel electrophoresis. This assay allowed also the detection of the mutation in the heterozygous state, as indicated by amplifications performed on samples containing equal amounts of DNA from the patient and a control. The 19-bp deletion was not found in 118 control DNA samples either in the homozygous or the heterozygous state (results not shown).

DISCUSSION

Molecular identification of aspartate N-acetyltransferase

Despite its discovery about 50 years ago [START_REF] Goldstein | Biosynthesis of N-acetyl-L-aspartic acid[END_REF], Asp-NAT had not yet been molecularly identified, owing to the difficulty of purifying this membrane-bound enzyme [START_REF] Ariyannur | N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes[END_REF]. The availability of databases comprising virtually all proteins encoded by the human and mouse genomes and providing information on their tissue distribution enabled us to identify two candidate proteins for this enzyme. Transfection studies in HEK293T cells showed that NAT8L-expressing cells synthesize NAA and that extracts of these cells display Asp-NAT activity. This activity had properties similar to those of brain Asp-NAT, including its K M value for aspartate, its specificity for this amino acid, its inhibition by the reaction product, its association with membranes and its inhibition by detergents [START_REF] Goldstein | The enzymatic synthesis of N-acetyl-L-aspartic acid by subcellular preparations of rat brain[END_REF][START_REF] Madhavarao | NAA synthesis and functional roles[END_REF][START_REF] Ariyannur | N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes[END_REF][START_REF] Madhavarao | Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain[END_REF]. These findings therefore indicate that NAT8L is responsible for NAA synthesis in brain. This conclusion is strongly supported by the finding that the NAT8L gene is mutated in the only patient reported to have undetectable levels of NAA in brain. Considering that NAA is restricted to neurons, where it is synthesized, our identification is also consistent with immunohistochemical studies showing that mouse shati (i.e. NAT8L) is exclusively present in neurons [START_REF] Niwa | A novel molecule "shati" is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference[END_REF].

As expected for Asp-NAT, close homologues of NAT8L were found in vertebrates, whose brain contains NAA, but not in insects and Caenorhabditis elegans, where NAA has never been reported. Unfortunately, the genome of the crayfish, in which NAA has been described [START_REF] Lieberman | Synthesis of N-acetylaspartylglutamate (NAAG) and N-acetylaspartate (NAA) in axons and glia of the crayfish medial giant nerve fiber[END_REF], has not yet been sequenced.

The molecular basis of hypoacetylaspartia

The finding that the NAT8L gene is mutated in the single known case of hypoacetylaspartia [START_REF] Martin | Absence of Nacetylaspartate in the human brain: impact on neurospectroscopy?[END_REF][START_REF] Boltshauser | Follow-up of a child with hypoacetylaspartia[END_REF][START_REF] Burlina | Hypoacetylaspartia: clinical and biochemical follow-up of a patient[END_REF] indicates that this disorder, in which no NAA is detectable in brain, is due to a lack of synthesis of this compound in brain. This excludes other potential causes, such as an increased degradation by a hyperactive aspartoacylase, or a deficiency in the transport or the formation of aspartate and/or acetyl-CoA in the compartment in which aspartate Nacetyltranferase is present. The finding that the mutation induces a change in reading frame indicates that it is a null mutation. Accordingly, no NAA synthase activity could be detected in cells that had been transfected with a plasmid carrying the mutated allele.

The homozygous mutation found in the patient suggests that he may be the offspring of consanguineous parents. Unfortunately, no information is available on the biological parents of this adopted child. Therefore one has to be very cautious before concluding that the phenotype of this unique patient is representative of hypoacetylaspartia, as he may suffer from other recessive genetic diseases.

It is likely that hypoacetylaspartia is underdiagnosed, and this for two reasons. Firstly, MRS is not systematically performed to investigate neurological problems in young children. Secondly, decreased levels of NAA are usually interpreted as being secondary to a loss of neuronal integrity, rather than as a primary cause of neuronal dysfunction. However, point mutations in the NAT8L gene that would not lead to a complete loss of enzymatic activity could result in low, yet detectable levels of NAA and be associated with milder forms of the disease than the one described in the only patient known so far. It would therefore be of interest to determine more systematically the brain NAA level in young patients with mental retardation, epilepsy of unknown origin, and/or ataxia, and to search the NAT8L gene for mutations if the NAA level is found to be decreased.

Subcellular localization of aspartate N-acetyltransferase

As mentioned in the introduction, the subcellular localization of Asp-NAT is still a matter of controversy. Fractionation studies of brain extracts have variously concluded to a microsomal [START_REF] Goldstein | The enzymatic synthesis of N-acetyl-L-aspartic acid by subcellular preparations of rat brain[END_REF] or a mitochondrial localization of Asp-NAT [START_REF] Patel | Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport[END_REF][START_REF] Ariyannur | N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes[END_REF]. However, one of the studies concluding to a mitochondrial localization shows in fact that the specific activity of Asp-NAT is higher in the microsomal than in the mitochondrial fraction [START_REF] Ariyannur | N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes[END_REF], which in fact argues for a microsomal localization. The finding [START_REF] Patel | Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport[END_REF] that purified brain mitochondria produce NAA when incubated with glutamate (a source of aspartate) and pyruvate (sources of acetyl-CoA in the mitochondrial matrix) is taken as evidence that intact brain mitochondria contain Asp-NAT. One has to note, however, that the assay used by Patel and Clark [START_REF] Patel | Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport[END_REF] for measuring the NAA produced by mitochondria employed a rather crude (7-fold purified) preparation of pig kidney aspartoacylase to convert NAA to aspartate. Therefore, another metabolite (asparagine, for instance) may have been measured. These contradictory findings are most likely the result of the inherent difficulty of studying the subcellular distribution by fractionation in such a heterogeneous tissue as brain.

Recent tracer studies comparing the incorporation of radioactivity into NAA from L- aspartate and L -malate in SH-SY5Y neuroblastoma cells have led Arun et al. [START_REF] Arun | Evidence for mitochondrial and cytoplasmic N-acetylaspartate synthesis in SH-SY5Y neuroblastoma cells[END_REF] to conclude that mitochondria are a major site of NAA synthesis. However, the technique used (thin layer chromatography) to isolate radiolabelled NAA and L -aspartate does not allow one to separate these metabolites from other radioactive metabolites that certainly form when cells are incubated for 3 hours with a radioactive precursor, making that the results of this work have to be interpreted with caution.

Low levels of NAA were found in the brains of mice deficient in ARALAR [START_REF] Jalil | Reduced N-acetylaspartate levels in mice lacking aralar, a brain-and muscle-type mitochondrial aspartate-glutamate carrier[END_REF], a glutamate/aspartate exchanger serving to export aspartate from brain mitochondria. This was taken to suggest that aspartate has to be transferred from the mitochondria to the cytosol before being acetylated by aspartate N-acetyltransferase. This argument, though pleading against a mitochondrial localization of Asp-NAT, is not decisive because one may not exclude that a defect in ARALAR also causes a decrease in the mitochondrial concentration of aspartate.

Our own data on the localization of NAT8L (i.e., Asp-NAT) indicate that this protein does not co-localize with mitochondria, consistent with the absence of a mitochondrial propeptide in the NAT8L sequence. The reticular pattern of the expression with labelling of the nuclear membrane and the (at least partial) co-localization with endoplasmic reticulum markers, indicate instead that NAT8L is associated with the endoplasmic reticulum. This localization is independent from the tag used for immunostaining and is observed not only in CHO cells, but also in neurons in primary cultures. These findings lead therefore to the conclusion that Asp-NAT is associated with the endoplasmic reticulum.

Perspectives

Our work opens the possibility of developing experimental models to better define the role of NAA. None of the roles proposed so far accounts for the very high concentration of NAA in neurons, since NAA does not appear to be a neurotransmitter [START_REF] Moffett | Preface: a brief review of N-acetylaspartate[END_REF] and since its role as an osmolyte is at best very minor compared to that of other solutes present in neural cells [START_REF] Verbalis | Control of brain volume during hypoosmolality and hyperosmolality[END_REF].

The role of NAA in myelination may even be questioned since this process is only moderately delayed in the patient with NAA deficiency [START_REF] Boltshauser | Follow-up of a child with hypoacetylaspartia[END_REF][START_REF] Burlina | Hypoacetylaspartia: clinical and biochemical follow-up of a patient[END_REF]. The creation of a mouse knock-out model may also permit to determine to what extent the symptoms found in the patient with hypoacetyaspartia are due to NAA deficiency. The availability of the Asp-NAT sequence will also allow for a better definition of the mechanisms that control the concentration of this compound, including the effect of pathological conditions [START_REF] Schuff | N-acetylaspartate as a marker of neuronal injury in neurodegenerative disease[END_REF] and antipsychotic drugs [START_REF] Arun | Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells[END_REF]. In this respect, the finding that Shati (i.e. NAT8L) is upregulated following metamphetamine treatment suggests that NAA plays a role, presumably an indirect one, in dopaminergic transmission [START_REF] Niwa | A novel molecule "shati" is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference[END_REF][START_REF] Niwa | A novel molecule "shati" increases dopamine uptake via the induction of tumor necrosis factor-alpha in pheochromocytoma-12 cells[END_REF]). This work confirms also the need to perform MRS spectroscopy in children with developmental problems, especially when microcephaly is present, to check for NAA deficiency.

7

  Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091024 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091024 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. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figure 1 .

 1 Figure 1. Multiple alignment of NAT8L sequences. The alignment shows the sequences from Homo sapiens (Hsap; NCBI accession nr EAW82541), Mus musculus (Mmus; NP_001001985) and Danio rerio (Drer; NP_001077308). Strictly conserved residues in the three sequences are indicated in bold. A 30 residue hydrophobic region is underlined in the human sequence.

Figure 2 .

 2 Figure 2. Effect of transfection of HEK cells with mouse NAT8L or NAT14 on the NAA concentration in the medium and the Asp-NAT activity in cell extracts.Cells were transfected with plasmids expressing NAT8L or NAT14, either as such (WT) or as C-terminal (CT) or N-terminal (NT) polyHis tag fusion proteins. Transfection with an empty plasmid (EP) was used as a control. After 48 h, the NAA concentration was measured by GC/MS in the medium (B) and the Asp-NAT activity was assayed in cell extracts (C). The indicated extracts were also analysed by Western blotting with an anti-polyHis tag antibody to evaluate the level of expression of the recombinant proteins (A). These data are representative (A) or the means ± SEM (B, C) of three independent transfection experiments.

Figure 3 .

 3 Figure 3. Kinetic properties of the Asp-NAT activity of NAT8L. HEK cells were transfected with untagged mouse NAT8L and the enzymatic activity was determined radiochemically on washed membrane pellets as described in the Materials and Methods. (A) Effect of two detergents on the Asp-NAT activity; assay medium contained 50 µM L-aspartate, 200 µM acetyl-CoA, and the indicated concentrations of CHAPS or octylglucoside. (B) Effect of CHAPS on the saturation curve for acetyl-CoA; assay medium contained 20 µM aspartate and the indicated concentrations of acetyl-CoA and CHAPS. (C) Effect of glutamate and (D) of N-acetylaspartate on the saturation curve for aspartate; assay medium contained 200 µM acetyl-CoA, 0.5 mM CHAPS and variable concentrations of aspartate. Panels (B), (C) and (D) show double reciprocal plots. The results shown are the means ± SEM for three determinations performed with extracts derived from independent transfections. In some cases, the SEM is not visible because it is smaller than the size of the symbol.

Figure 4 .

 4 Figure 4. Co-localization of myc-tagged NAT8L with the endoplasmic reticulum (ER) and complete segregation from the Golgi complex and mitochondria (mito). CHO cells were transfected to express low levels of C-terminal myc-tagged NAT8L and immunolabelled with an antibody against the myc-antigen (photographs of the central column; green in 'merge' photographs). (A) Double immunolabelling for KDEL-bearing proteins to label the ER (left photograph; red in 'merge'). (B) Cells were co-transfected with the PDIb-HA construct to immunolabel the Golgi apparatus (left; red in 'merge'). (C) Transfected cells were incubated with MitoTracker red before fixation (left; red in 'merge'). Notice the nuclear envelope labelling by NAT8L (arrows in the middle photographs) and its reticular pattern in the cytoplasm with substantial co-localization with the ER marker (arrowheads), contrasting with full segregation from Golgi and mitochondrial labelling. Scale bars, 5 µm. (D, E) Intensity profiles along the paths indicated by the arrows in the right-hand photographs of panels A and C, respectively.

Figure 5 :

 5 Figure 5: Co-localization of myc-tagged NAT8L with ER and its segregation from mitochondria in neurons. Primary cultures of rat fetal neurons were infected with lentiviruses encoding C-terminal myc-tagged NAT8L (see SI Text), fixed at a low level of expression, and immunolabelled for myc antigen (green in 'merge'). (A) Triple

Figure 6 .Figure 1 Biochemical

 61 Figure 6. Mutation found in the patient with hypoacetylaspartia and its effect on the activity and expression of NAT8L (A) Sequences of nucleotides 190-240 of the NAT8L open reading frame and deduced protein sequences in controls and in the patient. Nucleotide 1 corresponds to the adenine of the initiator ATG codon. The 19 nucleotides deleted in the patient are shown in small characters and the 11 bp repeats between which this deletion occurred is underlined. (B and C) HEK293T cells were transfected with an empty plasmid (EP) or a plasmid driving the expression of human wild-type NAT8L (Met1), the form with the 19-bp deletion (del19), and a shorter form corresponding to amino acids 169-302 (Met169). All proteins contained a Cterminal polyHis tag. (B) Asp-NAT activity in cell extracts. (C) Western blots with an anti-polyHis tag antibody.

Figure 2 BiochemicalFigure 3 Biochemical

 23 Figure 2

4 Biochemical 5 Biochemical

 45 Figure 4

Figure 6 Biochemical

 6 Figure 6

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

EW and FC are Chargés de Recherche of the Fonds National de la Recherche Scientifique. This work was supported by grants from the Interuniversity Attraction Poles Program-Belgian Science Policy (Networks P6/05 and P6/28), by the DIANE centre of excellence programme of the Région Wallonne, by the Région Bruxelloise, by the Fonds de la Recherche Scientifique Médicale, by the Fondation Saint-Luc, and by a grant from ASCO industries to MCN, MFV and EVS. The authors thank Ms. L. Thanh Lac for her excellent technical assistance and Didier Colau for his help in antibody purification. The PDIb-HA construct was a kind gift from L. Ruddock (University of Oulu, Finland) and the anti-MAP2 serum a kind gift from J.-P. Brion (Université Libre de Bruxelles, Belgium).


Pika Miklavc

Susanne Albrecht

Oliver H Wittekindt

Peter Schullian

Thomas Haller

Dr Paul Dietl
email: paul.dietl@uni-ulm.de

Existence of exocytotic hemifusion intermediates with a life time up to seconds in type II pneumocytes

Keywords: hemifusion, fusion, pneumocyte, lung, darkfield and fluorescence microscopy

Exocytosis proceeds through prefusion stages such as hemifusion, but hemifusion is still an elusive intermediate of unknown duration. Using darkfield and fluorescence microscopy in alveolar type II (ATII) cells containing large secretory vesicles ("lamellar bodies", LBs), we show that exocytotic fusion events were accompanied by a mostly biphasic scattered (darkfield) light intensity decrease (SLID) originating from the vesicle border. Correlation with the diffusional behavior of fluorescence markers for either content or membrane mixing revealed that the onset of the fast, second, phase of SLID corresponded to fusion pore formation, which was followed by vesicle swelling. In contrast, a slow, first, phase of SLID preceded pore formation considerably but could still be accompanied by diffusion of farnesylated DsRed, an inner plasma membrane leaflet marker, or nile red. We conclude that hemifusion is an exocytotic intermediate that may last for several seconds. SLID is a new, non-invasive approach by which a prefusion phase, including hemifusion, can be continuously recorded and distinguished from fusion pore formation and postfusion vesicle swelling.

INTRODUCTION

Regulated exocytosis denotes stimulated fusion between the limiting membrane of a storage vesicle and the plasma membrane, catalyzed by SNARE proteins under the influence of cytoplasmic factors such as Ca 2+ [START_REF] Almers | Exocytosis[END_REF][START_REF] Burgoyne | Secretory granule exocytosis[END_REF][START_REF] Chen | SNARE-mediated membrane fusion[END_REF][START_REF] Chieregatti | Regulated exocytosis: new organelles for nonsecretory purposes[END_REF][START_REF] Jahn | Membrane fusion[END_REF][START_REF] Neher | Vesicle pools and Ca 2+ microdomains: new tools for understanding their roles in neurotransmitter release[END_REF]. The fusion pore is the initial aqueous connection between the vesicle lumen and the extracellular space [START_REF] Chandler | Arrest of membrane fusion events in mast cells by quick-freezing[END_REF]. The initial fusion pore exhibits a channel-like behavior that may flicker between closed and open states, allowing partial release of stored materials [START_REF] Alvarez De Toledo | Release of secretory products during transient vesicle fusion[END_REF][START_REF] Breckenridge | Final steps in exocytosis observed in a cell with giant secretory granules[END_REF][START_REF] Chow | Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells[END_REF][START_REF] Takahashi | Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet[END_REF]. The diffusion of hydrophilic compounds through the pore can be preceded by the diffusion of amphiphilic and outer plasma membrane probes, suggesting that the initial lipidic pore is too narrow to enable passage of solutes [START_REF] Klauke | Frustrated Exocytosis"--a novel phenomenon: membrane fusion without contents release, followed by detachment and reattachment of dense core vesicles in Paramecium cells[END_REF][START_REF] Takahashi | Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet[END_REF]. Models of proteinaceous exocytotic pores have also been proposed [START_REF] Han | Transmembrane segments of syntaxin line the fusion pore of Ca 2+ -triggered exocytosis[END_REF]. In contrast to fusion pore formation, exocytotic pre-and hemifusion states are still much more refractory to experimental detection. Knowledge thereof is largely based on theoretical considerations, suggesting the "stalk model" as a relatively low energy cost intermediate that initiates hemifusion, through which fusion occurs ("fusion-throughhemifusion", reviewed in detail in [START_REF] Chernomordik | Membrane hemifusion: crossing a chasm in two leaps[END_REF][START_REF] Zimmerberg | Membrane fusion[END_REF]). Hemifusion is defined as the fusion between two opposing membrane leaflets, enabling diffusion of lipids and probably lipid-anchored proteins from one membrane to the other. Several studies on content vs. lipid mixing convincingly demonstrated the existence of hemifusion in viral and other models of fusion [START_REF] Giraudo | SNAREs can promote complete fusion and hemifusion as alternative outcomes[END_REF][START_REF] Jun | Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing[END_REF][START_REF] Kemble | Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion[END_REF]. In pure phospholipid bilayers, hemifusion was also postulated from electrophysiological evidence [START_REF] Chanturiya | Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers[END_REF]. However, hemifusion has not yet been demonstrated as an intermediate stage of exocytosis in a physiological setting, i.e. in an intact, secreting cell. A FRAP study on sea urchin eggs concluded that docked vesicles may be kept stably in a hemifused state by immobile SNARE proteins, but a transition between these stages, i.e. fusion-through-hemifusion, was not directly demonstrated [START_REF] Wong | Membrane hemifusion is a stable intermediate of exocytosis[END_REF]. Likewise, an electron tomography study on synaptic vesicles suggested docked vesicles to be in a hemifused state [START_REF] Zampighi | Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused[END_REF]. Docking and other processes, such as priming, that make a vesicle fusioncompetent, are considered to occur prior to hemifusion and may represent situations where a vesicle is spatially very close to the plasma membrane despite the lack of a lipid connection between both membranes. Strictly speaking, hemifusion is a morphological description, denoting a certain arrangement of membrane leaflets. However, due to methodological impediments to prove this condition, recent studies have used a more functional description, meaning the exchange of lipid-anchored components between opposing membranes [START_REF] Giraudo | SNAREs can promote complete fusion and hemifusion as alternative outcomes[END_REF][START_REF] Lu | Membrane fusion induced by neuronal SNAREs transits through hemifusion[END_REF][START_REF] Wong | Membrane hemifusion is a stable intermediate of exocytosis[END_REF]. Although this functional definition is certainly less accurate in a structural sense, it probably reflects the physiological implications of hemifusion (e.g. exchange of signaling molecules) better than the morphological one. Therefore, we use the term "hemifusion" here in its functional sense, bearing in mind the limitations to predict a clear structural state.

Major impediments to study exocytotic hemifusion are the extremely small diffusion distances that represent a methodological challenge to detect small time lapses between content and membrane mixing. Here we overcome these impediments by studying a cell model (the ATII pneumocyte) which is particularly suitable for the following reasons: 1. LBs are large secretory vesicles with more than 1 µm diameter. A c c e p t e d M a n u s c r i p t 2. LBs contain a surface active complex of lipids and proteins (surfactant), which can be selectively stained by FM 1-43, a membrane-impermeable amphiphilic dye [START_REF] Betz | Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction[END_REF][START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF]. Thereby, content mixing, i.e. the mixing of the extracellular fluid with vesicle contents, elicits a bright localized signal. This readout enables a high temporal resolution of fusion pore formation, as revealed by combined patch clamp (capacitance) and fluorescence experiments [START_REF] Mair | Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements[END_REF]. 3. In a previous report we showed that single LB fusion events were followed by actin coating of the fused LB, which was found to be a prerequisite for release [START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF]. Since the fusion pores in these cells open slowly [START_REF] Haller | Fusion pore expansion is a slow, discontinuous, and Ca 2+dependent process regulating secretion from alveolar type II cells[END_REF] and remain a mechanical barrier for release [START_REF] Singer | Mechanical forces impeding exocytotic surfactant release revealed by optical tweezers[END_REF], the shape of a fused LB is maintained for several seconds until the actin coat contracts, facilitating analysis of postfusion membrane mixing. In addition, surfactant is essentially insoluble in water, remaining in a compact form within the fused vesicle before active extrusion. 4. LB exocytosis is slow, sequential, and can be stimulated, facilitating analysis of single fusion events [START_REF] Frick | Secretion in alveolar type II cells at the interface of constitutive and regulated exocytosis[END_REF]. Here we present the first dynamic resolution of exocytotic prefusion stages including hemifusion and introduce darkfield microscopy as a direct and highly sensitive optical approach to distinguish between pre-and postfusion stages of exocytosis. A c c e p t e d M a n u s c r i p t EXPERIMENTAL Cell isolation and treatment ATII cells were isolated from male Sprague-Dawley rats (150-200 g) as described [START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF]. In short, rats were anesthetized with ketamin (10%) and xylazil (2%). Lungs were perfused, removed from the body, washed and incubated two times with elastase and trypsin at 37°C for 15 minutes. Lungs were immersed in DNase containing solution and minced with scissors into bits of approximately 1 mm size. Enzyme reaction was stopped by incubation with FCS and the tissue was filtered 3 times through gauze and nylon meshes (mesh width: 150, 20, and 10 µm). The final filtrate was centrifuged for 8 min at 130 x g, the cells were suspended in DMEM medium, and put on IgG coated plastic dishes for 15 min at 37°C. Non-adherent cells were centrifuged for 8 min at 130 x g, suspended in DMEM medium with 10% FCS and 1% Penicillin/Streptomycin (10.000 units/ml Penicillin, 10 mg/ml Streptomycin) and seeded on glass cover slips. ATII cells were cultured at 37°C, 5% CO 2 , and 95% humidity, and used for experiments 1-2 days after the isolation. In Lamp3-GFP and DsRed experiments, cells were transfected with adenovirus constructs after isolation. The composition of the experimental solution used for live cell imaging was (in mmol/l): 140 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 5 glucose, 10 Hepes; pH 7.4. LB secretion was stimulated with ATP (100 µmol/l). LB fusions were detected with FM 1-43 (300 nmol/l) added to the bath, as described earlier [START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF]. In the nile red experiments the cells were stained with 200 nmol/l nile red before the experiment, in DiI experiments the cells were incubated with 1 µmol/l DiI for 10 minutes, and in LTR experiments the cells were incubated with 100 nmol/l LTR for 10 minutes. All dyes were purchased from Molecular Probes. The experiments were made at room temperature (21-25°C).

Farnesylated DsRed and Lamp3-GFP adenovirus vectors

Lamp3-GFP adenovirus vector, cloning and cell transfection were recently described [START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF]. Vectors pDsRed-Monomer-F and pEGFP1-N1 were obtained from Clontech (Saint-Germaine-en-Laye, France). Vectors pDONR221 and pAd/CMV/V5-DEST were purchased from Invitrogen (Karlsruhe, Germany). cDNA encoding DsRed-Farn were amplified from the vector pDsRed-Monomer-F using the primers DsRed-Farn-F2 (ggggacaagtttgtacaaaaaagcaggcttcGTCAGATCCGCTAGCGCTACCGG) and DsRed-Farn-R2 (ggggaccactttgtacaagaaagctgggtcAGCTTGAGCTCGAGATCTGGATCC) using standard PCR methods. After isolation of PCR products, amplified cDNA were introduced into pDONR221 by in-vitro recombination using BP-clonase II enzyme mix (Invitrogen, Karlsruhe, Germany) and cloned in E. coli OmniMax TM 2-T1 R (Invitrogen, Karlsruhe, Germany). Thereafter, DsRed-Farn cDNA was transferred into adenoviral expression vector pAd/CMV/V5-DEST by in-vitro recombination using LR-clonase II enzyme mix (Invitrogen, Karlsruhe, Germany) and recombination products were cloned in E. coli OmniMax TM 2-T1 R (Invitrogen, Karlsruhe, Germany). Multi cloning site (MCS) and coding region of EGFP were amplified from plasmid pEGFP-N1 using primers MCSgfp-F1 (GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCTAGCGCTACCGGACTCAG) and MCSgfp-R1 Recombinant adenoviral expression vectors were isolated from E.coli cells using QIAGEN plasmid midi kit (Qiagen, Hilden, Germany). Plasmids were linearised using restriction endo-nucleases PacI (New England Biolabs, Frankfurt, Germany). Linearised plasmid was transfected into 293A cells (Invitrogen, Karlsruhe, Germany) using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) without further purification directly after heat inactivation of PacI. Transfected cells were cultivated for 5 to 8 days and virus particles were isolated using ViraBind Adenovirus purification kit (Cell Biolabs, Heidelberg, Germany) according to manufacturers' protocol. Virus particles were eluted in elution buffer (Cell Biolabs, Heidelberg, Germany), and stored in elution buffer containing 10% glycerol in aliquots at -80°C until use.

Imaging with the RTM 2.5 microscope

Darkfield and fluorescence imaging were performed on an upright Richardson RTM 2.5 microscope (Improvision, Germany). For darkfield illumination we used the red or the blue filter in the condenser; therefore the light-reflecting structures appeared red or blue on a dark background. Darkfield and fluorescence light was detected with a 3CCD color video camera (ExwaveHAD DXC-C33, Sony). Images were acquired with the 63x water immersion objective (Leica) and the RTM 3.0 blue filter cube (490 nm dichroic, 400-500 nm excitation filter, 510 LP emission filter) for FM 1-43, Lamp3-GFP, and CTG imaging, and with the RTM 3.0 green filter cube (560 nm dichroic, 540-550 nm excitation filter, 570 LP emission filter) for farnesylated DsRed, DiI, LTR, and nile red imaging. Volocity software was used for image acquisition and subsequent measurements. At the image acquisition rate of 5-10/s the darkfield and fluorescence signals were acquired simultaneously and were subsequently digitally separated from an RGB image based on their different color. In combined darkfield and FM 1-43 fluorescence experiments we used the red channel for darkfield and green channel for FM 1-43 fluorescence signals, and in combined darkfield and farnesylated DsRed, DiI and nile red experiments we used the blue channel for darkfield and red channel for fluorescence signals.

Imaging with the fluorescence microscope

The combined LTG and nile red measurements were performed with a 100x Neofluar oil, NA 1.3 on a Zeiss Axiovert equipped for epifluorescence (Polychrome II, TILL Photonics), emission beam splitting (DV-CC, Optical Insights LLC) and synchronous image acquisition (Sensicam, PCO) at a rate of 1/s. Wavelength and filters used were: 485 nm exc., 520 nm dichroic 1, 565 nm dichroic 2, 525 nm BP (LTG) and 630 nm LP (nile red). Cells were pre-incubated 30 min with LTG, 200 nm, nile red was added at 4 µmol/l 5 min prior to ATP, 50 µmol/l.

Image and statistical analysis

RESULTS

Measurement of single LB fusion with the plasma membrane by darkfield microscopy and FM 1-43 fluorescence was recently described in detail [START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF]. It exhibits the following key feature: The darkfield light intensity originating from the border of a fusing LB decreases (SLID), while vesicle contents appear dark (Figure 1). When FM 1-43 is present in the bath, this is accompanied by an increase of FM 1-43 fluorescence (F FM1-43 ) within the fused LB, starting at the site of the fusion pore (Figure 1). F FM1-43 originating from surfactant exceeds outer plasma membrane staining so manifold that the plasma membrane appears virtually dark (Figure 1).

To further investigate the obvious correlation between SLID and the fusion pore opening, we used higher image acquisition rates (10/s). These measurements revealed that SLID consists of two clearly discernible stages (Figure 2) -a slow component at the beginning (mean duration 2.24 s; s.d. = 2.19, n = 22) and a subsequent fast one (mean duration 0.92 s; s.d. = 0.49). The onsets of the two phases of SLID were measured as shown in Figure 2. The linear parts of the darkfield data were fitted and the crossing points of the linear regressions were considered as onsets of SLID phase 1 and 2. The onset of the F FM1-43 increase (indicating fusion pore opening [START_REF] Mair | Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements[END_REF]) corresponded to the start of the fast component of SLID (Figure 3). The highest observed discrepancy between the two events was 0.5 s. Therefore, it is reasonable to assume that the light intensity decrease gets faster when the fusion pore opens (either transiently or permanently). This assumption leads to the question what the initial slow component of SLID might reflect. Despite its variable duration, it was detectable in the majority (84.6%) of the observed fusion events. Theoretically, several explanations for the biphasic nature of SLID are possible, which will be discussed below. The most likely one is that the first phase of SLID reports the hemifusion stage ("membrane mixing"), where the proximal leaflets of the plasma and vesicle membranes merge but the fusion pore is not yet open. During this stage, a non membrane-permeable dye like FM 1-43 would not be able to enter the LB. The second phase of SLID corresponds to an open fusion pore and "content mixing".

To further test the hypotheses that hemifusion intermediates of considerable duration indeed exist in type II cells, we sought several independent experimental approaches, applying markers for either content or membrane mixing, as illustrated in Figure 4A: DiI was used as a marker of the outer plasma membrane leaflet [START_REF] Roddy | Identification of cellular sections with imaging mass spectrometry following freeze fracture[END_REF]. In contrast to FM 1-43, it was absent in the bath solution. Therefore, LB staining was exclusively due to lateral membrane diffusion, but not to bulk fluid entry. Conversely, LysoTracker Green (LTG) or LysoTracker Red (LTR) were used as fluid phase content markers. They are trapped in LBs by non-ionic diffusion and protonation, and rapidly discharged into the extracellular space upon the formation of an aqueous pore [START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF]. The onset of LTG discharge coincides (< 1 s) with uptake of FM 1-43 [START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF] and also with the dissipation of the proton gradient, the probably earliest diffusional event within the lumen of the pore (see supplementary data membrane to the outer leaflet of the LB membrane during hemifusion. Second, the unspecific lipid marker nile red, which is trapped within phospholipids [START_REF] Fowler | Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms[END_REF], resulting in stable staining of LBs (Figures 4 and6). In the course of hemifusion, the LB should lose nile red fluorescence intensity, possibly due to dye redistribution along the plasma membrane. Assuming the onset of the second, fast, phase of SLID as a marker for the instance of fusion pore formation, Figure 5A exhibits the temporal relation (negative values represent prefusion events) between all fluorescence and SLID phase 1 changes with reference to this event (= time 0). It is again apparent that the diffusion of content / outer leaflet markers (LTR, DiI, FM 1-43) followed the fusion pore formation. In contrast, nile red and DsRed fluorescence changes could start several seconds before fusion. There was, on average, a delay between the onset of SLID phase 1 and particularly that of DsRed, which could extend into the postfusion phase (Figure 5A). Hence, analysis of hemifusion by specific membrane diffusion probes is considerably limited by highly variable diffusion times and/or limitations of detection, as will be discussed below.

To further analyze a possible relation between SLID phase 1 and membrane merger (hemifusion), we plotted individual fluorescence changes as relative delays following the onset of SLID phase 1, shown in Figure 5B. The data reveal that although there was a clear tendency of nile red and DsRed to lag behind the onset of SLID phase 1, some events closely coincided (Figure 4B). Importantly, however, a fluorescence change was never observed before SLID phase 1. Hence, hemifusion cannot precede SLID phase 1.

To obtain a direct comparison between lipid and content mixing within a single vesicle, we used a combination of nile red and LTG. As indicated above, both dyes accumulate within LBs (Figure 6A). The spectral emission properties of both dyes allowed their synchronous recording with a beam splitting device in the emission path. Figure 6B demonstrates that the decline in nile red intensity precedes that of LTG (1.36 s, s.d. = 0.8, n = 7). Also here, the most reasonable explanation is a situation where vesicle and plasma membrane are physically conjunct, seconds before a water permeable pore is formed. The concept of hemifusion vs. fusion was further examined by analyzing vesicle swelling (Figure 7A). Postfusion swelling is a common finding, driven by fluid uptake through the fusion pore due to the physico-chemical properties of the vesicle matrix [START_REF] Finkelstein | Osmotic swelling of vesicles: its role in the fusion of vesicles with planar phospholipid bilayer membranes and its possible role in exocytosis[END_REF][START_REF] Marszalek | Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin[END_REF].

Postfusion swelling can therefore be taken as another parameter of "content mixing", independent of content marker diffusion. Fusion pore formation (measured as the start of 9 A c c e p t e d M a n u s c r i p t FM 1-43 staining) was followed by swelling of LBs (Figure 7B), measured as the surface change of the scattered light signal. Prefusion swelling, as reported in another cell type [START_REF] Jena | Gi regulation of secretory vesicle swelling examined by atomic force microscopy[END_REF], was never observed here. The median increase of the LB volume estimated from darkfield images was 17.5% (interquartile range 5.8 -24.3%, n = 50). Postfusion LB swelling occurred within seconds and was therefore several fold faster than the initiation of release [START_REF] Haller | Dynamics of surfactant release in alveolar type II cells[END_REF][START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF], (time constant of postfusion swelling: τ = 2.1 s, s.d. = 1.4, n = 6). To exclude the possibility that the diameter increase of the scattered light signal was just the result of a change in light diffraction properties, we transfected the ATII cells with Lamp3-GFP fusion protein. Lamp3 is localized at the membrane of lysosome associated organelles, and in ATII cells the Lamp3-GFP fluorescence and the darkfield signal overlap [START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF]. As in the darkfield illumination, the swelling was also detectable by measuring the Lamp3-GFP signal (Figure 7C; median increase in volume 30.9%, interquartile range 18.5 -38.4%, n = 5). Additional evidence for LB swelling is presented in supplementary data Figure 2.

These experiments indicate that content markers or water can enter the LB during the second phase of SLID exclusively, which makes this phase a sensitive postfusion indicator. The DsRed and nile red experiments indicate the existence of hemifusion intermediates that may last for seconds. SLID phase 1 is a newly described prefusion indicator that may represent the hemifusion state even more reliably and precisely than any diffusion marker, as discussed in the following.

DISCUSSION

Two opposing theories describe initial exocytotic fusion pores as being either proteinaceous (direct fusion pathway), or lipidic, emerging through transient hemifusion states (stalk model) [START_REF] Chernomordik | Membrane hemifusion: crossing a chasm in two leaps[END_REF]. Our study provides first experimental evidence in favor of the "fusion-through-hemifusion" model in a living, intact cell. It demonstrates prefusion and membrane merger seconds before content mixing in single secretory vesicles. Thereby, it confirms predictions from a multitude of non-exocytotic or artificial fusion systems, reviewed in detail elsewhere [START_REF] Chernomordik | Membrane hemifusion: crossing a chasm in two leaps[END_REF][START_REF] Chernomordik | Mechanics of membrane fusion[END_REF][START_REF] Zimmerberg | Membrane fusion[END_REF]. It also supports evidence in favor of "stable" hemifusion states of "docked" vesicles [START_REF] Wong | Membrane hemifusion is a stable intermediate of exocytosis[END_REF][START_REF] Zampighi | Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused[END_REF].

Decades of intensive research, including breath-taking advances in molecular and live cell imaging techniques, have yet failed to develop a method to continuously record exocytotic hemifusion states in living cells. SLID (phase 1) is a new approach in this direction. We are aware that at the time being, SLID is not more than a new phenomenon, but it yields a continuous recording of 2 phases, of which phase 1 can clearly be assigned to a prefusion stage. Its validation as a method that accurately reflects the morphological postulate of the hemifusion state will only be possible when hemifusion will be accessible to independent measurement at all. To this end, electron microscopy would be the method of choice, but this is far beyond the temporal resolution and technical feasibility, providing static information of the process only. SLID (phase 1) is a prefusion indicator that coincides fairly well, but not precisely, with diffusional membrane merger (Figure 5). Going beyond hemifusion, it could reflect any other conceivable pre-fusion condition, such as protein rearrangement and development of a tighter contact between the vesicle and the plasma membrane. Hence, the key question remains: What does SLID actually report? Darkfield microscopy has been extensively used to investigate the fusion and transformational properties of giant liposomes [START_REF] Nomura | Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion[END_REF][START_REF] Nomura | Capabilities of liposomes for topological transformation[END_REF]. These studies suggest that the light scattering intensity from a liposomal membrane reflects its thickness, and that the changes in scattered light intensity of liposomal membranes observed with the darkfield microscope might also be based on the cooperative movement of lipid molecules in a membrane [START_REF] Nomura | Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion[END_REF][START_REF] Nomura | Capabilities of liposomes for topological transformation[END_REF]. We also made the observation that extruded surfactant, in contrast to surfactant within LBs, generated a darkfield signal, probably because the highly organized lipid layers in LBs [START_REF] Perez-Gil | Structure of pulmonary surfactant membranes and films: The role of proteins and lipid-protein interactions[END_REF] became disordered. The origin of the darkfield signal from the limiting membrane of an LB is more difficult to interpret: The membrane of a secretory vesicle is not merely an even surface of free-floating phospholipids, but is probably endowed with a highly complex 3D topography due to abundant membraneassociated proteins [START_REF] Takamori | Molecular anatomy of a trafficking organelle[END_REF]. Although we do not know the exact composition, distribution and density of these proteins in LBs [START_REF] Wang | Proteomic analysis of lamellar bodies isolated from rat lungs[END_REF], they may affect the optical properties of the incident light. In any case, it can be assumed that SLID rather reports a change of LB surface properties than of vesicle content, since the first phase of SLID precedes measurable content mixing and LB swelling by seconds.

We have following reasons to favor the hypothesis that SLID phase 1 indeed reports the hemifusion state: 1. Lipid flow explains the biphasic nature of SLID much better than other prefusion processes, since the two phases of SLID would correspond with the two leaflets of the membrane, merging in sequence. In this case, the first phase of SLID would reflect a change of lipid composition in the outer leaflet of the limiting LB membrane. Although there is no direct way to test this hypothesis, it is consistent with other observations: First, darkfield microscopy gives the best high-contrast images of giant liposomes compared with other methods, indicating that darkfield images do not require the presence of membrane proteins [START_REF] Nomura | Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion[END_REF]. Furthermore, detergents like Triton X, which disrupt lipid domains, sequentially caused SLID in all LBs [START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF]. The lipid composition of a membrane is fundamental in determining its curvature and tension [START_REF] Chernomordik | The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition[END_REF]. In this way, specific lipids do not only determine the readiness of a secretory vesicle to hemifuse and fuse, but also create a force for lipids to flow when membrane leaflets merge [START_REF] Chizmadzhev | Dynamics of fusion pores connecting membranes of different tensions[END_REF]. Monck et al. demonstrated lipid flow from the plasma to the vesicle membrane during exocytosis, resulting in up to 40% loss of plasma membrane area [START_REF] Monck | Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore[END_REF]. It is likely that the inner plasma membrane leaflet and outer LB membrane leaflet have different lipid compositions [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF], which would be sufficient to explain substantial lipid flow during hemifusion.

2. The delay between the onset of SLID phase 1 and the change of DsRed or nile red fluorescence intensity does not exclude that membrane merger is the common underlying mechanism: Free lipid flow through the hemifusion stalk may be limited [START_REF] Chernomordik | Mechanics of membrane fusion[END_REF][START_REF] Zimmerberg | Membrane fusion[END_REF]. This could explain the observation that phase 1 of SLID does not occur abruptly (as a step), but rather linearly and slowly. If true, the slope of phase 1 would actually reflect diffusional limitations. Accordingly, hemifusion can present a barrier for the diffusion of proteins with the size of farnesylated DsRed [START_REF] Giraudo | SNAREs can promote complete fusion and hemifusion as alternative outcomes[END_REF]. In addition, we have to consider photobleaching (rapid decrease of DsRed fluorescence before and after fusion, Figure 4B) which can also explain the low detectability of a small DsRed increase before the fusion pore opening.

3. The existence of fusion events, where diffusion of farnesylated DsRed into the vesicle almost precisely coincided with the onset of SLID phase 1, indicates that membrane merger and SLID phase 1 can occur simultaneously. Hence, SLID phase 1 does not require any other or additional explanation than hemifusion alone. 4. In theory, both phases of SLID could occur after fusion pore formation, but the initial pore is so small that no "content mixing" (entry of extracellular fluid) would take place. This possibility, however, is very unlikely for the following reasons: First, we showed in simultaneous patch-clamp and FM 1-43 measurements that the fusion pore opening (measured as capacitance steps) and the increase in F FM1-43 are apart by no more than 1 s [START_REF] Mair | Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements[END_REF], which is considerably shorter than the duration of the first phase of SLID (2.24 s on average, 9 s maximum). Second, even if such "narrow pores" existed (small enough to escape detection by capacitance measurements), FM 1-43 should be able to enter the LB along the wall of the fusion pore, by lateral diffusion from the outer leaflet of the plasma membrane [START_REF] Klauke | Frustrated Exocytosis"--a novel phenomenon: membrane fusion without contents release, followed by detachment and reattachment of dense core vesicles in Paramecium cells[END_REF].

Our experiments revealed that there is no vesicle swelling prior to fusion pore formation. Hence, the prefusion phase was not accompanied by volume uptake. It does not appear, therefore, that vesicle swelling in ATII cells is a driving force for fusion pore formation [START_REF] Finkelstein | Osmotic swelling of vesicles: its role in the fusion of vesicles with planar phospholipid bilayer membranes and its possible role in exocytosis[END_REF][START_REF] Jena | Gi regulation of secretory vesicle swelling examined by atomic force microscopy[END_REF]. Independent of the prefusion marker (SLID phase 1, nile red, or DsRed), the hemifusion could last longer then 4 seconds (Figure 5A). Hence, considering the large variability in the duration of individual hemifusion states (not detectable to 9 s), these different methods yielded fairly consistent results. They suggest that hemifusion lifetimes cannot A c c e p t e d M a n u s c r i p t be solely explained on the basis of a different vesicle size. It rather appears that hemifusion does not proceed in a stereotypic way, but that specific lipid or protein domains in opposing membranes determine the transition to fusion. This prompts the question if hemifusion is an actively regulated process and which compounds are involved therein. It also prompts the question if transient hemifusion and LB retrieval exist in addition to kiss-and-run events [START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF]. In any case, the duration of the hemifusion state should influence the amount of membrane mixing. This may induce translocation of plasma membrane signaling molecules to secretory vesicles, triggering signaling cascades that induce -among others -actin assembly and influence the future mobility of the fused vesicle [START_REF] Malacombe | Exocytosis in neuroendocrine cells: new tasks for actin[END_REF][START_REF] Miklavc | Ca 2+ -dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run[END_REF][START_REF] Yu | Control of local actin assembly by membrane fusion-dependent compartment mixing[END_REF]. In the lung, these processes may determine polarized secretion, compound exocytosis and membrane recycling [START_REF] Dietl | Exocytosis of lung surfactant: from the secretory vesicle to the air-liquid interface[END_REF]. The potential of darkfield microscopy will be to disclose regulatory principles of hemifusion and possibly other prefusion events that are inaccessible by fluorescence probes, which are all subject to limitations caused by diffusion properties, photobleaching, staining specificity or spectral features.

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7 Biochemical

 7 A square region of interest was defined around the fusing LB, on which darkfield light and fluorescence intensity were measured. In darkfield and fluorescence measurements 6 Biochemical Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094 A c c e p t e d M a n u s c r i p t the lowest value was subtracted and the change in light intensity was expressed in arbitrary units. Unless otherwise stated, data are presented as mean ± s.d. Vesicle size was measured as the surface of the vesicle intersection in one focal plane. Vesicle volume change after fusion was calculated from the measured vesicle intersection in one focal plane, and median and interquartile ranges were calculated. Tau values were obtained by fitting the curve with a single exponential in Igor Pro 5.04B. Excel and SigmaPlot 10.0 were used for statistics and graphs. Some images were corrected for brightness and contrast with Adobe Photoshop CS2. Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094 A c c e p t e d M a n u s c r i p t

Figure 1 )

 1 . To probe for diffusion of components between the leaflets of the inner plasma membrane and outer LB membrane (hemifusion), we used two different dyes: First, farnesylated DsRed which selectively incorporates into the inner leaflet of the plasma membrane by posttranslational farnesylation of the protein through membranebound enzymes[START_REF] Fujiyama | S-farnesylation and methyl esterification of C-terminal domain of yeast RAS2 protein prior to fatty acid acylation[END_REF]. Under the assumption that the hemifusion stalk does not contain diffusion barriers for this dye, it should diffuse from the inner leaflet of the plasma 8 Biochemical Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094 A c c e p t e d M a n u s c r i p t

  Figure 4A exhibits the fluorescence stainings of individual LBs before and after both phases of SLID, i.e. the initial pre-and final postfusion stages. It illustrates that all tested dyes behaved according to the theoretical predictions outlined above with certain limitations regarding DsRed, which exhibited a wide scatter in its diffusional behavior (see below, Figure 5A, B and Discussion). In the absence of SLID, the fluorescence intensities of these markers were stable or subject to variable extents of photobleaching. A direct comparison of the earliest onsets of fluorescence changes between these dyes is shown in Figure 4B: DiI and LTR behaved like FM 1-43, exhibiting fluorescence change during phase 2 of SLID exclusively. The onset of DsRed and nile red fluorescence change, however, could start as early as the beginning of phase 1 of SLID (Figure 4B). Hence, the earliest observed onsets of fluorescence changes by markers of lipid vs. content mixing coincided almost precisely with the onsets of phase 1 and phase 2 of SLID, respectively.

Biochemical

  Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094 A c c e p t e d M a n u s c r i p t

Biochemical

  ABBREVIATIONS LIST

Figure 1 :Figure 2 :

 12 Figure 1: ATII cell imaged with darkfield illumination (left) and FM 1-43 fluorescence (right), before and after fusion (delay = 12 seconds) of one LB (arrowhead) with plasma membrane. Note that the darkfield light intensity of this vesicle decreased, which was not the case in other vesicles. Note also early staining of LB content with FM 1-43 at the presumptive site of the fusion pore. Scale bar = 8 µm.

Figure 3 :

 3 Figure 3: The time courses of darkfield light intensity change (filled squares) and F FM1-43 change (open squares) for 6 fusion events imaged with time resolution 10/s. The left dashed line on each graph indicates the start of SLID; the right dashed line indicates the transition of SLID from the first, slow to the second, fast phase. The latter corresponded to the start in FM 1-43 fluorescence increase, indicating fusion pore opening. Lowest light intensity values were set zero.

Figure 4 :

 4 Figure 4: SLID and its relation with fluorescence of membrane / content markers. A. Scheme of LBs in the presence of fluorescence indicators in the pre-(left), hemi-(middle) and postfusion (right) stages. Red color indicates postulated site of fluorescence during each stage. Gray circle denotes surfactant. Note that the upper 3 dyes report fusion pore formation, whereas the lower 2 should report lipid merger. Examples of fluorescence images of single LBs before and after fusion with the plasma membrane (arrowheads) are shown on the left and right side, respectively. Scale bar = 4 µm. B. Examples of earliest onsets of fluorescence changes (open squares) for 4 different dyes in comparison with SLID (filled squares). The time between the two dashed lines indicates SLID phase 1. The prominent decrease in LTR and DsRed fluorescence before and after fusion are due to photobleaching.

Figure 5 :

 5 Figure 5: Summary of SLID and fluorescence markers. A. Analysis of the onsets of SLID phase 1 and fluorescence intensity changes (shown as single data points for each fusion event) in comparison to fusion pore formation (defined as onset of SLID phase 2, time = 0, dashed line; Negative values indicate times before fusion pore formation, positive values times thereafter). B. Analysis of the onsets of fluorescence intensity changes (shown as single data points for each fusion event). Instance of fluorescence change is expressed as percent of SLID phase 1. 0 -100% denotes the entire duration of SLID phase 1 (space between dashed lines). Note that DsRed fluorescence exhibited the least precise correlation with a specific phase, which can be explained by restricted probe diffusion through the early hemifusion structures and by photobleaching (see Discussion).

Figure 6 :

 6 Figure 6: Comparison between content and membrane mixing. A. Co-staining of a single ATII cell with LTG (green) and nile red (red). Acidic vesicles without lipid contents appear green, acidic vesicles with lipid contents (LBs before fusion) appear orange, lipid contents (surfactant) alone (fused LBs) appear red. Scale bar = 10 µm. B. Time courses of LTG and nile red fluorescence intensities during fusion of a single LB. The average delay between the onsets of fluorescence changes was 1.36 seconds ± 0.8 (n = 7).

Figure 7 :

 7 Figure 7: Fusion pore formation was followed by LB swelling. A: Darkfield image series of several LBs, one fusing with the plasma membrane (arrowhead). Time = 0 s indicates last image before SLID. Double arrow marks initial LB size. Scale bar = 2 µm. B: Time courses of darkfield light intensity (filled squares), F FM1-43 (open squares) and vesicle size (filled circles). Image acquisition rate was 10/s. C: LB swelling in ATII cell transfected with the lysosomal vesicle membrane marker Lamp3-GFP. The swelling of the vesicle, observed under darkfield illumination (arrowheads; upper row), was independently

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  Biochemical Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094

  PCR products were introduced into pDONR221 by in-vitro recombination using BP-clonase II enzyme mix (Invitrogen, Karlsruhe, Germany) and cloned in E. coli OmniMax TM 2-T1 R (Invitrogen, Karlsruhe, Germany).

		Biochemical Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094
		(GGGGACCACTTTGTACAAGAAAGCTGGGTATTTACTTGTACAGCTCGTCC) by
		standard PCR techniques.
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094	A c c e p t e d M a n u s c r i p t
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  Biochemical Journal Immediate Publication. Published on 27 Aug 2009 as manuscript BJ20091094 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091094

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited