Catherine Thèves
email: catherine.theves@toulouse.inserm.fr

Christine Keyser-Tracqui

Eric Crubézy

Jean-Pierre Salles

Bertrand Ludes

Norbert Telmon

Detection and Quantification of the Age-Related Point Mutation A189G in the Human Mitochondrial DNA

ABSTRACT: Mutation analysis in mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine.

Among the difficulties linked to the mtDNA analysis one can find the detection of heteroplasmy which can be inherited or somatic. Recently, age-related point mutation A189G was described in mitochondrial DNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe and PNA/Real Time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/Real Time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies. KEYWORDS: forensic science, mitochondrial DNA, heteroplasmy, age-related mutation, detection methods The mitochondrial DNA (mtDNA) control region is a hypervariable sequence widely used in population genetic studies to investigate human evolution, history and patterns of migration as well as in forensic medicine for individual identification. Among the difficulties linked to mtDNA analysis is the detection of heteroplasmy (the presence of two or more mtDNA types in one individual), which can be inherited or somatic, and sometimes difficult to interpret. In recent years, numbers of reports have suggested that during individual natural aging, heteroplasmic point mutations accumulate in the control region of mitochondrial DNA (mtDNA) of some tissue types [START_REF] Michikawa | Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication[END_REF][START_REF] Murdock | The age-related accumulation of mitochondrial DNA control region mutation in muscle, but no brain, detected by a sensitive PNA-directed PCR clamping based method[END_REF][START_REF] Wang | Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication[END_REF][START_REF] Bo | Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondria patients[END_REF]. This accumulation of mutations is thought to be due to the oxidation of DNA bases by free radicals, generated especially during cellular energy production by the mitochondrion [START_REF] Harman | The biologic clock: the mitochondria?[END_REF][START_REF] Linnane | Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases[END_REF]. Among the reported heteroplasmic point mutations one can find the A to G transition at position 189 (A189G), which has been detected at significant levels in skeletal muscles from aged individuals by several authors [START_REF] Wang | Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication[END_REF][START_REF] Bo | Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondria patients[END_REF]. According to them, the A189G mutation accumulates with age in both healthy subjects and patients with mitochondrial diseases. However, because the level of heteroplasmy is lower in non-pathological cases, the mutation detection is technically difficult and requires a sensitive method [START_REF] Jazin | Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA[END_REF][START_REF] Wilson | A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes[END_REF][START_REF] Calloway | The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age[END_REF]. Moreover, the ratio of mutant (189G) to wild type (189A) has been reported as varying between different tissues from the same individual, which complicates the detection of low proportions of mutant mtDNA [START_REF] Chinnery | Nonrandom tissue distribution of mutant DNA[END_REF][START_REF] Calloway | The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age[END_REF].

In the present study we searched for the presence of the A189G heteroplasmic mutation in two tissue types, muscle samples and buccal cells, with the following aims: (i) determine whether the A189G mutation was really age-related; (ii) test its presence in buccal cells; (iii) evaluate the most efficient method for detecting low levels of heteroplasmy; and finally (iv) assess the possible implications of the A189G heteroplasmy in anthropological and forensic studies.

In order to prove the increase of mutant mtDNA molecules with age, buccal cells were collected from 37 individuals ranging from 4 to 85 years of age and belonging to 10 different maternal lineages. If the A189G mutation accumulates in an age-dependent manner, then it should be present in older subjects but not in younger ones of the same family. We also analysed muscle samples gathered from 69 subjects 1 to 97 years of age. These samples were analysed by means of three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe [START_REF] Holtke | The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acidsan overview[END_REF] and peptid nucleic acid (PNA) and quantitative Real Time PCR (qPCR) together [START_REF] Ørum | PCR clamping[END_REF][START_REF] Kyger | Detection of the hereditary hemochromatosis gene mutation by Real-Time fluorescence polymerase chain reaction and peptide nucleic acid clamping[END_REF].

Automated DNA sequencing is a technology widely and routinely used in research and forensic laboratories for population genetic studies or individual identification [START_REF] Rosenblum | New dye-labeled terminators for improved DNA sequencing patterns[END_REF]. Southern blot analysis was done using two probes (with the A or G base at position 189) labeled by a molecule of digoxigenin in 5'. The digoxigenin label can be detected with enzyme-linked immunoassays for antidigoxigenin protein conjugates, and visualized through a chemiluminescent reaction [START_REF] Holtke | The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acidsan overview[END_REF]. The PNA/qPCR combined method is a less employed methodology.

The PNA molecule is a DNA mimic, in which the negatively charged sugarphosphate DNA backbone is replaced by an achiral, neutral polyamide backbone formed by repetitive units of N-(2-aminoethyl) glycine [START_REF] Nielsen | Peptide nucleic acid (PNA), a DNA mimic with a peptide backbone[END_REF]. The PNA directed PCR clamping technique has been developed to detect single base-pair mutations [START_REF] Ørum | Single base pair mutation analysis by PNA directed PCR clamping[END_REF][START_REF] Ørum | PCR clamping[END_REF]. As shown in Fig. 1, PNA probe hybridizes perfectly to a target region and prevents PCR primer binding, whereas single base-pair mismatches containing PNA exhibit minimal primer binding inhibition. PNA directed PCR clamping was used in combination with the Real Time PCR [START_REF] Wittwer | Continuous fluorescence monitoring of rapid cycle DNA amplification[END_REF]. The amplification process is monitored in real time by fluorescent dye SYBR Green incorporation and allows a relative quantification of heteroplasmy [START_REF] Kyger | Detection of the hereditary hemochromatosis gene mutation by Real-Time fluorescence polymerase chain reaction and peptide nucleic acid clamping[END_REF]. Unlike other double-strand DNAselective dyes, SYBR Green is excitable by visible wavelength and provides excellent sensitivity. This method of mutation detection does not require post-PCR manipulations, reducing assay time and the probability of laboratory contaminations.

Materials and Methods

Samples and DNA Extraction

Buccal cell samples were collected by cytobrush on 37 individuals (4 to 85 years old) who gave their informed consent. Each one of the 10 maternal lineages included 2 to 4 members. Buccal samples were incubated for 2h at 50°C in 400µl extraction buffer (EDTA 5mM, SDS 2%, Tris HCl 10 mM (pH 7.5), sodium acetate 0.3M) and 0.2mg proteinase K. A phenol/chloroform/isoamyl alcohol (25/24/1) extraction was carried out on the supernatant. The aqueous phase was purified with the Cleanmix ™ Kit (Talent, Trieste, Italia). After purification, DNA was eluted into sterile water.

Muscle samples (psoas) were collected during forensic autopsies from 69 unrelated individuals ranging from 1 to 97 years in age. Individuals died accidentally and were exempt from any specific disease macroscopically detectable. Approximately 1cm 3 of muscle fragments were incubated in 3 ml extraction buffer (EDTA 10 mM, NaCl 50 mM, SDS 2%, Tris HCl 10 mM (pH 7.5) and 0.2 mg proteinase K) at 58°C overnight. Total DNA was extracted according to the saturated sodium acetate precipitation method [START_REF] Cattaneo | A simple method for extracting DNA from old skeletal material[END_REF]. An extraction reagent blank negative control accompanied all muscle and buccal samples extraction throughout the entire analysis.

Sequencing Analysis

PCR amplification was performed using 1µl total DNA in a 50µl reaction volume containing MgCl2 1.5 mM, BSA 0.2 mg.mL -1 , 0.2 mM of dNTPs, primers 200 nM, 1X buffer and 0.5 U of Taq HotGoldstar DNA polymerase (Eurogentec, Seraing, Belgium). Primer sequences are listed in Table 1. Amplification was carried out in a T3 Thermocycler (Biometra). After a 10 min pre-incubation step at 95°C, PCR amplification was performed by direct PCR (35 cycles) using the following conditions: 95°C denaturation for 45 s, annealing at 53°C for 1 min and extension at 72°C for 1 min, followed by a 7 min final extension at 72°C. PCR products were analysed by electrophoresis in 1% agarose low melting (Sigma-Aldrich, St Quentin Fallavier, France). Purification of amplicon products was carried out on Microcon ® PCR columns (Millipore, St Quentin-en-Yvelines, France). Sequencing reactions were performed using the BigDye  Terminator kit (Applied Biosystems, Courtabeuf, France). The products were purified using the protocol recommended by the manufacturer and submitted to capillary electrophoresis on an ABI Prism 3100 Genetic Analyser (Applied Biosystems).

The results were analysed using the Sequencing Analysis 3.7 Software (PE Applied Biosystems). Negative PCR controls accompanied samples and their extraction reagent blank negative controls throughout the sequencing process.

PNA Method and Real Time PCR

PNA sequences and complementary primers for the point mutation A189G are given in Table 1. PNA probes were purchased from Eurogentec (Seraing, Belgium). For PNA clamping reactions, PCR reactions contained: SYBR ® Green Jump Start TM Taq Ready Mix TM (Sigma-Aldrich, St Quentin Fallavier, France), 5 µl of DNA lysate, 300 nM of each primer and 2 µM PNA probe or H2O in 26 µl of final volume. The reaction mixture was subjected to a uracil-DNA glycosidase digestion step of 50°C for 2 min (the complex dissociates and the polymerase becomes fully active) followed by 95°C for 10 min and PCR cycling in the ABI Prism ® 7000 Sequence Detection System (Applied Biosystems, Courtabeuf, France). Real Time PCR was run in 40 cycles: denaturation (30 sec at 94°C), PNA hybridization (30s at 69°C), primer annealing (30 s at 60°C) and extension (30 s at 72°C).

The cycle threshold (Ct) value is defined by the cycle at which SYBR ® Green fluorescence increases significantly above the background as a result of specific amplification. Ct values were calculated by extrapolation to the x axis of the linear portion of the exponential growth phase. The reported cycle threshold (Ct) is the cycle number adjoining the x intercept [START_REF] Gibson | A novel method for Real Time Quantitative RT-PCR[END_REF].

Preliminary optimisation and validation of the PNA/qPCR combined method were carried out on two muscle DNA extracts. These samples (controls) were previously sequenced and identified as A (wild-type = WT) or G (mutant = M) at position 189. Linearity of template amplification relative to copy number was demonstrated. PCR efficiency (E) was calculated according to the equation: E = 10 (-1/slope) and showed PCR efficiency of 2.14. A dissociation protocol (Applied Biosystems) was performed, in order to identify the amplicon according to its melting temperature. Then a melting curve analysis confirmed the amplification of a single product with a specific melting temperature of 75°C.

Relative Quantification

A scale of G variant from 0 to 100 obtained by mixing in equimolar proportions DNA from M and WT controls established an experimental reference curve. In a first run, eleven points (in the following percentages: 0, 10, 20, …, 90 and 100%) were evaluated and amplification was repeated three times for each condition: without PNA probe, with PNA G probe or with PNA A probe (Table 1). Average amplification cycles were established for each point and an intra-assay precision test was calculated [START_REF] Gibson | A novel method for Real Time Quantitative RT-PCR[END_REF]. A second run was conducted including extra points situated between 0 and 20% and between 80 and 100% of G variant. Then the points common to each run enabled us to calculate an interassay precision test [START_REF] Gibson | A novel method for Real Time Quantitative RT-PCR[END_REF].

Once this experimental curve was established (x = mutant %; y = G/A Ct ratio), the Statistica v5.1 computer software (StatSoft, Tulsa, OK) calculated the percentage of mutants by its inverse relationship (x = G/A Ct ratio; y = mutant %).

For each sample on the same run an amplification triplicate was conducted for each PCR condition (without PNA probe, with PNA G probe or with PNA A probe). The cycle threshold obtained (Ct A for PNA A probe and Ct G for PNA G probe) was used to calculate the level of variant G or A present in the sample's mtDNA. A blank amplification as well as two M and WT controls were systematically included for each run.

Southern Blot

A 2 µl DNA was amplified by using primers listed in Table 1. PCR conditions were performed using a DNA thermal cycler (Perkin Elmer 2400) in a 26 µl reaction volume containing MgCl2 1.5 mM, dNTPs 200 µM, 400 nM of each primer, buffer 1X and 1U of Taq polymerase (Promega, Madison, WI). PCR steps were determined with an initial denaturation step at 94°C for 1 min, followed by 35 cycles including a denaturation step for 15 s at 94°C, a hybridization step for 30 s at 57°C, an extension step for 45 s at 72°C and a final extension step for 2 min at 72°C. Southern blot was performed using standard techniques. PCR products were blotted onto membranes (immobilon TM NY+, Millipore, Bedford, MA) and hybridized with 5'-digoxigenine labeled probes (Eurogentec, Seraing, Belgium) (Table 1). Detection of the digoxigenine-labeled hybridized probes was performed using DIG Luminescent Detection Kit as described by the manufacturer (Roche Applied, Penzberg, Germany). Detection of 5'-digoxigenine probes was performed by exposure to X-ray film and analysed using ImageQuant software v1.1 (Molecular Dynamics).

Results

Automated DNA Sequencing

All samples were initially analysed by automated DNA Sequencing. The region studied at position 189 is highly polymorphic, for example at sites 182 or 195.

The probes (for the PNA or Southern blot techniques) were synthesized on 15 or 20 bases according to the Cambridge Sequence [START_REF] Anderson | Sequence and organization of the human mitochondrial genome[END_REF] with a variation of bases A and G at position 189 (Table 1). It is necessary to know the samples' sequences underlying the probe in order to select the samples with the complementary Cambridge Sequence.

Of the 69 muscle samples studied, 13 were polymorphic between positions 182 and 195 and could not be analysed by the PNA/qPCR or Southern blot methods.

Their sequences were not complementary with those of the probes. Among the 13 polymorphic samples (Table 2), 9 were situated at the extremity of the probe (195 or 182) and 4 in a more central position (185 or 188). Among all samples analysed by sequencing, 11 (belonging to individuals of 38 to 97 years of age) had an A/G heteroplasmy at position 189 (Table 3). Among the 11 heteroplasmies detected, two individuals aged 68 and 77 years old, also had a polymorphism at position 195, and could not be analysed by the different methods using probes.

The 37 buccal samples from individuals of 4 to 85 years of age were also analysed by automated DNA sequencing. For 6 of them (belonging to two families of different maternal lineage) we were able to identify G at position 189 (Table 2) this transition was considered transmitted since it was found in all members of the family. Other polymorphisms were revealed by this sequencing method (Table 2)

and located either at the end or in the central part of the probe's hybridization region. Collecting buccal samples was done by family grouping; polymorphism detection excluded several families (representing a total of 14 individuals) from the PNA/qPCR or Southern Blot analysis. No A189G heteroplasmy was detected in any of the 37 buccal samples with sequencing (Table 3).

In order to determine the threshold of the automated DNA sequencing method, the experimental variant G scale was tested by this technique. A sequencing pattern G peak at position 189 was detectable for minimum 30% of G variants.

PNA/qPCR Tests

To test the linearity of the probe's PCR clamping according to the DNA concentration, serial-dilutions were performed between 100 and 0.001 ng from total DNA of WT and M controls. Differences in cycle threshold (ΔCt) between amplification with or without PNA were constant for total DNA concentrations between 100 and 0.1ng. Initial amplification of each sample without probe had to be included between 16 and 26 Ct. If the DNA concentration or quality was low, the samples could not be analysed because they stood outside the linearity of the probe's PCR clamping. For that reason, five muscle samples were excluded from the PNA/qPCR analysis.

To test the efficiency of the PCR clamping according to the percentage of mutants present in total mtDNA, we produced variant G ratios from WT and M controls.

We tested both PNA probes during qPCR amplifications (Fig. 2a and2b). Each graph demonstrated that the curves between the proportion of mutants and the PCR clamping were exponential and had inverse relationships. The Ct values included between 0 and 80% of G variant for the PNA G probe (Fig. 2a) and between 20 and 100% for the PNA A probe (Fig. 2b) varied only slightly. The differences between the Ct values with and without PNA probe (ΔCt) were noticeable only between 80 and 100% of mutants for the PNA G probe (Fig. 2a) and between 0 and 20% for the PNA A probe (Fig. 2b). Finally, the use of only one probe would only have enabled us to analyze levels of mutants ≤ or ≥ to 80 % of variant G for the PNA G probe and levels of mutants ≤ or ≥ to 20% of variant G for the PNA A probe. However, in order to obtain opposite and complementary Ct values between both PNA probes, the level of G variant had to be located between 0 and 20% or 80 and 100%. From the cycle threshold ratio G/A we established a variant G reference curve, to estimate the level of mutation in a more precise manner. Fig. 2c demonstrated that the G/A Ct values led to a better resolution of the mutant levels between 20 and 80%.

Intra-assay and Interassay Precision Tests

Amplification triplicates were conducted on the same run and for each different condition (without PNA probe and with PNA G or A probe) to test the precision of the variant G curve. As suggested by the manufacturer (Applied Biosystems) a value ≤ to 0.5 Ct was considered acceptable between triplicates of the same experimental condition in a same run. Also, the intra-assay precision test values on WT and M controls in each of the three PCR conditions was situated between 0.1 and 0.5% [START_REF] Gibson | A novel method for Real Time Quantitative RT-PCR[END_REF]. The test's precision on 2 runs for 2 different days, representing two triplicates for each control condition was also measured. The interassay precision test was situated between 1.5 and 4.4% for the 3 PCR conditions [START_REF] Gibson | A novel method for Real Time Quantitative RT-PCR[END_REF].

The experimental reference curve of the controls' G/A values (Fig. 2c) was reversed in order to obtain an estimated level of mutants in an unknown sample.

We have excluded 0% and 100% points in this inverse relationship and their corresponding G/A Ct ratios (0.7 for 0% and 1.6 for 100%) because they lacked complementary information. Statistica v5.1 software calculated a polynomial function (Fig. 3) from measured G/A Ct ratios and the values of mutant levels were obtained with a 1.4% average standard deviation (SD) (minimum SD: 0.02%, maximum SD: 4.1%). The function then was applied to the Ct G/A ratios of the samples.

PNA/qPCR Samples

In order to be statistically representative, minimum variation of ≤ 0.5 Ct was considered acceptable for all amplification triplicates of a given condition. The intra-assay precision test carried on both tissue types were comprised between 0.06% and 1.68% [START_REF] Gibson | A novel method for Real Time Quantitative RT-PCR[END_REF]. Samples presenting unstable amplification in a run were either re-analysed or excluded.

Among the 23 buccal samples analysed by the PNA/qPCR method, 18 presented a level of heteroplasmy at position 189 superior to 5%, the maximum being 12.6% (Fig. 4a). These heteroplasmies were especially noticeable in 3 out of 4 members (4, 37, 60 and 85 years old) of the same maternal lineage (Fig. 4b; family 1).

While the heteroplasmic mutation was detected in the three adults (at levels of 8 to 9.5%), it was not observed for the child. Similarly the heteroplasmic mutation was detected in the older members (73 and 80 years old at levels of 12.6% and 11.5% respectively) in families 2 and 4, and more slightly observed in the younger members (at levels of 1.5 to 8.9%) (Fig. 4b). From these family observations even though the level of mutation was low, we noticed that the A189G transition in its heteroplasmic form was a somatic mutation because it was little or not observed in young individuals. Considering all the individuals (Fig. 4a) heteroplasmy levels were low and there didn't seem to be an accumulation threshold age for this specific mutation.

The 50 muscle samples from individuals of 1 to 97 years old were analysed by the PNA/qPCR method. Fig. 5 represented the accumulation of the 189G mutation according to the subject's age. The levels of heteroplasmy were higher in the muscle tissues compared to the buccal samples. In fact, for individuals 60 years old or more, the level of mutants was ≥ to 20% for 10 out of 12 individuals (against 12.6% for buccal cells). On the contrary, for the individuals of less than 40 years old, the percentage of mutants was low, ≤ to 10% for 18 out of the 23 individuals analysed. The PNA/qPCR method also detected the 189G polymorphisms identified in the 6 buccal samples by sequencing (Table 2). As expected, the G/A Ct values were similar to the control M (i.e. ≥ 1.6).

Southern Blot

All buccal and muscle samples with the complementary Cambridge Sequence to the probe's hybridization region were analysed by Southern blot. The detection of the 189G heteroplasmic form in muscle tissues was important in individuals of more than 60 years old (Fig. 6a). The labeling's relative quantification was done by the ImageQuant software and the percentage of mutants detected in the samples was calculated compared to M control (an equal quantity of control or samples DNA was deposited). Concerning the individuals under 60 years old (Fig. 6b), with a lower percentage of G variants, detection was slighter but present up to 40 years old. The individuals of less than 40 years old (with a few exceptions) were lightly or not labeled. The method's detection limit of the percentage of G variant seemed to be 10%. On the 50 muscle samples analysed by the Southern blot method, detection of the 189G heteroplasmic form was demonstrated in 27 individuals in levels close to 10% and quantified by the ImageQuant software (Table 3). On the contrary, detection of mutant molecules was almost impossible in buccal cells and did not allow sample discrimination.

Moreover, we obtained from two deceased individuals of 63 and 70 years old both types of tissue samples and as shown in Fig. 6c, the 189G heteroplasmy was clearly detected in the muscle tissues of each one of them (with a percentage of 12% and 22% respectively) and was slightly present in buccal cells (≤ 5%).

Discussion

In order to minimise possible contaminations, which can be disastrous in the research of heteroplasmies, negative controls were done at each of our method's steps (extraction, PCR, sequencing, real time PCR). Our laboratory followed the working conditions suggested by the EDNAP group in order to prevent any possible contamination [START_REF] Tully | Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclatures and interpretation of mitochondrial DNA profiles[END_REF]. Pre and Post PCR areas were separated and all disposables treated by UV cross-linking. Concerning the sequencing method, samples were analysed on both their DNA strands to confirm heteroplasmies.

They were analysed only when both bases appeared visible above the background and on both strands. A positive control as well as the laboratory personnel's sequence were determined in order to test the absence of sample contamination.

Our DNA samples were of good quality and their amount sufficient for a good PCR amplification. That is why the number of PCR cycles was limited to 35 for the initial PCR amplification of the specific strand before its analysis by sequencing. Concerning the three detection methods, all PCRs were conducted with a number of cycles between 35 and 40. Indeed, when the amount of DNA is low, the number of cycles needs to be increased, for example in hair mtDNA analysis, and heteroplasmic artefacts should occur [START_REF] Budowle | Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair[END_REF]. The five muscle samples whose amplification happened late by the PNA/qPCR method (Ct without probe was over 30) were excluded from our study because they were not situated within the linearity of the PCR clamping. Consequently, the levels of heteroplasmies found on our healthy muscle samples were similar to those previously published [START_REF] Bo | Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondria patients[END_REF]. We also excluded from our studies individuals whose autopsies revealed any mitochondrial or neurodegenerative pathologies. The muscle samples all came from the psoas, a muscle with low energetic needs on working or living conditions. Within the individuals chosen for the buccal samples none presented pathologies linked with oxidative stress.

Analysis by sequencing of PNA probes hybridization region polymorphism enabled us to evaluate the effects of these polymorphisms on the PNA/qPCR method. The PNA probe's sequence mismatch leads to instability and an important loss in the probe's hybridization temperature. Loss in hybridization temperature is more or less important depending on the mismatched base and its location in the probe's sequence [START_REF] Ørum | Single base pair mutation analysis by PNA directed PCR clamping[END_REF].

If the mismatch is central, the instability is greatest and hybridization temperature can rise up to 15°C. We decided to locate the nucleotides A and G at position 189 in the centre of the probe so that an eventual mismatch could lead to an important temperature loss. However, when the mismatch occurs near the probe's extremities, temperature loss is less significant (around 7 or 8°C) and primer or PNA hybridization can take place in various proportions independently from the base at the position 189. This kind of probe or primer hybridization can be observed in polymorphic samples near the extremities of the PNA probes and can lead to important Ct variations during real time PCR cycles. Such polymorphic samples presented higher Ct variations than the accepted value (0.5 Ct) between triplicates of the same run. In consequence, we couldn't take into consideration the polymorphic samples' Ct values. Concerning the samples containing central polymorphisms (for example: positions 185 or 188), the amplification result was different. Whatever the PNA probe, the mismatch occurred systematically because of the central polymorphism leading to an important loss in hybridization temperature. With or without PNA probe, the amplification slightly varied (≤ 1

Ct).

It is interesting to notice that two polymorphic samples in the position 195 had a heteroplasmy in position 189 detectable by sequencing, in rates higher than 30%, and were from individuals 77 and 68 years old. In order to analyse them completely, and samples where heteroplasmy was not detected by the sequencing method, we would need to synthesise PNA probes whose sequence would correspond to the most common polymorphisms. Among all our samples (muscle and buccal) 195 and 188/185 positions are the most polymorphic sites.

Comparing automated sequencing and PNA/qPCR methods demonstrated two facts. Where the levels of 189G mutant molecules were > 30%, including 9 muscle samples submitted to both techniques, the percentage of G variants obtained, either by the peak heights on the sequencing pattern or by the qPCR's Ct G/A ratio, was the same. This observation revealed that our evaluation method was correct if certain precautions like minimal variations between same sample triplicates (≤ 0.5 Ct) were respected and if a standard deviation of 1.4% was included. Concerning the lower levels of heteroplasmy, our study demonstrated that the PNA/qPCR method was more sensible. In fact, 44 muscle samples were identified as heteroplasmic in position 189, when only 9 were detected in the older individuals by automated sequencing (Table 3). This sensibility difference between sequencing and PNA/qPCR methods, incited us to use a third detection method, the Southern blot in order to evaluate and confirm the previously detected heteroplasmies by the PNA/qPCR method. The Southern blot method confirmed the PNA/qPCR results in detecting levels of heteroplasmy as low as 10%. As shown in Fig. 6a and6b, on individuals of 40 years old or more, this technique detected the 189G form of heteroplasmy in proportions of 50 to 10%. Moreover, relative quantification of the labeling by the probe on mutants using ImageQuant software confirmed the proportion of mutant levels analysed through both methods. For example, in Fig. 6a, individuals of 87 and 74 years old had an estimated percentage of mutants respectively of 44% and 21% whereas the 77 year old individual's labeling was estimated at 1% (considerably under this technique's the level of detection). Estimated percentage of G variants with the PNA/qPCR method on these same individuals was of 48% for the 87 year old, 19.7% for the 74 year old and 5.3% for the 77 year old. This was also true for lower levels as shown in Fig. 6b where the 43 year old individual presented a Southern blot labeling of 15.5% with the M probe and of 13.2% with the PNA/qPCR method.

However, concerning the buccal samples, the Southern blot technique did not reveal, the really low heteroplasmy levels detected by the PNA/qPCR technique, because, the variant G rates remained very low in buccal cells with a maximum of 12.6%. As shown in families 2 and 4 (Fig. 4b) the 73 and 80 year old individuals represented an estimated 12.6% and 11.5% of mutants with the PNA/qPCR method, these individuals showed a very weak labeling with the Southern blot technique, estimated ≤ to 5% by the ImageQuant software. The difference in detection can be explained by the detection limits of the Southern blot technique, but also by an estimated percentage of errors of the PNA/qPCR technique and/or experimental differences.

Our study exerts another important fact: the accumulation of mutations is higher in skeletal muscle (20 to 50% in the older individuals) than in buccal cells (12.6% maximum) (Fig. 4a and5). These high mutation levels in muscle tissue have been described previously [START_REF] Wang | Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication[END_REF][START_REF] Bo | Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondria patients[END_REF]. This difference could be explained by the important mitotic activity in buccal cells. MtDNA mutations, associated with aging, are frequently found to accumulate themselves rapidly in tissues with high energetic needs and slow mitotic activity, like skeletal muscle, heart and brain [START_REF] Linnane | Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases[END_REF][START_REF] Nagley | Ageing and mammalian mitochondrial genetics[END_REF][START_REF] Wallace | Mitochondrial diseases in man and mouse[END_REF]. This could explain why the mutation level in buccal cells did not have an accumulation threshold age (Fig. 4a) and that the variation in mutation percentage between a 40 year old individual and a 60 year old one, cannot be found, because the rapid cell turn-over could conceal the mutation accumulation. Therefore, even in advanced ages (3 individuals are over 80 years old among the buccal samples), the mutation level did not exceed 10%, cellular turn-over overcoming the cumulating mutational process.

The fact that the A189G mutation accumulates itself in a tissue-specific manner is clearly established by double sampling on two individuals (of 70 and 63 years old). For both, we collected muscle and buccal samples and analysed them by the PNA/qPCR method. Concerning the 70 year old, the percentage of mutant molecules was of 8.9% in buccal cells and of 21.2% in muscle tissue. A similar situation was observed for the 63 year old since the level of mutant was 3.5 times higher in muscle tissues than in buccal cells (3.9% versus 14.3%). These results were confirmed by the Southern blot method (Fig. 6c) where the 70 year old individual presented a higher labeling level in muscle tissue than in buccal cells. This was also confirmed by ImageQuant software, the level of mutation in muscle tissue was of 22% compared to buccal cells where it was ≤ to 5%. The variation in the mutation frequency between different tissues has been previously established in mitochondrial pathologies [START_REF] Chinnery | Nonrandom tissue distribution of mutant DNA[END_REF], implying once again the cellular turn-over.

Simultaneous use of three technologies in our work, besides demonstrating better sensitivity of the PNA/qPCR method, revealed an interesting technical point.

Automated DNA sequencing is a widely employed technique to detect mtDNA mutations; nevertheless, it has been demonstrated by serial-dilution experiments that low levels (≤ 20%) of mtDNA heteroplasmy could not be reliably detected by this technique [START_REF] Chinnery | Point Mutations of the mtDNA control region in normal and neurodegenerative human brains[END_REF]. In this study, we also showed that automated DNA sequencing did not allow a quantitative estimation of the proportion of A189G mutations < to 30%. This technique usually uses dye-labeled terminators, which are dideoxynucleotide triphosphates (ddNTPS) labeled with different fluorophores [START_REF] Rosenthal | New protocols for DNA sequencing with dye terminators[END_REF]. A major drawback of the dye-terminator chemistry is the highly uneven peak pattern produced after the amplification reaction, this latter being largely dependent on the local sequence context and of the DNA polymerase used [START_REF] Parker | AmpliTaq ® DNA Polymerase, FS Dye-Terminator sequencing: analysis of peak height patterns[END_REF].

Dideoxynucleotides labelled with BigDye ™ Terminator show more uniform peak heights and less G suppressions [START_REF] Rosenblum | New dye-labeled terminators for improved DNA sequencing patterns[END_REF]. However, Zakeri et al. [START_REF] Zakeri | Peak height pattern in dichlororhodamine and energy transfer dye terminator sequencing[END_REF] have analysed the context dependency of peak height variations to improve data interpretations.

A common problem occurs when a G follows an A. The size of dye-labeled G peaks was lower than the height of the preceding A peak in the sequencing pattern. This was true for sites of mixed A/G bases in heterozygous mutations [START_REF] Korch | Improved DNA sequencing accuracy and detection of heterozygous alleles using manganese citrate and different fluorescent dye terminators[END_REF]. This variability in peak height reduced base-calling accuracy of software and hindered heterozygous mutation detection. In the case of the 189 position, where the local sequence was either GAA or GAG, the G BigDye ™ Terminator was incorporated with difficulties in comparison with the A BigDye ™ Terminator, giving rise to an A peak with an underlying G peak in this sequence. These problems, low levels of heteroplasmy and G BigDye ™ incorporation after or with A BigDye ™ , could explain detection differences between automated sequencing and the PNA/qPCR method as well as the differences in detection levels of 20% for Chinnery et al. [START_REF] Chinnery | Point Mutations of the mtDNA control region in normal and neurodegenerative human brains[END_REF] and of 30% in our study.

An early study already demonstrated difficulties to detect heteroplasmies according to different techniques employed [START_REF] Tully | A sensitive Denaturing Gradient-Gel Electrophoresis assay reveals a high frequency in hypervariable region 1 of the Human mtDNA control region[END_REF]. The sensitive Denaturing Gradient-Gel Electrophoresis (DGGE) revealed heteroplasmies detection at levels as low as 1%. This way, the mtDNA heteroplasmy frequency was more common in comparison with those reported by sequencing analysis. Very recently, Hancock and colleagues (31) evaluated the sentivity of other detection techniques.

Particurlarly, the automated DNA sequencing, including several chemistries and genetic analyzers, allowed detection at 30% levels. At 20% or 10%, the heteroplasmy was difficult to distinguish from the baseline noise, and was detectable only if the position was known. On the contrary, the PNA technique was able to detect the mutation at 5% level.

As seen in our work, the PNA-directed PCR clamping technique is a very specific and highly sensitive method to identify single nucleotide changes in DNA molecules [START_REF] Ørum | PCR clamping[END_REF]. According to Murdock's team, it allowed the detection of mutant molecules at a level of 1% of total molecules by direct PCR [START_REF] Murdock | The age-related accumulation of mitochondrial DNA control region mutation in muscle, but no brain, detected by a sensitive PNA-directed PCR clamping based method[END_REF][START_REF] Murdock | PNA-mediated PCR clamping[END_REF]. Individual nucleotide bases are attached to each peptide unit to provide a molecular design that enables PNA to hybridise to complementary nucleic acid targets according to the Watson and Crick base-pairing rules [START_REF] Elghom | PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules[END_REF]. PNA-DNA hybrids are more stable than DNA-DNA hybrids and PNA hybridisation kinetics is less influenced by sodium concentration. PNAs are more sensitive to internal base-pair mismatch with their DNA complement; this can result in a 10 to 18°C lower melting temperature. Moreover, PNA is not recognized by polymerases and therefore cannot be copied or used as a primer. PNA probes are more robust detector molecules for Real Time PCR methods than many of their DNA-derived counterparts, such as the hybridisation probes which are degraded during PCR by the endonuclease activity of Taq DNA polymerase [START_REF] Wilhem | Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridisation probes[END_REF]. This technique combined with Real Time PCR allowed a very sensitive quantification and an excellent reproducibility.

The PNA/qPCR sensitivity and accuracy compared to that of automated DNA sequencing could be interesting in forensic investigations where detection of heteroplasmy is not unusual. Technically, the advantage of the PNA/qPCR method is that it requires very few pre PCR manipulations and none post PCR.

One run including amplifications without and with both probes is enough to obtain fast and reliable results on the level of heteroplasmy in a sample with minimum contamination possibilities. In order to limit the lack of precision, we can incorporate both WT and M controls in the run but also the experimental scale of G variant on the sample's amplification run, adding internal precision to the method with a limited margin of error. It is nonetheless necessary to know ahead of time the studied samples' sequences in order to apply the corresponding PNA probe for the 189 position. Other age-related heteroplasmies have been described at positions 414 and 408 [START_REF] Michikawa | Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication[END_REF][START_REF] Murdock | The age-related accumulation of mitochondrial DNA control region mutation in muscle, but no brain, detected by a sensitive PNA-directed PCR clamping based method[END_REF][START_REF] Bo | Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondria patients[END_REF], their investigation could benefit from this technique and their hybridization region is less polymorphic sites requirering only two PNA probes. The PNA/qPCR method is rapid and flexible, avoids laboratory contaminations by limiting the number of manipulations and enables a fine detection in levels ≤ to 10%.

Moreover, the fact that the level of heteroplasmy varies in the different tissues of an individual, and that it can be expressed at very low levels in some, (i.e. buccal cells versus muscle tissue) increases interpretation difficulties. That is where a very sensible method can come in use to obtain very low levels of detection.

Interpretation in forensic cases can sometimes be complicated because of the presence of heteroplasmies in the given sample (hair for example) and of its absence in others (blood for example). Consequently, demonstrating the presence of mutant molecules in two different samples can help eliminate uncertainties. On the contrary, if the number of mutants is higher in the muscle samples, other tissue samples should be preferred (like buccal cells or blood) for automated mtDNA sequencing in forensic cases to minimise interpretation uncertainties.

Because a correlation was clearly established between the age of the studied individual and the level of mutant molecules at position 189, the age of the subject tested should be considered when interpreting mtDNA typing results. As previously underlined by Calloway et al. [START_REF] Calloway | The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age[END_REF], age and heteroplasmy should be considered in missing person cases when a significantly older individual is used as a reference for a missing younger maternal relative. In the same way, this information is useful for interpretation of mtDNA caseworks including mass disaster biological remains.

It is obvious, as shown in Fig. 5, that evaluating only one heteroplasmic mutation point cannot help determine the analysed individual's age and this was not the aim of this study. Nonetheless, we could suggest evaluating several heteroplasmic mutation points and/or mitochondrial deletions. In fact, mitochondrial deletions accumulate with age, and were quantified using Real Time PCR by von Wurmb-Schwark et al. [START_REF] Wurmb-Schwark | Quantification of human mitochondrial DNA in a real time PCR[END_REF]. Counter-crossing the values of several heteroplasmic mutation points or deletions, could help recognize these age-related mutations in relation with human aging. We should also investigate a possible link between these mutations and osteologic markers of normal and pathological aging widely used in forensic medicine, increasing label performances in identification cases.

Conclusion

The analysis of heteroplasmic A189G mutation by PNA/qPCR method permitted a sensitive detection as well as reliable and reproducible quantification of mutant level. This technique allowed us to demonstrate the absence of the A189G transition in buccal cells in young individuals and its presence in older individuals from the same maternal lineage, concluding that it is a somatic mutation. Moreover, we demonstrated for the first time the accumulation with age of the A189G mutation in mitotic buccal cells in levels lower than 13%. In post-mitotic muscle tissues, mutation accumulation was age-related and reached very high levels in 60 year-old or more individuals. Our results could have many implications in heteroplasmy interpretation in forensic caseworks but also in anthropological studies, for example in the investigations of great names of history. The PNA/qPCR method allowed detection of low levels of the A189G heteroplasmy in two cell types. The automated DNA sequencing did not detect the heteroplasmy level inferior to 30%. In buccal cells, heteroplasmy were globally inferior to 10% and were not detected by automated sequencing or by Southern blot. In muscle samples, Southern blot detected heteroplasmy level superior to 10% and PNA/qPCR method could determine all levels of heteroplasmy.

Figures legends

FIG.

1 -FIG. 3 -

 13 FIG.1-The PNA directed PCR clamping methodology. Arrows show PCR primers.

FIG. 6 -

 6 FIG. 6-Southern blot. WT: Wild-type control; M: Mutant control; the samples

TABLE 1 -

 1 Sequences of the primers and probes used in this study.

	Sequence (5'-3') by Anderson and al. (20)

TABLE 2 -

 2 Polymorphisms characterization in hybridization region of PNA probes.

	Cambrige		1	1	1	1	1	1
	Sequence		8	8	8	8	8	9
	Reference (20)		2	4	5	8	9	5
		n	C G G A	A	T
		7	.	.	.	.	. C
		1	.	.	.	.	G C
	muscle	1	T	.	.	.	.	.
	samples	2	.	.	A G	.	.
		1	.	.	A	.	.	.
		1	T	.	C	.	.	C
		6	.	.	.	.	G	.
	buccal	1	.	.	.	.	.	C
	samples	2	T	A	.	.	.	.
		6	.	.	A G	.	.

TABLE 3 -

 3 Characterization of the position 189 between three technologies on muscle tissues and buccal cells.

				Automated
					PNA / qPCR method	Southern blot
				sequencing
				A189G		A189G		A189G
	Samples	n	n		n		n
				heteroplasmy		heteroplasmy		heteroplasmy
	Muscle tissues	69	69	11	50	44	50	27
	Buccal cells	37	37	0	23	18	23	0

  Accumulation in buccal cells of the A189G heteroplasmy level with age. (n = 23). The level of 189G mutants was determined by G/ACt ratio within the PNA/ Real Time PCR run. None of the G/A Ct ratios measured surpassed 0.92, thus giving a maximum percentage estimation of G variants of 12.6%. b: A189G heteroplasmy in buccal cells of individuals belonging to the same maternal lineage (from three different families). Mutation 189G in levels inferior to 12.6% was detected in subjects of the same family by PNA/qPCR. Mutation was absent or weakly present in the younger individuals compared to the older ones of the same family. FIG. 5-Accumulation in muscle samples of the A189G heteroplasmy with age. The level of 189G mutants was determined by G/A Ct ratio within the PNA/ Real Time

	35
	FIG. 4-a:

2 + 11918x -19242.5x 2 + 13574.1x 3 -3486.6x 4 ). It calculated the level of mutants present in a sample by it's G/A Ct ratio. PCR run. After 50 years of age, the levels are generally ≥ to 10% (15/19); for those of 60 years old or more, the levels are closer to 20% and over (10/12). The individuals 40 years old or less, except a few individuals (5/23) have levels of mutations ≤ to 10%.

Acknowledgements

We are grateful to Pr. D. Rouge and B. Martinez (Service de Médecine Légale de Rangueil, Toulouse) for the access to muscle tissue materials.

We would like to thank S.Romac (Laboratoire d'Anthropologie Moléculaire, Strasbourg) for technical help, C.Cariven and N.Malet (INSERM, U563 Toulouse) for technical advices and S.Laurencin for helpful English writing. Grant Sponsor: ACI « Espaces et Territoires : le Complexe Spatial Altaï-Baïkal. Plaque tournante des flux géniques en Haute Asie de la période protohistorique à l'époque moderne »


Here we describe an experimental technique, termed Plasmon Waveguide Resonance (PWR) Spectroscopy that enables the characterization of molecular interactions occurring at the level of anisotropic thin films as lipid membranes and therein inserted or interacting molecules. PWR allows one to characterize such molecular interactions at different levels: 1) acquire binding curves and calculate dissociation constants; 2) obtain kinetic information; 3) obtain information about associated anisotropy changes; 4) get insight about lateral homogeneity. When applied to membrane proteins as the G-protein Coupled Receptor (GPCR) family, both ligand-induced conformational changes of the receptor can be followed as well as interactions with effectors (e.g. G-proteins). Additionally, by either altering the lipid composition in cellular membranes or specifically controlling its composition in the case of lipid model membranes with reconstituted proteins, the role of the lipid environment in receptor activation and signaling can be determined. Additionally, the eventual partition of receptors in different lipid microdomains can be followed. Such information can be obtained with cell membranes fragments expressing the protein of interest and/or lipid model systems where the protein under investigation has been reconstituted. Moreover, PWR can also be applied to directly follow the reconstitution of membrane proteins in lipid model membranes. The measurements are performed directly (no labeling of molecular partners), in real time and with very high sensitivity.

Here we will discuss different aspects on GPCR activation and signaling where PWR brought important information in parallel with other approaches. The utility of PWR is not limited to GPCRs but can be applied to any membrane protein. PWR is also a great tool to characterize the interaction of membrane active molecules with lipids. A brief section is dedicated to such applications. To finalize, as PWR is a homemade technology, ongoing instrument developments aiming at breaking current experimental limitations are briefly discussed.

Those established between membrane receptors and environmental stimuli or via external therapeutic agents play major roles in several cellular processes: signal transduction, nutrient uptake, motility, etc [START_REF] Uings | Cell receptors and cell signalling[END_REF] . One important and large class of membrane receptors are GPCRs, 7 transmembrane proteins that sense molecules outside the cell, activate intracellular signal transduction pathways and modulate cellular responses. Since they are activated by a large panoply of extracellular stimuli of varied size and nature such as light, odorants, hormones, and neurotransmitters, these receptors are extremely crucial therapeutic targets [START_REF] Kobilka | The structural basis of G-protein-coupled receptor signaling (Nobel Lecture)[END_REF][START_REF] Lagerstrom | Structural diversity of G protein-coupled receptors and significance for drug discovery[END_REF] . Moreover, because they can finely tune cellular responses, they are targets of choice for specific pharmacological treatments. Over the last decades, a huge effort has gone into the identification of molecules that target specific receptors and even subtypes of downstream signalling events, aimed at developing highly-selective treatments. Such development requires a deep understanding of the physico-chemical determinants of their behaviour and the interplay between the different actors: ligand, receptor, lipid, effector. Studies on this class of proteins are hampered by the difficulties of working with them due to their inherent properties: hydrophobicity, low natural abundance, difficulty in maintaining their functionality when isolated from their natural lipid environment, among others.

Here we describe a tool, termed PWR, enabling the quantitative characterization of molecular interactions directly (receptor/ligand, receptor/effector) or indirectly (modulation of protein activity by lipids, lipid partitioning) implicated in GPCR activation and signalling [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF][START_REF] Harte | Probing the kinetics of lipid membrane formation and the interaction of a nontoxic and a toxic amyloid with plasmon waveguide resonance[END_REF][START_REF] Salamon | Chapter 6. Plasmon resonance methods in membrane protein biology applications to GPCR signaling[END_REF] . PWR allows the characterization of such molecular interactions in terms of affinity (K D from pM to mM) and kinetics (msec) directly (no labelling needed) and with a very high sensitivity (femtomole). Such studies can be performed both ex-cellulo, that is with cell membrane fragments over expressing the receptor under investigation, and in vitro with the receptor reconstituted in lipid model membranes of controlled lipid composition.

PLASMON WAVEGUIDE RESONANCE METHOD

While Surface Plasmon Resonance (SPR) is routinely employed in many laboratories to characterize various molecular interactions, supported by the development of specific sensors for the different applications [START_REF] Homola | Surface plasmon resonance sensors for detection of chemical and biological species[END_REF][START_REF] Karlsson | SPR for molecular interaction analysis: a review of emerging application areas[END_REF][START_REF] Nguyen | Surface plasmon resonance: a versatile technique for biosensor applications[END_REF][START_REF] Lee | Exploring Molecular-Biomembrane Interactions with Surface Plasmon Resonance and Dual Polarization Interferometry Technology: Expanding the Spotlight onto Biomembrane Structure[END_REF] , PWR has emerged as a powerful SPR variant for the study of molecular interactions occurring in uniaxially oriented anisotropic thin films as proteolipid membranes [START_REF] Harte | Probing the kinetics of lipid membrane formation and the interaction of a nontoxic and a toxic amyloid with plasmon waveguide resonance[END_REF] . One particular and important difference concerns the sensor design: while SPR uses sensors consisting of a plasmon generating material (usually gold), PWR sensors consist in a metal layer (50 nm of silver is used because it provides sharper resonances than gold) over coated by a thick dielectric layer (460 nm of silica) that function as a waveguide. Plasmon and waveguide modes are coupled in PWR measurements [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF] . This has important consequences, namely the fact that with SPR only p-polarized light (perpendicular to the sensor surface) can be used to create resonances, while in PWR resonances can be obtained with both pand s-(parallel to the sensor) polarized light. SPR is sensitive to changes in mass occurring upon molecular interactions which allows to obtain affinity constants between the molecular partners. Due to the possibility of PWR to obtain resonances with both pand s-pol, the method becomes sensitive to both changes in mass and anisotropy (see further) that occur as a result of molecular interactions (Fig. 1) [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF] . Taking into consideration that both lipids and GPCRs are anisotropic molecules, anisotropy data provides insight about their molecular orientation. This information is crucial for such studies, as explained below. Additional advantages include: 1) higher spectral resolution of PWR compared to SPR supported by the increased electromagnetic field due to the silica. 2) sensitivity of the p-pol, s-pol and the total internal reflection (TIR) signal to the distance from the sensor surface is different. Indeed, while s-pol and p-pol decrease exponentially with the distance from the surface (the s-pol sensitivity dropping at considerably lower distance than p-pol), the TIR signal is constant all the way from the sensor surface to the bulk. This has important consequences in data analysis as the TIR signal can be indicative of non-bound (bulk) material.

3) The dielectric layer serves as mechanical and chemical shield for the metal layer, allowing reactive metals as silver to be used in aqueous environment [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF] . PWR sensors are quite resistant and can be reused for months, decreasing running costs.

PWR DATA ANALYSIS

Different types of PWR data analysis are possible. In any case, important control experiments are necessary to ensure proper data analysis. Most commonly and routinely, to determine the affinity of either protein/ligand or lipid/ligand interactions, a plot of ligand concentration (total ligand) versus resonance minimum position obtained at each particular concentration and fit with appropriated equation allows determination of K D values (further described below) [START_REF] Alves | Different structural states of the proteolipid membrane are produced by ligand binding to the human delta-opioid receptor as shown by plasmon-waveguide resonance spectroscopy[END_REF] .

To get further insight into the nature of the changes that occur upon specific molecular interactions, graphical analysis can be applied. The method is based on the fact that PWR spectra are determined by two physical properties of a thin film such as a lipid membrane: an average surface mass density and the spatial distribution of mass within the system that results from the structure of the deposited film [START_REF] Salamon | Graphical analysis of mass and anisotropy changes observed by plasmon-waveguide resonance spectroscopy can provide useful insights into membrane protein function[END_REF] . The separation of mass changes from those caused by structure can be obtained by transforming the measured spectral changes (magnitude and direction of the changes in the resonance minimum position obtained for both pand s-pol) from an (s-p) orthogonal coordinate system into one reflecting (mass-structure) (see for details [START_REF] Salamon | Graphical analysis of mass and anisotropy changes observed by plasmon-waveguide resonance spectroscopy can provide useful insights into membrane protein function[END_REF] and applications [START_REF] Alves | The interaction of cell-penetrating peptides with lipid model systems and subsequent lipid reorganization: thermodynamic and structural characterization[END_REF] ).

The most complete analysis involves spectral fitting. This type of analysis cannot always be applied, exceptions due to the complexity of the system, poor spectra quality, etc. This analysis is supported by the thin-film electromagnetic theory based on Maxwell's equations that provide an analytical relationship between experimental spectral parameters (spectral position, width and depth) and the optical properties of the system (refractive index n, extinction coefficient k and thickness t). As only n and k have different values along the measurement axes: 5 optical parameters must be considered: n p , n s , k p , k s and t. Nonlinear least-square fitting of the experimental to theoretical spectra can be performed to obtain the optical parameters [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF] which can be used to further characterize the system. From the optical parameters, the anisotropy of the refractive index can be calculated to provide information about the molecular orientation and ordering. The surface mass density of the system can also be calculated from Lorentz-Lorentz equation (for details see [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF] ) allowing the number of proteins in a lipid membrane to be determined.

PROTOCOLS FOR RECEPTOR IMMOBILISATION IN THE SENSOR

Plasmon resonance methods require the immobilization of one of the molecules under study on the sensor surface. A large choice of sensor chips with functionalized surfaces has been developed for SPR allowing the target molecule to be attached covalently or non-covalently.

While SPR is uncontestably valuable and routinely used to characterize diverse molecular interactions, its application for uniaxially oriented films as lipid bilayers is rather limited. Indeed, since SPR does not provide information about anisotropy or molecular orientation it is unable to ascertain proper membrane formation. Non-ideal lipid membranes (unproperly formed or oriented, with defaults, etc) can compromise results due to surface heterogeneity. Other approaches providing anisotropy information as Dual Polarization Interferometry 10 or PWR, among others, are thus ideal.

Solid-supported planar membranes

Solid-supported lipid membranes can be formed by two alternative procedures: 1) by the same principles that govern the spontaneous formation of freely suspended lipid bilayers, a method developed by Mueller and Rudin 14 to form bilayers across holes in teflon. In PWR studies, such bilayer is anchored to a teflon orifice by an annulus of lipid solution (the plateau Gibbs border) that allows lipid molecules to move into and out of the bilayer in response to protein insertion and/or conformation changes. The protocol has been thoroughly described elsewhere [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF][START_REF] Harte | Probing the kinetics of lipid membrane formation and the interaction of a nontoxic and a toxic amyloid with plasmon waveguide resonance[END_REF] . 2) by fusion of small unilamellar vesicles (SUVs) with the silica surface. Due to their high membrane curvature, upon contact with silica they spontaneously burst to form a lipid membrane [START_REF] Reimhult | Intact vesicle adsorption and supported biomembrane formation from vesicle sin solution: influence of surface chemistry, vesicle size, temeprature, and osmotic pressure[END_REF] .

Cell membrane fragments

Two different protocols might be used to immobilize cell membrane fragments: 1) following isolation of membrane fraction from cells (expressing or not the protein of interest) they are incubated with the sensor in presence of 5 mM CaCl 2 . This protocol was successfully applied to membrane fragments from mammalian cells in culture and from animal tissue [START_REF] Salamon | Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells[END_REF] . 2) immobilization of cell fragments on the sensor pre-coated with polylysine (PLL) by electrostatic interaction between sugars in cell membranes and PLL (adapted from Vogel et al [START_REF] Perez | Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains[END_REF] ). It allows cell membrane fragment immobilization from cells in culture (detailed protocols found here [START_REF] Boye | Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR)[END_REF][START_REF] Boye | The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors[END_REF] ).

STUDY OF GPCR SIGNALING IN RECONSTITUTED LIPID MODEL MEMBRANES

Despite the difficulties in working with reconstituted systems, they are mandatory to totally control lipid membrane composition and restrain to one single protein component.

GPCR reconstitution in lipid model membranes

Following GPCR isolation from cellular membranes using detergents, reconstitution in lipid model membranes is necessary to maintain their stability. Two different strategies can be used to follow GPCR reconstitution: 1) the detergent-solubilized receptor is reconstituted in SUVs following different reconstitution procedures [START_REF] Goddard | Reconstitution of membrane proteins: a GPCR as an example[END_REF] (dilution or detergent removal using dialysis, gelfiltration, hydrophobic absorption using Bio-beads, etc) and the proteolipid membrane is formed by spontaneous proteoliposome fusion with the silica surface; 2) a planar lipid model membrane is formed on the PWR sensor first by one of the two methods presented above, then receptor is then reconstituted in this pre-formed lipid bilayer by detergent dilution below the critical micelle concentration (cmc). While this second procedure is advantageous as it allows one to directly monitor GPCR reconstitution while evaluating associated problems (partial or total detergent membrane solubilisation, no protein insertion, protein insertion in non-expected orientation, etc), the first method might be necessary when the second fails (detergent concentration or nature is too harsh for the lipid membrane, unfavorable detergent to protein ratio, sample protein concentration is too low, etc). Most reconstitution procedure require: first separation of reconstituted species from non-reconstituted ones by sucrose gradients or other purification methods, then validation by SDS-PAGE and Western-blot or by biophysical methods (NMR, IR and Raman, EPR, CD, etc), depending among others on the lipid model system. Alternatively, fluorescence microscopy or other imaging methods are used to analyze the reconstituted protein in giant unilamellar vesicles (GUVs), which requires labeled material.

The reconstitution of the chemokine receptor CCR5 into planar lipid membranes has been directly monitored by PWR and rate constants for the process obtained [START_REF] Calmet | Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids[END_REF] . To do so, a small volume of detergent-solubilized receptor is inserted in the PWR chamber filled with buffer with a pre-formed lipid bilayer. Detergent concentration decreases below the cmc, favoring spontaneous protein insertion in the membrane. Ideally, for the process to proceed well and to avoid unwanted deleterious detergent effect on the lipid membrane (leading to large defaults due to partial membrane solubilisation), the protein is in a detergent with not too low cmc (detergents as dodecylmaltoside are quite deleterious for the membrane) as octylglucoside. Since octylglucoside often fails in keeping most receptor biological activity, an exchange of detergents just before the reconstitution process should be envisaged. Alternatively, if the protein is at considerably high concentration and the addition of a very small volume of detergent-solubilized protein to the PWR chamber for reconstitution has no impact in membrane integrity. Both approaches have been applied for the reconstitution of the chemokine CCR5 receptor and showed to be successful [START_REF] Calmet | Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids[END_REF] . The added value of PWR for this particular application resides on the fact that within the same experiment both the reconstitution and receptor activity, in terms of ligand recognition, can be directly determined with very high sensitivity. Indeed, most approaches do not allow for both aspects to be investigated, following receptor reconstitution, the activity of the protein is measured in yet a diff erent experimental setup with often a tedious and long procedure relying in high quantities of material and labeled proteins. PWR provides a good alternative with the only limitation being that the detergent in which the protein is solubilized must not perturb membrane integrity.

Receptor interaction with ligands and effectors

Once the receptor has been properly reconstituted in the lipid membrane, its activation by ligand can be monitored and ligand affinity measured. By comparing the data with reported K D values receptor activity can be validated, following isolation and reconstitution procedures. To determine ligand binding affinity, ligand is incrementally added to the proteolipid system and resonance spectral changes measured for each concentration. By plotting ligand concentration (total ligand) versus resonance shifts obtained for each concentration when the system reached equilibrium (no spectral changes observed with time; this reflects the R/L complex) and fitting by hyperbolic function that describes classical ligand binding, the dissociation constant for the process can be obtained [START_REF] Alves | Different structural states of the proteolipid membrane are produced by ligand binding to the human delta-opioid receptor as shown by plasmon-waveguide resonance spectroscopy[END_REF] . Since the majority of GPCRs ligands are quite small and the affinities are very high, the small amounts of low molecular weight ligands do not contribute to mass changes measured by PWR. Therefore, spectral changes observed are mainly attributed to ligand-induced receptor conformational changes. PWR has successfully been applied to monitor ligand binding of several GPCRs. Studies with the hDOR involving a large panoply of ligands revealed that different classes of ligands (agonists, antagonists, inverse agonists) lead to very different receptor conformational states [START_REF] Alves | Different structural states of the proteolipid membrane are produced by ligand binding to the human delta-opioid receptor as shown by plasmon-waveguide resonance spectroscopy[END_REF] .

Besides receptor/ligand interaction, PWR was also applied to study G-protein interaction (1 st effector following receptor activation) with receptor, either in the unbound or ligand-bound state [START_REF] Alves | Selectivity, cooperativity, and reciprocity in the interactions between the delta-opioid receptor, its ligands, and G-proteins[END_REF][START_REF] Alves | Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy[END_REF] . Most approaches employed to monitor receptor/G-protein interaction, besides FRET or BRET assays, are rather indirect as they measure further downstream signaling (GTPS and cAMP). Moreover, such assays reflect the response of a mixture of G-protein subunits naturally expressed in the cell system used. Pioneer PWR studies of G-protein/receptor interaction reported in 2003 were indeed the first studies able to directly measure such interaction and associated affinity. The affinity of the G-protein to the receptor/ligand complex and that of the GTPS for the ligand/receptor/G-protein was separately determined showing no direct correlation between the efficacy of a receptor to recognize a G-protein and to induce GTP/GDP exchange [START_REF] Alves | Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy[END_REF] . It also showed that while both unoccupied, agonist and antagonist-occupied hDOR are all able to bind to G-proteins, only the agonist-bound receptor induces GTP/GDP exchange.

Moreover, by investigating the interaction of different hDOR ligand-bound states with different G-protein subunits (G iα1 , G iα2 , G iα3 , G oα ), the selectivity of such interactions was evidenced. Indeed, certain ligands have a high degree of selectivity in terms of the G-protein they activate while others don't [START_REF] Alves | Selectivity, cooperativity, and reciprocity in the interactions between the delta-opioid receptor, its ligands, and G-proteins[END_REF][START_REF] Alves | Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy[END_REF] . This has important implications for agonist-direct trafficking and rational drug design whereby ligands could be designed that target specific pathways, avoiding known detrimental side effects of certain drugs.

Lipid modulation of receptor activity

Since GPCRs are almost completely immersed in the lipid membrane, the composition of such must impact their structure and function [START_REF] Lee | Biological membranes: the importance of molecular detail[END_REF] . Lipids can exert their action in a "specific" manner whereby bound lipids act as chemical partners, or in a "bulk" manner by modulating the physical lipid properties (fluidity, curvature, surface tension, etc) and thereby affecting protein function.

Cholesterol is highly abundant representing more than 20% of lipids in mammalian cell membranes [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF] . Its presence modulates the fluidity of the lipid membrane and also the formation of lipid domains (lipid rafts that are enriched in cholesterol and sphingomyelin, SM) consisting of highly ordered and packed lipid regions that co-exist with less ordered ones mainly composed of unsaturated phospholipids [START_REF] De Meyer | Effect of cholesterol on the structure of a phospholipid bilayer[END_REF] . Studies have indicated that cholesterol is required for the entry of HIV virions into cells expressing the co-receptor CCR5 and/or CXCR4 (co-receptors implicated in HIV-infection), and that cholesterol depletion inhibits HIV entry [START_REF] Ablan | The role of cholesterol and sphingolipids in chemokine receptor function and HIV-1 envelope glycoprotein-mediated fusion[END_REF] . Furthermore, cholesterol depletion leads to failure of CCR5 to inhibit cAMP activity [START_REF] Cardaba | CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity[END_REF] . As most studies performed relied in the use of β-cyclodextrin to deplete cholesterol from the membrane, a molecule associated with several artifacts [START_REF] Plazzo | Uptake of a fluorescent methyl-beta-cyclodextrin via clathrindependent endocytosis[END_REF] , more direct studies are needed. Ligand binding to CCR5 in model lipid membranes containing or not cholesterol was investigated by PWR. The data indicates an enhancement of the binding affinity of maraviroc, one of the first commercialized anti HIV agents, to membranes containing cholesterol, and that independently of the presence of sphingomyelin, therefore independent of the presence or lipid-ordered domains [START_REF] Calmet | Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids[END_REF] .

In another study the effect of the membrane curvature on the light-activation of rhodopsin was investigated. For such, rhodopsin was reconstituted in lipid membranes composed of lamellar lipids (phosphatidylcholine) in absence or presence of non-lamellar lipids (phosphatidylethanolamine, PE) that form negative membrane curvature. By PWR the pH dependent transition from the photointermediate metarhodopsin I to metarhodopsin II was followed and the pKa of the transition determined. An increase of the pka of 1 unity was observed when rhodopsin was reconstituted in PE enriched membranes. Moreover, the affinity of the light-activated rhodopsin for transducin (the rhodopsin G-protein) was also impacted by the lipid membrane composition, with a 25-fold increase in affinity when PE was present [START_REF] Alves | Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy[END_REF] . While the impact of PE in the meta I/meta II transition was determined by flash-photolysis experiments [START_REF] Botelho | Conformational energetics of rhodopsin modulated by nonlamellarforming lipids[END_REF][START_REF] Botelho | Curvature and hydrophobic forces drive oligomerization and modulate activity of rhodopsin in membranes[END_REF] , it was the first time that a study suggested that lipid impact could propagate beyond the receptor level (at rhodopsin-transducin interaction).

Receptor partition in lipid domains

Lipid membranes are not laterally homogeneous assemblages but possess nano or microdomains due to the spontaneous segregation of certain types of lipids in the plane of the membrane [START_REF] Simons | Model systems, lipid rafts, and cell membranes[END_REF][START_REF] Dietrich | Lipid rafts reconstituted in model membranes[END_REF] . Indeed, lipid microdomains form spontaneously in mixtures of SM and unsaturated phospholipids (as POPC or DOPC), in both presence and absence of cholesterol [START_REF] Simons | Model systems, lipid rafts, and cell membranes[END_REF] . Microdomains could provide more specificity and diversity in the signaling cascade by providing compartmentalization of the different components that are involved or by modulating the intrinsic activities of proteins therein located [START_REF] Insel | Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae[END_REF] . Therefore, it is very much pertinent to understand how GPCRs partition between such domains, and more importantly how such partition is impacted by receptor occupancy and bound-state. In the beginning of 2000, we ask ourselves whether by PWR measurements, one could discriminate between the lipid-ordered and disordered domains and eventually follow receptor partition between such domains. PWR measurements of two-component lipid membranes, known to laterally separate to form microdomains (namely, POPC/SM at 1/1 mol/mol), performed with the classic PWR sensor (50 nm of silver overcoated by 460 nm of SiO 2 ) resulted in wider PWR spectra but rather symmetric (non-published data). This lead us to think that this sensor did not possess high enough resolution to discriminate lipid microdomains. Another sensor was developed with a more elaborated composition that resulted in much sharper spectra [START_REF] Salamon | Plasmon-waveguide resonance studies of lateral segregation of lipids and proteins into microdomains (rafts) in solid-supported bilayers[END_REF] . With this sensor, spectra obtained for the binary lipid mixtures could be ascribed to a superposition of two resonances corresponding to microdomains consisting of POPC-and SM-rich phases coexisting within a single bilayer. Indeed, spectra deconvolution and simulation, allowed to attribute part of the spectra to the PCrich region (at lower resonance angle) and the other part to the SM-rich region (at higher resonance angle). Optical parameters were obtained for each of these regions, demonstrating that the PC-rich region is less thick and less packed than the SM-rich region. This data is in very good agreement with AFM literature that demonstrate these two lipid domains to have about 1nm thickness difference. In such binary lipid membranes, the partitioning and functioning of the hDOR in the two domains was investigated as well as the impact of the ligand on receptor partitioning. The study demonstrate that the partitioning was highly dependent on the type of ligand bound to the receptor: in the unbound-state the receptor preferentially partitions in the PCrich domain, while when activated by agonist it partitions preferentially in the SM rich domain (with two fold-greater propensity for this microdomain). Moreover, the addition of ligand to an unbound receptor leads to its trafficking from the PC-rich to the SM-rich domain. This study revealed that receptor partitioning impacts the interaction with the G-protein, a 30-fold higher affinity was observed for its interaction with the receptor present in the SM-rich domain [START_REF] Alves | Ligand modulation of lateral segregation of a G-protein-coupled receptor into lipid microdomains in sphingomyelin/phosphatidylcholine solid-supported bilayers[END_REF] . While these studies did not go far enough to determine the membrane property that was at the origin of such partitioning and impact in first receptor signaling events, one could suppose that the lipid environment impacts the receptor conformational state. The preferential partition of the agonist-bound receptor into the thicker SM-rich domain could be triggered by hydrophobic matching, as the activated receptor-state should be more elongated than the non-activated one.

STUDY OF GPCR SIGNALING IN CELL FRAGMENTS

Certain aspects on GPCR activation and signaling can alternatively be performed on the natural GPCR environment, namely the cells overexpressing the receptor of interest. As explained above, protocols have been established and validated for the immobilization of cell fragments on the PWR sensor. With this approach the affinity of ligands to the chemokine receptors CXCR3 and the ligand-induced receptor conformational changes have been evaluated. The CXCR3 receptor regulates several biological functions and plays important roles in angiogenesis, inflammation and cancer [START_REF] Belperio | CXC chemokines in angiogenesis[END_REF][START_REF] Groom | CXCR3 in T cell function[END_REF] . One peculiarity of this receptor is that there are two distinct isoforms that have antagonistic effects in cancer development and angiogenesis: the CXCR3-A isoform is implicated in cell proliferation, migration and cell survival and the CXCR3-B isoform (that differs from the A form by an additional 52 residues in the N-term) displays angiostatic, anti-proliferative and apoptotic functions [START_REF] Billottet | CXCR3, a double-edged sword in tumor progression and angiogenesis[END_REF] . Extended literature supports the idea that the activation of these two CXCR3 isoforms leads to distinct biological behaviors and clinical outcomes, which could be explained by very early changes in the two signaling pathways. With the objective of understanding the origin of such diverging responses in the signaling mediated by the two receptor isoforms, PWR experiments were performed to measure the affinity of ligands to each receptor isoform and associated conformational changes. The studies revealed that the affinity of an agonist to the two receptors was substantially different and most interesting that the conformational changes associated were completely different [START_REF] Boye | Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR)[END_REF] . This could explain why different signaling cascades are activated by ligand-binding to the two receptors, as recently reported [START_REF] Smith | C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways[END_REF] . PWR along with calcium-flux experiments were able to define a very narrow window of a receptor antagonist concentration that specifically inhibited the agonist binding and activation of the CXCR3-A isoform, while having negligible effect on the CXCR3-B isoform. This could have important pathological perspectives as the CXCR3-A isoform is ultimately triggering tumor progression, its specific inhibition relative to the B isoform (triggering tumor regression) is desirable. Moreover, this reveals the very high fine-tuning of GPCRs.

PWR AS A TOOL TO INVESTIGATE THE INTERACTION OF MEMBRANE ACTIVE

MOLECULES WITH LIPIDS

Besides GPCRs, our research group is focused on the understanding of the dynamics and the mechanisms of interaction between membrane active peptides and biological or biomimetic membranes.

We employ biophysical approaches as polarized ATR-FTIR, PMIRRAS to evaluate structural changes of peptides and proteins and lipid organization. PWR allows a very complementary description by bringing the kinetic aspect and determination of binding affinities. PWR has been applied to investigate the interaction of cell penetrating peptides with both model membranes and cell membrane fragments from cells expressing or not certain glycosaminoglycans allowing both the lipid and sugar role to be determined [START_REF] Alves | Cell biology meets biophysics to unveil the different mechanisms of penetratin internalization in cells[END_REF] .

For 10 years, a strong interest of the team has been on the relation between morphology and structure of amyloid peptides, and toxicity expressed by their deleterious effects on the biological membranes [START_REF] Berthelot | What does make an amyloid toxic: morphology, structure or interaction with membrane?[END_REF] . Our first study was to develop in the yeast Saccharomyces cerevisiae a model of amyloid toxicity based on toxic mutants of the HET-s prion of Podospora anserina. Our collaborators have created 40 mutants of varying toxicity. A reported highlight was that toxic species, deleterious for the membranes are small objects (oligomers), structured in anti-parallel -sheets while fibers structured in parallel -sheet, had in turn no disruptive effect on membrane models [START_REF] Berthelot | What does make an amyloid toxic: morphology, structure or interaction with membrane?[END_REF] . In this project, PWR made it possible not only to establish the kinetic interaction constants of the HET-s and mutant peptides with different model membranes, but above all to highlight the presence of different forms or sizes of aggregates interacting with these membranes [START_REF] Harte | Probing the kinetics of lipid membrane formation and the interaction of a nontoxic and a toxic amyloid with plasmon waveguide resonance[END_REF] . The addition of the toxic peptide to the lipid membrane lead to the appearance of additional resonances in the s-pol that was attributed to heterogeneity in peptide assembly with the presence of regions (domains) possessing different mass and organization. Taking into account the ability of amyloid peptides to oligomerize and to form fibers and plates the multiple resonances seen can correspond to different oligomeric states of the peptides.

Similarly, more recent work on the peptide A 1-42 involved in Alzheimer's disease by PWR allowed us to establish that the least toxic species (monomers) interact with the membrane but that the oligomeric species induce a reorganization of the lipids of the membrane. The interaction was decomposed into two phases with two different kinetic constants between the anchorage of the oligomers on the membrane and the induced reorganization of the lipids [START_REF] Henry | Interaction of Abeta(1-42) amyloids with lipids promotes "off-pathway" oligomerization and membrane damage[END_REF] .

CONCLUSIONS

PWR provides an interesting technique to characterize molecular interactions occurring in thin anisotropic films as lipid membranes and herein inserted membrane proteins (as GPCRs) or interacting molecules as membrane active peptides. For studies with model lipid membranes, other systems besides supported bilayers can be used (polymer cushioned, partially suspended, hybrid, etc) depending on the system under investigations. Moreover, immobilisation strategies (implicating chemical modification of the surface) allowing controlled and homogeneous orientation of the target molecule in the sensor surface can be applied.

As any other method, PWR has also its limitations. One limitation regards the distinction of the spectral contribution of each molecular partner in complex systems. For example, when a ligand binds and activates a receptor, with the current PWR setup and by measuring at a single wavelength it is impossible to determine if the spectral changes reflect conformational changes of the receptor alone or lipid reorganization contribution. To circumvent this limitation, there are two possibilities: 1) to fluorescently label one of the partners and to measure the PWR signal at two wavelengths. Both the anisotropy of the refractive index and that of the extinction coefficient (see for details [START_REF] Salamon | Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties[END_REF][START_REF] Alves | Introduction to Plasmonics Advances and Applications[END_REF] ) are determined allowing one to distinguish each molecular contribution [START_REF] Salamon | Optical anisotropy in lipid bilayer membranes: coupled plasmonwaveguide resonance measurements of molecular orientation, polarizability, and shape[END_REF] . 2) Another possibility that we are currently starting to explore concerns the expansion of PWR measurements from the visible into the IR-region. Since lipids and proteins have inherent and specific IR absorption bands, by measuring at appropriate wavelengths, specific molecular information can be obtained.

Another limitation concerns the lack of information regarding lipid membrane electrochemical properties. To follow changes in membrane electrochemical properties upon pore formation or opening of a channel could be highly interesting. Such development is important because it would: help establish the mechanism of action of membrane active peptides (e.g. pore formation); allow protein ionophore properties to be followed, allow the role of membrane potential in receptor activity to be determined; to follow electrochemically the activity of certain proteins as redox enzymes. While this has been pioneered by Tollin, that included an electrode in the sensor design PWR spectra quality is yet poor (depth loss) [START_REF] Salamon | Plasmon-waveguide resonance and impedance spectroscopy studies of the interaction between penetratin and supported lipid bilayer membranes[END_REF] . The design of a new sensor with improved PWR signals, while keeping optimal electrochemical potential, is currently under development.

FIGURES

  Study of G-protein coupled receptor signaling in membrane environment by plasmon waveguide resonance Isabel D. Alves* and Sophie Lecomte* CBMN UMR 5248 CNRS, U. of Bordeaux, Bat. B14 allée Geoffroy St. Hilaire, 33600 Pessac, France KEYWORDS: G-protein coupled receptors, plasmon waveguide resonance, lipid model membrane, ligand-receptor interactions, protein reconstitution INTRODUCTION Molecular interactions play a central role in the regulation of several physiological responses.

Figure 1 .

 1 Figure 1. PWR setup. On the left the optical and mechanical components, the incident

with the lipid bilayer, embedded receptor and ligand or effector binding. On the right bottom is a typical PWR spectra showing the total internal reflection angle (TIR), pand s-resonances.

AUTHOR INFORMATION

Corresponding Author *Corresponding authors: Isabel D. Alves (i.alves@cbmn.u-bordeaux.fr) and Sophie Lecomte (s.lecomte@cbmn.u-bordeaux.fr). Address: CBMN UMR 5248 CNRS, U. of Bordeaux, Bat B14, allée Geoffroy St. Hilaire, 33600 Pessac, France

Author Contributions

The manuscript was written through contributions of both authors. All authors have given approval to the final version of the manuscript.

BIOGRAPHIES