We attribute variations in observed G peak asymmetry to a combination of a ‘stiffening’ of the E2g optical phonon where the graphene is supported by the underlying MRR waveguide structure, as a result of this increased hole concentration, and a lowering of the degeneracy of the same mode as a result of localized out-of-plane ‘wrinkling’ (curvature effect), where the graphene is suspended. Examination of graphene integrated with two different MRR devices, one with radii of curvature r = 10 μm and the other with r = 20 μm, indicates that the device geometry has no measureable effect on the level of doping.We present a Raman mapping study of monolayer graphene G and 2D bands, after integration on silicon strip-waveguide-based micro-ring resonators (MRRs) to characterize the effects of the graphene transfer processes on its structural and optoelectronic properties. Analysis of the Raman G and 2D peak positions and relative intensities reveal that the graphene is electrically intrinsic where it is suspended over the MRR but is moderately hole-doped where it sits on top of the waveguide structure. This is suggestive of Fermi level ‘pinning’ at the graphene-silicon heterogeneous interface, and we estimate that the Fermi level shifts  In factreported , 9–11.−1 [−1 [−1 [q = 0) specifically by an electron-phonon DR scattering process, whereas the D peak requires highly localized electron-defect scattering to conserve momentum [Graphene exhibits three principal Raman scattering peaks, each with a distinct physical origin: the doubly resonant (DR) D peak appears around 1350 cm−1  and is r−1 [−1 , 12, andmomentum –16. The momentum , 13.Graphene integration with the silicon photonics platform is interesting from a number of device applications perspectives, e.g. for demonstrating enhanced biochemical sensors in which the graphene acts as a high affinity surface functional layer for adsorbed species that may be probed by evanescent optical fields in the underlying silicon photonics device. The two-dimensional nature of graphene also leads to an optoelectronic band structure, the charge filling of which can be tuned by very low power electrostatic gating. In this case, the ‘Pauli blocking’ effect can alter the opacity of the material to incoming photons, providing the possibility of very fast (GHz) optical modulation or switching, which is likely to be of use in telecoms applications. Previous reports –20 of thThe Si MRR devices in this study were fabricated in a commercial Si foundry  and comprise of strip-waveguides of 335-nm width, lithographically formed from commercial 220-nm silicon-on-insulator with a 2-μm-thick buried oxide layer. These waveguide dimensions, specifically, the relatively narrow waveguide width , were selected to ensure good modal overlap with surface integrated graphene, post-transfer. Two ‘racetrack’-type MRR devices are studied, one in which the radial component is 10 μm and the other is 20 μm and both having identical 20-μm-long linear sections. Prior to graphene transfer, the devices were washed with acetone, isopropyl alcohol (IPA), de-ionized water and resist stripper (NMP: 1-methyl-2-pyrrolidone). This was followed by an oxygen plasma etch (for 40 s) immediately before transfer. The graphene was grown by chemical vapour deposition (CVD) on copper foils  and then transferred onto the pre-cleaned waveguides using a polymer-mediated wet transfer procedure . The grar = 10 μm and 20 μm. The maps were obtained from a 120 × 120 point array with a step size between each point of 0.25 μm, and the precise frequency, intensity and width of the Raman G and 2D peaks were determined by fitting with Lorentzian line-shapes to the spectral peaks. From measurements of a single crystal silicon sample using the same instrument configuration , we estimate a spectral resolution from the bandwidth of the main Si scattering peak of 4.6 cm−1 or better.The Raman spectral mapping was performed at room temperature in back scattering configuration, using a Horiba LabRAM HR Evolution Spectrometer with a 600 g/mm grating. The scattering signal was collected confocally and detected with an integrated Peltier cooled charge coupled device (CCD) camera. The samples were excited by a 633-nm helium neon laser light, and mechanical movement of the sample during mapping was provided by a Marzhauser motorized microscope XYZ stage. The incident laser light was focused on the sample surface using a × 50 objective lens with numerical aperture of 0.75. In order to avoid laser heating, the laser power density on the sample was kept below 2 mW . Raman m−1. This combination of a relatively intense, symmetric 2D scattering peak and a G peak frequency close to the predicted value, ωG (n) = 1581.6 + 11/(1 + n1.6) where n is the layer number [r = 10 μm) is presented in Fig. −1, respectively) where the graphene sits on top of the MRR waveguide structure relative to where it is suspended.In order to check if we had transferred the single layer graphene, prior to the Raman mapping study, we also measured the single point Raman scattering signal, Fig. r number , confirmr number . The optωD2/∂ε) is approximately six times that of the G peak (∂ωG/∂ε) [n) and hole (p) doping, the frequency of the G peak always increases from the intrinsic value, meaning that a plot of G peak position with Fermi level is nearly symmetric about zero. However, for the 2D peak, whilst the frequency is up-shifted considerably for a moderate increase in p-doping level (~ 15 cm−1 for 3 × 1013 cm−2), it remains virtually unchanged from its intrinsic position up to an electron concentration of ~ 3 × 1013 cm−2, above which it down-shifts rapidly. This leads to a highly asymmetric curve for the 2D peak position with the Fermi level about zero. That we observe shifts that are both similar in magnitude and in the same direction for the G and 2D peaks strongly suggests that the graphene is moderately p-doped, where it sits on the waveguide, compared to where it is suspended. In order to quantify this effect, we used the following empirical relations , the strain-related shift of the 2D peak (∂∂ωG/∂ε) . That we∂ωG/∂ε) . For botω0(G) (=1580 cm−1 [ω0(2D) (=2640 cm−1 [ω(G) and ω(2D) are the G and 2D peak positions we have determined for each point in our maps and EF is the Fermi level in units of eV. In Fig. EF ~ 0) but that the hole concentration is increased  where the graphene sits atop the waveguide structure. A similar analysis of an MRR with radius r = 20 μm (not shown here) gave a very similar result, indicating that the effect is not dependent on the waveguide geometry, rather that it is purely a material-dependent (substrate) doping effect. The source of this doping is almost certainly the result of locally trapped, static ad-charges at the interface between the silicon/SiO2 and graphene. The density of these ad-charges is known to be increased in samples which have received more aggressive cleaning treatments (such as the O2 plasma etch we have employed) [where 580 cm−1 ) and ω0640 cm−1 ) are thecm−1 [ω0D (=2640 mployed) . AlthougRepresentative Raman scattering spectra (from the mapping) are shown in Fig. R2 = 0.993) by a single, symmetric Lorentzian line-shape, a signature of single layer graphene [The 2D peak is well described (graphene . We notegraphene .R2 > 0.995) by a double Lorentzian line-shape, indicative of two distinct scattering processes. We note that the width of the main (G+) peak decreases by ~  25%  going from the suspended graphene to where it is supported by the MRR waveguide structure. This is consistent with current understanding and prior observations of the ‘stiffening’ of the graphene E2g optical phonon, as a result of doping [−), responsible for the asymmetry, also exhibits a significant decrease in width of ~ 35%  going from the suspended graphene to where it is supported by the MRR waveguide structure. Asymmetry in the graphene Raman G peak has previously been attributed to highly localized charge inhomogeneity within the laser probe area [E2g optical mode can be split between phonons along the nanotube axis, −) scattering mode itself can be broadened and even split when nanotubes buckle and collapse under high pressure loading. We note from fitting the graphene G band spectra here that both the frequency difference ΔωG and the line-width of the G− mode  doping level, principally because of a quenching of the 2D mode with increasing carrier-phonon scattering [I2D/IG, from ~ 3 where the graphene was suspended to ~ 2.5 on the waveguide structure, we note that this change is small relative to the degree of G peak shift we observe, when compared with other reports [I2D/IG as a function G peak position, which appears to increase with excitation wavelength suggesting this alone may not be the most reliable indicator of absolute doping level, especially in the low doping limit.We also examined the ratio of peak intensities, attering , 32. How reports  for the  reports , there iAG/A2D, which takes account of the peak widths as well as the variations in peak heights can be used to obtain the carrier concentration directly from Eq.  ~ 0.069 when νf is taken to be the electron velocity, 1.17 × 108 cm/s. Our measurements indicate that where mined in  to be ~ ed in [2 , which in through Eq.  in a so-called vector decomposition plot, introduced by Lee et al. , Fig. 5.ωD2/∆ωG ~ 2.2) are very different for those associated with doping (∆ωD2/∆ωG ~ 0.7) [p-type doping vectors. With increasing tensile strain or p-doping, the ωG, ωD2 coordinate values will move from the origin , either along the doping-free (strain) or strain-free (p-doping) lines, respectively. The G-2D coordinate space is divided into four quadrants, Q1–Q4 by these strain and doping vectors, and so any significant deviation of the coordinate data from these lines, say into region Q1 (Q4), would indicate that the peak shifts are the result of a combination of compressive (tensile) strain and p-doping. Scattering of data within Q2 and Q3 is forbidden because both n- and p-doping manifests only in increases in the G peak position.Representing the data in this type of plot enables us to determine to what degree the peak shifts may be influenced by strain. This is based on the fact that the rates of variation in peak position ratios for strain (∆G ~ 0.7) . Any coop-doping) vector (dashed line) and doping-free (strain) vector (solid line), after [p-doping) vector for the ON-ring with the average ON-ring coordinate value (purple dot) being . The increased scatter for the ON-ring data along the strain-free line indicates a greater range of doping levels detected from the relative peak shifts, likely because of the uncertainty in probing a highly localized substrate doping effect produced by the underlying, sub-micron waveguide width, compared with the probe laser spot size (> 1 μm). Despite the apparent scatter in the data, into both Q4 and Q1, we discount any significant global strain effects because the average ON-ring coordinate lies so close to the strain-free line. We suggest that the peak shifts we observe are only due to silicon substrate induced hole-doping and the average ON-ring G-2D coordinate confirms this to be in the range of (2 to 3) × 1012 cm−2.We define the intrinsic, unstrained graphene peak frequency coordinate as the origin (red dot) , 26 and ), after . Data fo2 waveguide (substrate doping effect). The data for the suspended region reveals no measurable distinction from intrinsic graphene, but for the supported region, a maximum down-shift of the Fermi level of ~ 0.2 eV is determined, which corresponds to a peak hole concentration of ~ 3×1012 cm−2. An asymmetry in the Raman G peak, which varies according to whether the graphene is suspended or supported, indicates a combination of doping-induced ‘stiffening’ and lifting of the degeneracy of the E2g optical mode. These effects should be taken into account when graphene is combined with silicon photonics platforms, certainly when attempting to use such platforms to determine graphene’s characteristic properties and for optimization of future graphene-integrated silicon photonics devices, such as optical modulators and sensors.In summary, monolayer CVD graphene was integrated with silicon waveguide-based MRR photonic devices. Frequency shifts and integrated intensities of the characteristic graphene Raman G and 2D peaks were determined for mapped regions, and these indicate a Fermi level ‘pinning’ where the graphene sits on the Si MRR structure as a result of unintentional hole-doping from the underlying silicon/SiO


The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts. TDP-43 binds tightly the (UG)n motif4 and helps regulate several aspects of mRNA biogenesis including transcription, pre-mRNA splicing and export, and mRNA stability5. TDP-43 is one of the causative gene products of amyotrophic lateral sclerosis (ALS) that is an adult-onset neurodegenerative disorder characterised by progressive degeneration of upper and lower motor neurons The majority of TDP-43 mutations that cause ALS are found in the C-terminal region, although some are found in the RRMs10. ALS mutant TDP-43 proteins, including those with mutation of the RRMs have an increased half-life in cell models12.The trans-activating response region DNA-binding protein 43 (TDP-43) is a member of the family of heterogeneous nuclear ribonucleoproteins and contains two highly conserved RNA recognition motifs (RRMs) and a non-conserved C-terminal region that mediates protein-protein interactions13. TDP-43 is cleared from the nuclear compartment and is incorporated into cytoplasmic ubiquitinated and/or hyperphosphorylated inclusions in patients with familial or sporadic ALS and in patients with frontotemporal lobar degeneration16. Several animal models over-expressing wild type TDP-43 leads to neurodegeneration similar to that observed in ALS24, accumulate mitochondria in cytoplasmic inclusions in motor neurons23, lack mitochondria in motor axon terminals24, or have abnormal juxtanuclear aggregates of mitochondria in spinal motor neurons22, suggesting that wild type TDP-43 affects mitochondrial function. Supporting this, TDP-43 localises to mitochondria of NSC34 cells, a hybrid cell line that has several motor neuron features25. TDP-43–induced cytotoxicity also strongly correlates with mitochondrial functions, including respiratory capacity and respiratory chain activity, in yeast cells expressing human TDP-4326. In addition, TDP-43 co-localises with mitochondria in motor neurons, and ALS-associated mutants enhance its localisation in mitochondria28. The enhanced localisation of mutant TDP-43 to mitochondria also disrupts mitochondrial dynamics and function28. The mitochondrial fusion protein mitofusin 2 (Mfn2) has also been demonstrated to be involved in mitochondrial impairment, but the functional relationship between TDP-43 and Mfn2 is unknown27. In this regard, TDP-43 mutants also disrupt the vesicle-associated membrane protein-associated protein B (VAPB)– protein tyrosine phosphatase-interacting protein-51 (PTPIP51) interaction and cellular Ca2+ homeostasis, which disrupts mitochondria–endoplasmic reticulum interactions that are implicated in several physiological processes including ATP production, mitochondrial biogenesis, and apoptosis29; however, TDP-43 is involved only indirectly in these processes through its ability to activate glycogen synthase kinase-3β29. In addition, it was recently proposed that TDP-43 and its mutants impair the assembly of electron transfer complex I by binding to mitochondrial mRNAs encoding NADH-ubiquinone oxidoreductase chain 3 (ND3) and ND6 and inhibiting their translation28. Despite the obvious involvement of TDP-43 in mitochondrial function, the direct mitochondrial target of TDP-43 remains elusive.Although TDP-43 shuttles between the nucleus and the cytoplasm, it mainly resides in the nucleus under physiological conditions32. We established human Flp-InTM T-RExTM 293 (T-REx 293) cell line expressing doxycycline-inducible wild-type human TDP-43 carrying a triple affinity-purification tag  sequence replacements were made corresponding to a specific region of mt-tRNALeu  as well as mt-tRNALeu as a negative control , we first investigated the association of RNA with exogenously expressed TDP-43 using mass spectrometry (MS) and the Ariadne search engineAGE Fig. . The MS-nts Fig.  and are nts Fig. , and theion Fig. . To deteion Fig. , and sho-43 Fig. .Figure 1Asn, mt-tRNAGln, and mt-tRNAPro present within the mitochondria of T-REx 293 cells.To assess localisation of TDP-43 in mitochondria further, we treated a mitochondrial fraction with proteinase K and detected both endogenous and exogenous TDP-43; TDP-43 was not detected, however, if the mitochondrial membrane was permeabilised with Triton X-100 prior to proteinase K treatment Fig. . Further33. The L-strand encodes one oxidative phosphorylation system subunit (ND6) and eight mt-tRNAs , Tyr, Cys, Asn, Ala, and Gln)  to inhibit mtDNA transcription and first examined the effect of EtBr on stability of L-strand coded mt-RNAs in T-REx 293 cells. EtBr reduced the levels of all of the L-strand-encoded mt-tRNAs with time but it did not affect a genomic tRNAMet   and mt-12S rRNA by inhibiting mtDNA transcription with EtBr before and after doxycycline induction of TDP-43. Overexpression of TDP-43 slowed the degradation of mt-tRNAAsn and mt-tRNAGln , whereas it had only a negligible effect on the degradation of mt-12S rRNA  upon overexpression of TDP-43 cannot be attributed to stabilisation mediated by direct binding of TDP-43.In human mitochondria, large polycistronic RNA transcripts are generated from the L- and H-strand promoters of mtDNAGln Fig. . MoreoveRNA Fig. . These rMet, U1 snRNA, and 5.8S rRNA) as the references. Overexpression of TDP-43 significantly increased the cellular levels of all L-strand-encoded mt-tRNAs  , and mt-tRNALeu(UUR) appeared slightly larger than the mature transcripts upon overexpression of TDP-43  Fig. . In addi-43 Fig. . These dTyr (Y)-mt-tRNAAla (A), the region complementary to COXI mRNA (mirrorCOXI)-Y, mirrorND2-mt-tRNAGln (Q), and mirrorCYB-mt-tRNAGlu (E) to test for cleavage within these boundary regions. All boundaries were found to increase in abundance with time after doxycycline induction compared with GAPDH mRNA  and confirmed the direct binding of the precursor RNA to recombinant TDP-43 . The RT-PCR primers were designed to amplify the boundaries among mt-tRNAs or between mt-tRNAs and mt-mRNAs in the region containing mt-tRNARNA Fig. . In addi-43 Fig.  and the -43 Fig. . These rLeu(UUR)  Fig. , we alsoUR) Fig. . To deteUR) Fig.  and showUR) Fig. , W-COXII34. Therefore, accumulation of large precursor transcripts was also investigated by northern blotting after knockdown of MRPP3 or ELAC2 , and W-COXII (detected by probes 6–9) relative to 28S rRNA staining compared with a mock knockdown with a sequence-scrambled RNA -mediated cleavage of precursor molecules at the 5′ end, and tRNase Z (ELAC2)-mediated cleavage at the 3′ endAsn and mt-tRNAGln  did not inhibit cell proliferation  of this L-strand transcription unit, although we cannot exclude the possibility that the entire L-strand transcript is stabilised by the binding of TDP-43 because the region containing mt-tRNAPro is located in the first part of the L-strand mtDNA that is transcribed, and the region containing mt-tRNAAsn/mt-tRNAGln is located at the 3′ end. It is also likely that TDP-43 binds to regions of mt-transcripts other than the regions corresponding to the three mt-tRNAs because of its general RNA-binding properties2. We also propose that the increase in the L-strand transcript and/or its processing products induced by TDP-43 overexpression may stabilise all or part of the H-strand transcript and/or its processing products through complementary association, resulting in increased levels of these processing products  seems to be corresponded to RNA 19 that is increased in diseases associated with mt-tRNALeu(UUR) gene mutations39.In human mtDNA, the genes are located in three different transcription units (one in the L-strand and two in the H-strand) that are transcribed to different extents. The one H-strand transcription unit that includes only the mtcts Fig. . Rackham4, our findings provide important insight into the physiological roles of this negative feedback control and indicate that the strict control of TDP-43 expression is critical for maintaining mitochondrial homeostasis. In this context, mutations in TDP-43 that affect its stability or expression are expected to reduce mitochondrial function. Our present data provides a new mechanism underlying disruption of mitochondrial function by TDP-43 that differs from the mechanisms that involve perturbation of Mfn227, the VAPB–PTPIP51 interaction29, or translation of ND3 and ND628.Because TDP-43 strictly controls its own expression through a negative feedback loop in which TDP-43 binds to the 3′ untranslated region in its own mRNAg for 30 min at 4 °C, the supernatants were used as cell lysate for the following immunoprecipitation. Cell lysate (5 mg) prepared from 2 × 107 cells were incubated with 15 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich) for 2 h at 4 °C. The beads were washed five times with 1 ml of lysis buffer, and proteins and RNA were eluted with 150 µl of Protein-RNA extraction buffer , 10 mM EDTA, and 2% 2-mercaptoethanol) for 5 min. The eluted proteins and RNA were subjected to SDS-PAGE or denaturing (8 M) urea PAGE, respectively, as described previously42.Confluent cells were washed with PBS and suspended in five packed cell volumes of lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% (w/v) IGEPAL CA-630) containing 2 mM ribonucleoside-vanadyl complex, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin for 30 min on ice. After removal of any insoluble residue by centrifugation at 20,000 × 31. LC-MS analysis of RNA, and database search and interpretation of MS/MS spectra of RNA were performed as described41.In-gel RNA digestion was performed as described previously2. For mt-mRNAs analysis, the membranes were stained with methylene blue. Hybridizatioin with biotin-labeled DNA probes and detection of RNA signals were performed as described previously41. The oligonucleotides used as probes are shown in Supplementary Table Total RNA was isolated using the RNAgents Total RNA Isolation System (Promega). For analysis of mt-mRNAs, 5 µg of total RNA was subjected to 2% agarose/formaldehyde gel electrophoresis in 3-(N-morpholino)propanesulfonic acid running buffer. For analysis of mt-tRNAs, 1 µg of total RNA was subjected to denaturing (8 M) urea PAGE in Tris-borate-EDTA running buffer, stained with SYBR Gold (Invitrogen) for 10 min, and visualised using the LAS4000 Luminescent Image Analyzer System (Fujifilm). The separated total RNAs were transferred to a Hybond N + membrane . The membrane was dried and subsequently cross-linked using an ultraviolet cross linker  at 120 mJ/cm2, and DAP-TDP-43-associated complexes were immunoprecipitated with anti-FLAG M2 magnetic beads. After heating for 5 min at 65 °C in SDS sample buffer, DAP-TDP-43-associated complexes were released from the anti-FLAG M2 magnetic beads, and separated by SDS-PAGE using a Bis-Tris gel (Running gel: 360 mM Bis-Tis-HCl (pH 6.8), 8% acrylamide, 0.1% tetramethylethylenediamine, 1% ammonium persulfate; Stacking gel: 360 mM Bis-Tis-HCl (pH6.8), 5% acrylamide, 0.1% tetramethylethylenediamine, 1% ammonium persulfate) and Bis-Tris running buffer propanesulfonic acid, 5 mM EDTA, 0.5% SDS). The separated proteins were electrophoretically transferred to a Protran BA 85 Nitrocellulose membrane . The membrane was blocked with 5% non-fat dried skim milk in TBST for 30 min, washed with TBST, and then incubated with HRP-conjugated streptavidin  for 30 min. After washing three times with TBST for 10 min, the Chemiluminescent Nucleic Acid Detection Module kit (Thermo Fisher Scientific) was used for detecting biotin of DAP-TDP-43. Stained parts of the membrane were excised and incubated in 250 µl of Proteinase K buffer  at 37 °C for 30 min. After addition of 250 µl of Protein-RNA extraction buffer, RNA was isolated by phenol-chloroform extraction and isopropanol precipitation and subjected to northern blot analysis.T-REx 293 cells expressing DAP-TDP-43 were cross-linked by ultraviolet irradiation at 400 mJ/cmin vitro transcription of mt-tRNA(Asn), AC-stem(Leu), D-Loop(Leu), pAntiCdn(Leu), Var-R(Leu), T-Loop(Leu), and Leu(UUR) RNA were generated by annealing the primer sets shown in Supplementary table 2, respectively. In vitro transcription was performed with 0.2 pmol of the template DNA using CUGA® 7 in vitro transcription kit (NIPPON GENE) according to the manufacturer’s instructions. Transcripts were collected by isopropanol precipitation, and purified using reverse-phase liquid chromatography. To synthesize biotinylated mirrorCOXI-YCNA RNA, template DNA for in vitro transcription was prepared. We amplified DNA fragments corresponding to mtDNA between mirror mt-tRNAAsn to COX I with the primer set (mirrorANCY-COXI-for/ mirrorANCY-COXI-for) using KOD neo plus (TOYOBO), and inserted them into the BamHI/PstI site of the pSPT19 vector (Roche) (mirrorANCY-COXI pSPT19). mirrorANCY-COXI pSPT19 linearlized by digesting with SmaI was used as template DNA for in vitro transcription. Biotin-labeled mirrorCOXI-YCNA was in vitro transcribed from the DNA template (0.06 pmol) in the presence of 7.5 mM of ATP, 7.5 mM of CTP, 7.5 mM of UTP, 7.5 mM GTP, 0.5 mM of biotin-UTP (Roche) using CUGA® 7 in vitro transcription kit , and purified by 1% agarose gel electrophoresis and the subsequent gel extraction using Zymoclean Gel RNA Recovery Kit (ZYMO RESEARCH).The template DNAs for 2, 0.01% IGEPAL CA-630, 1 mM DTT, 10 µg of yeast tRNA (Ambion), 10 µg BSA, 10 ng of synthetic RNA, 200 ng of recombinant protein), and electrophoresed on a 6% non-denaturing polyacrylamide gel at 100 V for 50 min in 0.5× Tris borate/EDTA buffer (44.5 mM Tris-borate and 1 mM EDTA). The separated RNA-protein complexes were transferred to a Hybond N+ membrane that was dried and UV-crosslinked using the FS-1500 crosslinker (Funakoshi) at 120 mJ/cm2. The synthetic RNA was detected by northern blot analysis using the indicated probes.Each binding reaction was performed at room temperature for 20 min. Each reaction was performed in 10 µl binding buffer (40 mM Tris-HCl (pH 7.4), 30 mM KCl, 1 mM MgCl7 cells expressing DAP-TDP-43 was used for this assay. Synthetic RNA (sRNA) (1 pmol) was added to the total cell lysate and subjected to immunoprecipitation using anti-FLAG magnetic beads as described above. RNA isolated from DAP-TDP-43 complex was subjected to denaturing urea-PAGE and northern blot analysis.Total cell lysate from 1.0 × 102 dishes. Cells washed twice with PBS were sedimented (800 × g for 5 min at 4 °C), and resuspended in 9 ml of ice-cold Solution A . Cells were homogenized by passing ten times with a 25 G needle. The nuclei from this suspension were sedimented (800 × g for 10 min at 4 °C), and the centrifugation step was repeated for the supernatant once more. Mitochondria were sedimented from the supernatant by centrifugation , and washed once with 1 ml of Solution A. Protein concentration was measured using NanoDrop. For Proteinase K protection assay, the isolated mitochonaria was diluted into 1 mg/ml with Solution B . 100 µg of mitochondria was treated with the indicated concentration of Proteinase K  for 20 min on ice. Mitochondria were treated with 0.5% (w/v) of Triton X-100 along with the treatment of Proteinase K for a control of Proteinase K protection assay. Proteinase K reaction was stopped with the addition of 1 mM PMSF, and the mitochondria were centrifuged , resuspended with SDS sample buffer, and subjected to western blot analysis.Mitochondria were prepared from T-REx 293 cells or 293 T cells grown in 5 × 15 cm100 µg of Mitochondria isolated from 293 T cells were resuspended with 500 µl of 20 mM Hepes-KOH pH 7.4, and incubated for 20 min on ice. After sedimenting mitochondria by centrifugation , pellet were washed once with Solution B, resuspended with 50 µl of 20 mM Hepes-KOH pH 7.4, and disrupted twice for 30 sec using Bioruptor UCD-250 (Cosmo Bio). After sedimenting them by centrifugation , the supernatants were used as a mitoplast extraction. Protein concentration was measured using BCA assay.41. The eluted proteins and RNA were subjected to SDS-PAGE or northern blot analysis using 1.5% denaturing agarose gel. Biotinylated RNA was detected using HRP-conjugated Streptavidine.mtRNA degradation assay was performed at 25 °C for 5, 10 or 30 min. Each reaction was performed in 20 µl of reaction buffer (20 mM Tris-HCl (pH 7.4), 60 mM KCl, 12.5 mM MgCl2, 0.1 EDTA, 2 mM DTT, 10% Glycerol, 10 µg mitoplast extract, 10 fmol biotinylated mirrorCOXI-YCNA RNA, 600 ng TF-fused protein). Reaction was stopped by the addition of 150 µl Protein-RNA extraction buffer , 10 mM EDTA, and 2% 2-mercaptoethanol), and protein or RNA in the reaction was purified as described previouslySupplementary Information


Mage = 23.35; SDage = 4.93), leading to a nine-item scale (Study 1). Third, in Study 2, the internal factor structure, temporal stability, and concurrent validity were examined through a series of structural hypothetical modelisation in a sample of 129 male athletes . The results indicated that the scale has good psychometric properties. Specifically, the PCA, and the series of structural hypothetical modelisation, suggested two theoretical factors , and more, with a bi-factor model in a SEM. The results also indicated sufficient concurrent validity with the Male Body Dissatisfaction Scale (MBDS) and adequate internal consistency . The findings overall suggest good reliability and construct validity for this French version of the DMS (DMS-FR), which will be useful for future research and clinical practice in French-speaking countries.The purpose of this paper was to adapt and examine the psychometric properties of a French-language Drive for Muscularity Scale (DMS). First, a preliminary version of the scale, based on both the English-language version of the DMS and the literature on the drive for muscularity, was developed following a committee validation process. Second, the factor structure of the DMS-FR was investigated with principal component analysis (PCA) in a sample of 114 male athletes ( Social values have changed in the past few decades, and appearance issues now affect both men and women . StudiesSeveral questionnaires have been developed to measure DM or its associated constructs. Some scales assess DM, such as the Drive for Muscularity Scale (DMS) , the DriThe most extensively used questionnaire, with good levels of reliability and validity, is the DMS . In the I think about taking anabolic steroids") but other recent studies have found that this item loads onto the MB subscale [In line with the recommendations of McCreary et al. , some stsubscale .The two-factor model of DMS scores of the parent study has been confirmed by McCreary et al.  and suppEscoto et al.  first foA good factorial structure was found in the study of McCreary et al. , revealiThe DMS has been translated into several languages: Brazilian Portuguese , ItalianAnother research avenue concerns the construct validity of the scale. Despite its extensive use and the finding of Tod et al.  that theThis study is not just a transcultural validation of the DMS. In link with previous transcultural studies of the DMS and observed limitations, we have chosen to adapt a short version of the questionnaire with two subscales based on the English version of the DMS. The authors of the present study have removed some items to realize validation of a questionnaire only on two concepts: body dissatisfaction and muscularity. The purpose of this study was to adapt and validate a French-language measure of the drive for muscularity to establish a more theoretically based scale for future research. In the first step, a preliminary version of the DMS was developed in French; in the second step (Study 1), the factor structure of the DMS-FR was investigated using principal component analysis; and in the third step (Study 2), the internal factor structure was examined through a series of hypothetical equation modelisation and the temporal stability and concurrent validity were assessed.These three steps were conducted following the validation procedure of Vallerand  and MyerThe purpose of this step was to adapt a preliminary short version of the DMS in French and assess the clarity of its items.The development of the preliminary version followed the usual recommendations . Five stThe DMS scale was initially translated from English into French-language by the first and third authors, both of whom are fluent French-speakers. Two criteria guided the translation process: (a) conformity with the original questionnaire intentions and (b) clarity of the items in French.The two translated versions were compared and the two translators agreed on the preliminary version of the DSM-FR.Based on this preliminary version, a back-translation was performed by a translator unaffiliated with the study and with no prior knowledge of the original instruments. The first and third authors compared the original English version of the DSM, the back-translation, and the preliminary version of the DSM-FR and worked together to ensure a clear final French-language version, equivalent to the English one in terms of semantics and concept. The two other authors did a back-translation of this final preliminary version. The back-translation was satisfactory since the back-translated questionnaire was identical to the original English version.I wish that I were more muscular"; "I think that my arms are not muscular enough"; "I think that my chest is not muscular enough"; "I think that my legs are not muscular enough") and muscularity behaviors . Items related to other constructs such as self-efficacy  or subjective norms/social approval  were removed for conceptual clarity. After the committee validation process, the final preliminary version of the DSM-FR was composed of 10 items classed into two subscales: Body Dissatisfaction Muscularity (5 items) and Muscularity Behaviors (5 items).A bilingual review committee was set up with three sport and health psychology researchers and one PhD student (the four authors). The committee first worked on the 15 basic items of the English version of the DSM and selected items which were clearly related to muscularity body dissatisfaction  recruited through a social network. A 6-point Likert scale ranging from 1  to 6 (item quite clear) was chosen to remove the neutral option at the mid-point. Questionnaire completion was carried out under standardized conditions  and did not exceed more than 10 minutes. Then, meetings were held to elicit feedback on problems understanding the questionnaire items or questions about them .The clarity of the items of the preliminary version of the DMS-FR was assessed by 20 males ranging in age from 21 to 41 years . The clarity of each item was satisfactory for the two subscales of Muscularity Body Dissatisfaction and Muscularity Behaviors . A few minor changes were made on the basis of comments. The final 10 translated items of the DMS-FR are presented in The descriptive statistics related to clarity assessment showed that the preliminary version of the questionnaire was very clear for the participants  using principal component analysis (PCA).Mage = 23.35; SDage = 4.93) and had been practicing sport from 1 to 30 hours per week for 9.76 years on average (SDnb of year = 5.44). All were French-speaking athletes practicing mostly bodybuilding or strength training. In this sample, 25 male athletes were not competitors and 89 male athletes competed at different levels. All participants were from France or the French-speaking part of Switzerland. A coding system was adapted to ensure no duplicate data.The exploration of the factorial structure of the DMS-FR was conducted with 114 men, all of whom were eligible to participate in the study because they met the following criteria: (a) a minimum age of 16 years, (b) more than one hour of physical training per week, and (c) more than one year of sports experience. Participants were 18–48 years old  and that all responses would be used for research purposes only. Participation was entirely voluntary and full confidentiality was guaranteed. The authors collected information about nationality in order to ensure that the participants were French-speakers.At the beginning of the questionnaire, participants were first invited to give personal information before responding to the DMS-FR: their age and main sport .Drive for Muscularity Scale (DMS-FR): At this stage, the DSM-FR was composed of 10 items. Participants answered each item with a Likert scale ranging from 1  to 6 (absolutely).Parallel Analysis: The factorial structure was examined by principal axis factor analysis (Promax-type rotation). In order to extract the most appropriate factors, parallel analysis [analysis  was usedanalysis . The iteI lift weights to build up muscle"). The nine remaining items all showed item-total factorial weights greater than .40. Cronbach’s alphas were .87 for the Muscularity Body Dissatisfaction subscale and .85 for the Muscularity Behaviors subscale, demonstrating high levels of internal consistency of the subscales [Based on several indicators from this analysis, including the scree plot, eigenvalues, and proportion of variance, a two-factor solution accounting for 69.2% of the common variance was found. The first factor was labeled Muscularity Body Dissatisfaction and the second factor was labeled Muscularity Behaviors . The facubscales .The aim of study 1 was to explore the factorial structure of the DMS-FR. The PCA produced a two-factor model that replicated the original DMS subscales  and thosThe aim of study 2 was to examine the psychometric properties of the DMS-FR by conducting a series of structural hypothetical modelisation according the four steps of Myers et al. . We thenMage = 27.03; SDage = 7.81) and had been practicing sport from 1 to 20 hours per week for 7.76 years on average (SDnumber of years = 6.82). The participants were French and Swiss male athletes practicing mostly bodybuilding or strength training. In this sample, 91 male athletes were not competitors and 38 male athletes were competitors at different levels. Correlations between age, sport experience and the DMS did not show significant results. When considered as covariates, the results of the exploratory structural equation modelling (ESEM) did not change. This yielded no significant effect for these two variables. This sample size was considered appropriate according to the 1:10 subjects-to-variables ratio generally adopted in the literature for CFA or SEM sample size calculations [The 129 selected participants were 16–57 years old  from the total sample were contacted four weeks after responding to the first DMS-FR and agreed to complete it a second time under the same conditions.To test the temporal stability, 61 male athletes : At this stage, the DMS-FR consisted of nine items and two factors: Muscularity Body Dissatisfaction  and Muscularity Behaviors . The items were answered with a Likert scale from 1  to 6 (absolutely).Male Body Dissatisfaction Scale (MBDS): The MBDS [The MBDS  was valiThe MBDS . It is cFor the factorial validity, because of the significant multivariate non-normality of the data , the analysis were performed with the AMOS 7.0  using thp < .001; TLI = .99; CFI = .99; RMSEA = .01. Comparison of acceptable models  revealed that model d provided the best goodness of fit indices, as well as the lowest ECVI and AIC indices [The results are presented in  indices . Satisfar = .86, p < .01, and for Muscularity Body Dissatisfaction, and Muscularity Behaviors, the correlation coefficients were .83, p < .01 and r = .86, p < .01, respectively. Estimates of internal consistency  in Time 2 were .81 and .74 for Muscularity Body Dissatisfaction and Muscularity Behaviors, respectively.The DMS-FR demonstrated adequate test–retest reliability. The Pearson product-moment correlation coefficient between the two sets of total scores (Time 1 and Time 2) was r = .35, p < .01. The two subscales of the DMS-FR (Muscularity Body Dissatisfaction and Muscularity Behaviors) were significantly and positively correlated with the Muscularity Body Dissatisfaction subscale of the MBDS . The Muscularity Body Dissatisfaction subscale of the DMS-FR and the General Body Appearance Dissatisfaction subscale of the MBDS were negatively related in the expected directions . However, the General Body Appearance Dissatisfaction subscale of the MBDS and the Muscularity Behaviors subscale of the DMS were not significantly related  with a view to establishing a more theoretically based scale for future research. Two studies were carried out following the validation procedure of Vallerand . The valContrary to studies where participants’ age had a significant effect on the overall DMS and younger men are more likely to desire a more muscular body , age didOur results indicated a two-factor structure that mirrored the model proposed by McCreary et al.  and agreThe DMS has been previously used in many populations: males , male adThis study presents some limitations and points to directions for future research. First, the data of studies 1 and 2 were collected online, which may have limited participation to those with access to a computer, smartphone, and/or stable internet connection. Second, a typical limitation of research based on self-report measures is the potential for social desirability bias. Third, our sample included only male athletes, and it is thus now necessary to test this survey in non-athlete males to generalize our results. An interesting question is whether the DMS scores are multi- or one-dimensional. Only two previous transcultural studies have examined fit of the higher-order dimensionality of DMS scores and have found equivocal results. Nerini et al.  reportedThe drive for muscularity has been linked to significant physical and psychological problems in males . The avaTo conclude, the drive for muscularity is an important component of men's self-image. Theorists and researchers are currently at the beginning of the long path that will lead to a better understanding of the individual differences in men's body image perceptions and body-related behaviors. The DMS-FR can be used to measure the drive for muscularity in French-speaking male athletes and this should facilitate future research on the antecedents, consequences, and correlates of DM in this population. Greater insight into the drive for muscularity should also aid in the development of better prevention strategies for coaches and male athletes.S1 DatasetData used for the realization of the different analyses.(XLSX)Click here for additional data file.


Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in  Escherichia coli, Salmonella, and Shigella species.Type III secretion systems (T3SSs)Shigella species are the etiological agent of bacillary dysentery in humans  form a flat, elongated structure   .Yersinia YopN .We generated an x et al. . In our degraded B.A) , indicating deletion of the N terminus affects its ability to become secreted. However, secretion of IpaB, IpaC, and IpaD was unaffected in mxiCΔNterm; small differences between ΔmxiC/mxiC+ and mxiCΔNterm were not statistically significant (A and C). Thus, the ability of MxiC to prevent effector secretion and to promote inducible translocator secretion are not coupled, and only the former requires its secretion signal.As described previously, MxiCΔNterm is not secreted after Congo red (CR) induction A 23) an anA) , which are equivalent to the last 11 residues of SepL  ; translocators were only weakly induced, and effector proteins were leaked. In addition, MxiCΔCterm itself was only poorly secreted  even in ΔipaB . Therefore, unlike for SepL, MxiC's C terminus is essential.The 11 C-terminal residues of the EPEC MxiC homolog, SepL, are required for regulation of effector secretion but not for translocator secretion . As a coFig. S3) . The restop) to generate mxiCΔCBD, lacking residues 32–72. This construct was stably expressed, but it was leaked and only weakly secreted upon CR induction (A). Interestingly, secretion of MxiCΔCBD was enhanced in a ΔipaB background . This indicates the chaperone-binding domain influences the regulation of MxiC secretion rather than the intrinsic ability of the protein to become secreted. Furthermore, although mxiCΔCBD could not block effector secretion, it was able to promote translocator secretion (A).The chaperone-binding domain of YopN has a chaperone-independent role in secretion regulation . We thusnduction A. Intereecretion A.supplemental Fig. S5), we made several single and combined charge-swap mutations. mxiCK66E was unable to complement ΔmxiC. Paradoxically, the mutant protein was detected in culture supernatants and, to a lesser degree, in the supernatants after CR induction, but only low levels were detected in whole culture lysates (C). mxiC had a wild-type-like phenotype, except for some premature MxiC secretions. mxiC leaked MxiC at high levels and was unable to prevent effector secretion (data not shown). A similar phenotype was observed in mxiCK68E . Translocator IpaD and early effector IpgD were secreted at intermediate levels of ∼40–50%. Thus, either only a small proportion of intracellular MxiC is involved in blocking secretion and/or its secretion per se is not required to release this block.MxiC lacking its secretion signal is not secreted and is also unable to prevent effector secretion . In an mreleased , 16. ThuYersinia YopN needs its heterodimeric chaperone SycN/YscB for efficient secretion and regulation  and a triple deletion (ΔipgEΔipgAΔspa15). However, even the deletion of multiple chaperones had no effect on MxiC . This suggests that none of these chaperones influences MxiC function. Therefore, either there are no such chaperones binding to the CBD of MxiC, or we have not identified them because of low sequence similarities. However, we think the latter is unlikely because MxiC is primarily monomeric in cytosol, and no known class 1 chaperone was identified, even using mass spectrometry after cross-linking , suggesting it as a site for protein-protein interactions. Cherradi et al.  do not effect its function. We mutated residues Leu-222 and Leu-242 into serines and found that neither mxiCL222S and mxiCL242S nor the double mutant mxiC had any effect on MxiC stability or function (data not shown).Deane et al.  noticed i et al.  changed mxiC mutant was stably expressed (C) and able to induce translocator secretion after induction (A). However, mxiC was unable to prevent effector secretion (A and B). Thus, the hydrophobic patch is involved in preventing effector secretion.As change to a polar side chain may not have been enough to disrupt interactions in this area, we introduced two charges. The xpressed C and ablnduction A. Howeveecretion , A and Btop), is not conserved. However, it contains multiple serines, aspartates, valines, and threonines that are not classically helix favoring . We generated a model of the “bent” wild-type protein using the YopN/TyeA structure . In comparison with the crystal structure, the bent MxiC model is not only folded at the hinge region but undergoes a slight twisting motion. We then made three mutants as follows: one mutant introducing a proline  that would likely cause a break in helix 9 and thus a conformation similar to that of YopN/TyeA; a second mutant introducing three glycines  in the area to favor switching between both putative forms; and a third mutant that might stabilize a straight helix . MxiC Ile-251 is structurally equivalent to Phe-268 of YopN, which is sandwiched between hydrophobic residues in the core of TyeA, thus likely stabilizing the bent conformation.The sequence in MxiC helix 9, its putative “hinge” region , top, isfavoring . Multiplode 1XL3 ) as tempC). The triple glycine mutant mxiC behaved like ΔmxiC. MxiC was not secreted efficiently, and translocator secretion was reduced (43 ± 23% of the complemented strain), and effector proteins were leaked. Under the straight mutant mxiC, induced secretion of both IpaB and IpaC was reduced, whereas that of IpaD was as efficient as in the complemented strain or even increased (D). This mutant did not affect secretion of MxiC or effector proteins. The bent mutant mxiCV256P leaked high levels of a low molecular weight protein. We identified this band by mass spectrometry using a sample equivalent to the “−CR” sample in Fig. 5A. The top T3S-related protein identified was effector IpgB1 . The similarly sized effector/anti-activator OspD1 was not detected in this band. The equivalent band from a ΔmxiC “+CR” sample was analyzed in parallel; again, IpgB1 was the top T3S-related hit . These results suggest that a “straightened” MxiC favors the earliest step in the induction hierarchy, i.e. IpaD secretion, whereas a bent MxiC cannot prevent or favors, independently of its own secretion, the final one, i.e. effector release. In the wobbly mutant, three residues are altered that are initially identical to those changed in the straight one, but its phenotype is opposite, i.e. more like the bent one. This indicates that it is not the chemical nature of the amino acids in this surface patch that leads to these phenotypes but rather the stability of the secondary structure they form.All mutant proteins were expressed at wild-type levels or better C. The trncreased D. This mated hit . The mxiThe secretion patterns observed in the helix 9 mutants suggested movement in this area of MxiC is required for its function. We decided to examine the molecular conformation(s) of MxiC using double electron-electron resonance (DEER), also known as pulsed electron double resonance (PELDOR). This is an increasingly popular electron paramagnetic resonance (EPR) technique used to measure distances between paramagnetic spin labels on a nanometer scale .2VJ4 , we chose residues Ala-247 and Ser-290 to introduce paramagnetic centers into the molecule as the distance between these residues was predicted to be in the measurable range for both forms, and the difference in distance between both forms was predicted as significant enough to be detectable . To covalently couple the paramagnetic nitroxide label MTSL to these residues, Ala-247 and Ser-290 were mutated into cysteines. At the same time, the two endogenous cysteines Cys-184 and Cys-233 were mutated into alanine and serine, respectively, to finally generate a quadruple mutant mxiC or mxiC(Cys). Alternatively, complementary mutant MxiCs, with cysteines for labeling inserted at two sets of different but equally suitable locations, could not be purified in sufficient amounts (data not shown).Based on the MxiC structure (chain A of PDB entry 2VJ4 ) and themxiC(Cys) behaved like the complemented strain in a CR secretion assay . A His-tagged version of the cysteine mutant (His-MxiC(Cys)) was soluble when expressed in E. coli . We also combined the quadruple cysteine mutant with the previously generated bent (mxiCV256P) and straight ) mutants. However, neither His-MxiC nor His-MxiC were solubly expressed in E. coli (data not shown). Thus, we could only analyze the “wild-type” His-MxiC(Cys).The m sample (A). Using a previously obtained standard curve, the spin concentration was calculated to be 66 μm. The amount of residual unbound label was calculated to <1%; thus a spin labeling efficiency of close to 100% could be extracted. The spectrum also indicated the protein was folded; because of increased mobility in a disordered structure, an unfolded protein would result in a spectrum similar to that of the free label.MTSL was covalently coupled to His-MxiC(Cys) at residues 247 and 290. The protein was concentrated and mixed with deuterated glycerol for EPR/DEER experiments . The labeling efficiency was assessed using continuous wave room temperature EPR on a 35 μm sample A. Using B and C). Reliability of the two peaks comes from the high signal-to-noise F(t) trace. We simulated the expected distance distribution for the bent MxiC model (C) and the MxiC structure  using MMM . Although the modeled form factors all differ in their overall shapes, they all show distances in the 1.5–3-nm range, in line with the experimental setup. However, the simulated peak at 3 nm was not as prominent in the experimental data. We thus conclude that the experimental data are not consistent with the structures nor with the bent model. When modeling the possible observable distances in the structure and the bent model using the “all rotamers” function in MMM  , a protein that is not known to interact with MxiC (A), indicating Spa15 does not influence the rotational freedom of the labels in MxiC. Consistently, the DEER form factor and resulting distance distribution were also unchanged (data not shown). Thus, Spa15 does not affect MxiC's conformation in vitro.As EPR only detects signals from paramagnetic centers, one can add potential binding partners to the sample without affecting the signal as long as they are diamagnetic. To determine whether other T3S-related proteins influence MxiC's conformation, we mixed labeled His-MxiC(Cys) with a 10-fold molar excess of the proposed interaction partner IpaD  or Spa15ith MxiC . Spa15 aith MxiC A, indicaCsupplemental Fig. S11) changed the continuous wave (cw) spectrum obtained (C), indicating the conformational freedom of the spin-labeled side chains has been affected by IpaD-MxiC complex formation, either due to direct interaction with IpaD residues or to subsequent conformational changes in MxiC. To confirm IpaD was responsible for signal modification, we mixed MxiC with different concentrations of IpaD (D). We found that the larger the stoichiometric fold of IpaD versus MxiC, the bigger was the spectral change detected. This shows the signal modification is specific to IpaD addition and that, in the concentration range used, we can modify the ratio of the MxiC-IpaD complex versus MxiC alone in the equilibrium ensemble. We also diluted the 10:1 IpaD/MxiC mix 1:4 (E). This confirmed that in preventing the interaction between MxiC and IpaD by dilution, the MxiC alone signal is recovered, meaning the interaction is reversible.In contrast, addition of a 10-fold molar excess of purified His-IpaD  (B) or when added in the presence of a 10-fold molar excess of IpaD (C). Taken together, these data indicate that in solution only IpaD displays an affinity for MxiC. However, we found no evidence of a distance change between the two spin-labeled side chains by DEER upon addition of any protein partners (data not shown).Given the evidence that MxiC family proteins can interact directly with translocator chaperones , 36–38, 36–g. S11D) . Neitherg. S11D) B or when of IpaD C. Taken mxiC mutants that leak effectors do so because MxiC's function(s) in blocking effector secretion is compromised or because they cannot retain MxiC intracellularly. We examined this by combining them with a mutation that prevents MxiC secretion, mxiHK69A.To dissect at which point they are deregulated, we wanted to understand whether our mxiHK69A mutant in the needle protein does not secrete effector proteins (mxiHK69A mutant lacking MxiC (ΔmxiHΔmxiC/mxiHK69A) can secrete effectors  is affecting function should continue to leak effectors even if MxiC is retained inside bacteria. For instance, the hydrophobic core mxiCF206S mutant . We focused solely on MxiC mutants displaying premature secretion of effectors and MxiC leakage. For completeness, we also included mxiC, which carries mutations in a negatively charged surface patch (top), secretes translocators and MxiC prematurely, and is defective in IpaD secretion upon activation , and its inducible secretion was also slightly reduced compared with wild type but of a similar level to that of ΔmxiCΔmxiH/mxiCmxiHK69A (top panels). Indeed, slightly fewer functionally mature T3SSs are assembled in ΔmxiH/mxiHK69A relative to wild type (mxiCΔmxiH/mxiCmxiHK69A could not leak or induce secretion of the effectors IpaA or IpgD nor of MxiC (bottom panels), respectively. However, it secreted slightly reduced amounts of the translocators inducibly, as observed previously . We then examined how each mxiC mutant behaved in an mxiHK69A background. We focused on leakage in the absence of induction because activation of MxiC secretion is blocked in an mxiHK69A background but effector secretion might be allowed by a functionally defective MxiC.We were unable to detect leakage from Δild type . Howevereviously . In ΔmximxiCK68E was unable to leak or induce effector secretion in the presence of mxiHK69A but not of mxiH. This indicates it is the only mutant where MxiC would be functional in blocking effector secretion, if it were not secreted prematurely. All other mutants leak effectors similarly whether mxiH or mxiHK69A is present, indicating that in these mutants MxiC's function(s) in blocking effector secretion are affected, either directly as in mxiC or indirectly, via premature secretion of translocators as in mxiC.Only i.e. regulation of translocator, MxiC, or effector secretion. This indicates the three regulatory functions of MxiC are interlinked. Therefore, the mutants were classified according to where their main initial defect lies, assuming the regulatory steps occur in this order.Our mutants can be organized into classes according to their phenotypes . HowevermxiC, mxiCΔCterm, which we therefore placed in class 0. However, unlike mxiCΔCterm, mxiCΔNterm and mxiCΔCBD were still largely able to induce translocator, but not MxiC, secretion, suggesting they are not full loss-of-function mutants (class 1a). Mutant mxiCV256P also could not secrete MxiC well inducibly or in a ΔipaB background, suggesting it has some defect in MxiC secretion. This mutant leaks effectors, but as it can still stimulate secretion of translocators, it is also not a complete loss-of-function mutant (class 1a). Both classes of mutants readily by-passed the block on effector secretion imposed by mxiHK69A, although none secreted MxiC efficiently in this background, indicating they are unable to establish all or some of MxiC's regulatory functions.Only one mutant showed such severe loss-of-function as to resemble ΔmxiCK66E and mxiCK68E are phenotypically similar in that they secrete MxiC prematurely and hence show reduced translocator induction and increased effector leakage. However, MxiCK68E is secreted much more efficiently than MxiCK66E. Furthermore, among all mutants, only MxiCK68E does not leak effectors in an mxiHK69A background. This indicates that the mutations in MxiCK68E and MxiCK66E, despite their proximity and chemical similarity, lead to different defects. K68E (class 1c) primarily deregulates MxiC secretion, leading it and effectors to be secreted prematurely. In addition to some premature secretion, K66E (class 1b) seems to confer more fundamental defect(s) leading to effector leakage. Finally, the only defect seen in mxiC is effector leakage (class 1d).Class 2 mutants are primarily, if oppositely, affected in translocator release. MxiC cannot induce IpaD secretion but leaks IpaB, IpaC, itself, and effectors , althougi.e. mxiCΔCterm and mxiCΔNterm, but still lead to full or partial null phenotypes, respectively.Using the mutant classes, we conclude that several physiological features of MxiC are regulated as follows: 1) its secretion, because several mutants up- or down-regulate it; 2) hierarchical secretion of translocators and effectors, and even of proteins within those two categories; and 3) the ability to switch from translocator to effector secretion. There are also uncharacterized fundamental properties of MxiC as indicated by mutations that most likely do not affect its overall structure, et al.  might localize the YopN-TyeA complex to the secretion apparatus. Ferracci et al. , these regions may also be involved in binding it to and releasing it from that site.MxiC's secretion signal and putative CBD are dispensable for stimulation of translocator secretion, which indicate domains 1–3 alone mediate this function. Multiple residues in the C-terminal helix of MxiC are highly conserved. Schubot et al.  suggestei et al.  found Tyi et al.  of MxiC i et al. . In contecretion , 33. Howi et al.  were oveV mutant  also led ) or not at all ). Thus, the action of MxiC on translocators and effectors can be uncoupled. MxiC acts as if it had already received the activation signal  leaks IpaB and IpaC and is unable to induce IpaD secretion  instead displays reduced induction of IpaB and IpaC, although IpaD secretion is slightly increased. These opposite effects correlate with the mutations being on opposite faces of MxiC (top). These faces are also the ones that may undergo a conformational change from flat to bent . Furthermore, the predicted bent mutant (mxiCV256P) and the predicted straight mutant show opposite phenotypes; although the former leaks effectors and is only weakly affected in translocator secretion, the latter shows differential induction of translocators. Thus, reduced secretion of the repressor IpaD could affect secretion of the hydrophobic translocators. Alternatively or additionally, MxiCV256P was only poorly secretable and could be affected in more fundamental functions. Although we cannot exclude that these mutations mainly affect MxiC interactions by altering its surface, the type of mutations, the fact that the mutations are directly adjacent, and their opposite phenotypes suggest that we have modified MxiC's ability to undergo conformational changes.Even hydrophilic h et al. ). Intere of MxiC , top. Th to bent . In factmxiC lies on the face of the molecule that is flat in MxiC but concave in YopN/TyeA and the E. coli homolog SepL , or both. The latter is supported by the work of Archuleta and Spiller  and IpaD were mixed have two possible explanations as follows: (i) the increased molecular weight of the complex(es), which is reflected in slower overall rotational correlation times; (ii) a direct interaction between the spin-labeled probes and residues in IpaD. Although the reason of the spectral effects observed cannot be clarified, it corroborates the notion that complex formation between MxiC and IpaD occurs. From Fig. 7ipaDL99P and mxiC (A), with the negatively charged patch of MxiC  at their interface, yielding an overall elongated form. The interacting surface is relatively flat and contains several charged residues on the edges and stacked tyrosine residues in the center giving an interface formed by two charge-complementary surfaces (B). This model is consistent with the data of Lee et al.  ), we buiK,E293K) A, with tsurfaces B. This me et al. , who fouiCE201K,E6K,E293K mxiCΔNterm mutation in the constitutive secretor background ΔipaB, we confirmed that this mutant is unsecretable. Similar to the N-terminal secretion signal, the putative chaperone-binding domain is required for efficient MxiC secretion. However, this domain did not affect secretability. Thus, MxiC secretion is merely deregulated in mxiCΔCBD. Interestingly, two different mutations in the CBD of MxiC led to increased MxiC leakage, mxiCK68E and mxiCK66E. Taken together, these data suggest that the putative chaperone-binding domain positively regulates MxiC secretion.By analyzing the ipaB background. This is reminiscent of the lack of requirement of MxiC for translocator secretion in the same background. Similar to the repression of translocator secretion  has gained the ability to activate its own secretion  is transmitted to the cytoplasm, most likely through the needle.The mechanism preventing premature translocator secretion is counteracted by MxiC. Secretion of IpaD requires the negatively charged patch and probably a flat conformation of the molecule. Removal of IpaD may initiate a conformational change in MxiC, which bends to release the IpgC chaperone and its bound translocators allowing these to become secreted in turn. IpaB and secreted IpaC form a pore, connected to IpaD at the needle tip and also inserted in the host cell membrane, which transmits “Signal 2” to the cytoplasm. Reception of this signal may allow finalization of a conformational change in MxiC that releases it for secretion. Then, early effectors are secreted. Moreover, as both negative regulators are now removed, the secretion rate is enhanced , possiblmxiC, we dissected at which stages of the regulatory cascade MxiC acts, including which of its functional steps can be uncoupled. This also allowed us to determine how the different steps are connected. Finally, we provide the first evidence that a directionally defined conformational change in MxiC is involved in controlling the secretion hierarchy. The conservation of the components involved indicates the importance of this regulatory pathway to T3SS-carrying bacterial pathogens of animals.By using structure- and sequence-based mutagenesis of supplemental Tables S1 and S2 list the strains and plasmids used. S. flexneri strains were grown in Trypticase Soy Broth (BD Biosciences) at 37 °C with appropriate antibiotics the following final concentrations: ampicillin 100 μg ml−1, kanamycin 50 μg ml−1, tetracycline 5 μg ml−1, and chloramphenicol 10 μg ml−1. IPTG was used at the final concentrations indicated in the figure legends. The supplemental Table S3 lists the primers used.The Plasmids were generated as described below and verified by sequencing.mxiC) was generated by amplifying mxiC from the Shigella virulence plasmid pWR100 using primers mxiC_SacI and mxiC_BamHI. The purified PCR product was digested with SacI and BamHI and cloned into pWSK29* . The resulting PCR products were digested with SacI and BamHI. Fragments mxiCΔNterm and mxiC(1–341) were cloned into pACT3 digested with SacI/BamHI yielding pDR60 and pDR73, respectively.To express the terminal mxiCΔCBD, an internal deletion mutant that was created by two-step PCR and lacks residues 32–72. The mxiC gene was amplified from pIMA208 using primer pairs mxiC_SacI/mxiC_D32–72_R and mxiC_D32–72_F/mxiC_BamHI. The obtained PCR fragments were used as template for the second PCR step using primers mxiC_SacI and mxiC_BamHI. The PCR product was purified, digested with SacI/BamHI, and ligated into pACT3 digested with the same enzymes.pDR80 contains mxiCK66E was generated by two-step PCR. First, mxiC was amplified from pIMA208 using primers mxiC_SacI and mxiC_K66E_R and/or mxiC_K66E_F and mxiC_BamHI, respectively. These fragments were combined and reamplified using mxiC_SacI and mxiC_BamHI. The PCR products were purified, digested with SacI and BamHI, and cloned into pACT3 digested with the same enzymes yielding pDR96. pDR100 contains mxiCK68E cloned via SacI/BamHI, equivalent to amplification of mutant mxiC using primers mxiC_SacI and mxiC_BamHI. This mutant was obtained by chance and retained as the mutation was in a relevant area of the molecule.Mutant mxiC was generated by two-step PCR. pIMA212 was used as template for reactions with primer pairs mxiC_SacI/mxiC_M226K_R and mxiC_L242D_F/mxiC_BamHI. The obtained fragments were combined and reamplified using primers mxiC_SacI and mxiC_BamHI. The product was purified, digested with SacI and BamHI, and cloned into pACT3 digested with the same enzymes, yielding pDR72.Double mutant mxiC(Cys) (C184A (TGT to GCA), C233S (TGT to TCT), A247C (GCA to TGT), and S290C (AGT to TGT)) required for EPR experiments was synthesized by MWG Eurofins and supplied as pEXA-mxiC_EPR2. For pDR104 (pET28bmxiC(Cys)), mxiC(Cys) was amplified from the pEX-A-mxiC_EPR2 using primers mxiC_NdeI_F/mxiC_EcoRI_R. The PCR product was purified and digested with NdeI/EcoRI before cloning into pET28b (Novagen).Mutants in helix 9 were generated by two-step PCR; first, mxiC was amplified from pIMA208 using primer pairs mxiC_SacI/mxiC_V256P_R, mxiC_V256P_F/mxiC_BamHI, mxiC_SacI/mxiC_wobble_R, mxiC_wobble_F/mxiC_BamHI, mxiC_SacI/mxiC_straight_R2, and mxiC_straight_F2/mxiC_BamHI. The obtained PCR fragments were used as template for the second PCR using primers mxiC_SacI and mxiC_BamHI. The products were purified and digested with SacI/BamHI before ligation into pACT3 digested with the same enzymes. The resulting plasmids were named pDR91 (pACT3mxiCV256P), pDR92), and pDR93), respectively. The background quadruple cysteine mutant gene Shigella wild type yielding ΔipgA, ΔipgE, and Δspa15, respectively. For ΔipgE Δspa15 (abbreviation of ΔipgE::FRT Δspa15::kan) and ΔipgE ΔipgA (abbreviation of ΔipgE::FRT ΔipgA::kan), the kanamycin cassette was removed in ΔipgE by FLP-mediated recombination using the introduced FRT sites yielding ΔipgE::FRT. The same fragments used for the deletion of spa15 and ipgA in the wild-type background were now used for λ Red recombination in ΔipgE::FRT. This step was of low efficiency as the recombination preferentially occurred at the FRT scar at the ΔipgE site and not up- and downstream of spa15 and ipgA, respectively. Colonies were prescreened by colony PCR using primers annealing ∼200–300 bp upstream and downstream of the original chaperone gene, seeking to obtain a PCR fragment with a size compatible with the insertion of the kanamycin cassette versus the chaperone gene. ΔipgE ΔipgA Δspa15 (abbreviation of ΔipgE::FRT ΔipgA::FRT Δspa15::kan) was generated by removing the kanamycin cassette in ΔipgE ΔipgA by FLP recombination yielding ΔipgE::FRT ΔipgA::FRT and subsequent λ Red recombination using the ipgA::kan PCR fragment. Again, this step was of low efficiency as now two FRT sites were available on the virulence plasmid and colonies were prescreened by colony PCR as described for the double knockouts. All insertions and cassette removals were verified by sequencing.Deletions of the chaperone genes were performed using the method of Datsenko and Wanner . A kanammxiC plasmids detailed above were then combined with plasmids carrying mxiHK69A or wild-type mxiH as control in ΔmxiCΔmxiH  were mixed with 4× Laemmli sample buffer (“whole culture lysates”). Samples from equivalent cell numbers (∼3 × 106 cfu) were separated by 10% SDS-PAGE and immunoblotted.Whole cultures of S. flexneri strains grown to A600 ∼1 were collected by centrifugation at 15,900 × g for 10 min at 4 °C. The supernatants were mixed with 4× Laemmli sample buffer, normalized for equivalent cell numbers, separated on 10% SDS-PAGE, and visualized by silver staining or immunoblotting. On silver-stained SDS-PAGE, labels indicate the position of proteins as determined by mass spectrometry or from deletion strains.Shigella culture leads to a burst of Ipa protein secretion called “induction”  was added to a final concentration of 200 μg ml−1. After incubation at 37 °C for 15 min, samples were centrifuged at 15,900 × g for 10 min at 4 °C. 20 μl of the supernatants denatured in Laemmli sample buffer were subjected to 10% SDS-PAGE and visualized by silver staining or immunoblotting.CR, a small amphipathic dye molecule, is an artificial inducer of T3S. Its addition to a duction” , 48. S. et al. (Proteins were transferred onto Immobilon FL (Millipore) membrane using a semi-dry method. Primary antibodies used were as follows: anti-IpaA mouse monoclonal, gift from Kirsten Niebuhr ; anti-Ipet al. ; anti-Ipet al. ; and antet al. . Near-inShigella were grown to A600 ∼1 and resuspended in PBS to an A600 of 15. The suspension was brought to 37 °C in a water bath before CR was added at a final concentration of 200 μg ml−1, and the cultures were incubated for 8 min. For secreted proteins, samples were centrifuged at 15,000 × g for 10 min at 4 °C, and supernatants were denatured in Laemmli sample buffer. For whole cultures, the bacterial suspension was directly denatured in sample buffer. To calculate the secreted percentage of each protein, we compared protein amounts in supernatants and whole cultures by Western blotting. Undiluted and 1:4 diluted supernatant was compared with a dilution series of the whole cultures. Near-infrared fluorescent secondary antibodies were quantified on a LI-COR Odyssey imaging system. A linear fit of the dilution series allowed us to determine the concentration of protein in the supernatant in comparison with the whole culture.Wild-type 2VJ4  and thet et al. ) were uset al.  were extracted so that only a single polypeptide chain was present in each PDB file. This was necessary as the labeled residues (247 and 290) are in close proximity to the other polypeptide chains in the original PDB files. The neighboring chains thus also influence the rotamer modeling; however, MxiC is most likely monomeric in solution C322S was amplified from pUC18 ipaDC322S , and cloned into pET15b (Novagen) via NdeI/BamHI. The changes to the protocol for each protein are detailed in supplemental Table S4.Each protein was purified first by nickel affinity chromatography and then by size-exclusion chromatography. The protocols for these purifications were based on articles herewith: Spa15 ; IpgC/IppaDC322S , using psupplemental Table S4). Bacteria were grown to an A600 of ∼0.6 before cooling to 20 °C and induction with a final concentration of 1 mm IPTG. Cultures were then left shaking at 20 °C overnight.All protein concentrations, measured with a Nanodrop Lite spectrophotometer (Thermo Scientific), were adjusted using molecular weight, extinction coefficient (as obtained from the ExPASy server) of the protein, and consideration of the path length. Prior to each protein purification, the appropriate bacterial strain was streaked out on an Lysogeny Broth (LB) agar plate containing the appropriate antibiotics and grown overnight at 37 °C. The next day small LB cultures were made overnight. In the morning, cultures were made with overnight cultures (g for 15 min), washed in 10 ml of PBS (3500 × g for 5 min), and resuspended with ∼30 ml of Binding buffer with protease inhibitors . Bacteria were lysed by sonication (Sonics Vibra CellTM) at amplitude 60%, pulse 1-s on and 1-s off, time of 30 s, and variable cycle numbers .Cells were harvested by centrifugation (5000 × g at 4 °C. Supernatants were filtered through a 0.45-μm and then 0.22-μm syringe filter (Sartorius) and applied to a 5-ml HisTrap FF column  equilibrated in binding buffer  using a peristaltic pump . The entire supernatant was passed over the column three times at 2.5–5 ml/min at 4 °C. The column was then connected to an ÄKTA  and washed with 10–15 column volumes of Binding buffer, followed by elution buffer . Elution of bound proteins was carried out in the presence of protease inhibitors, with an imidazole gradient from 20 mm to 1 m  to 3 ml/min for 25 min. 5-ml fractions were collected. Peak fractions potentially containing proteins of interest had their concentration determined at A280 and were examined using Coomassie-stained SDS-polyacrylamide gels. The appropriate fractions were pooled and concentrated using Amicon Ultra spin concentrator .Lysates were clarified by centrifugation for 30 min at 20,000 × m Tris-HCl, pH 7.5, 100 mm NaCl, and 100–250-μl samples were applied, and the column was run at 0.5 ml/min collecting 1-ml fractions. Location and purity of proteins of interest were verified by Coomassie-stained SDS-PAGE. After the final gel filtration step, all samples were concentrated as above  and flash-frozen in aliquots in liquid nitrogen and stored at −80 °C until use.These concentrated fractions were run on a Superdex75 10/30 size exclusion chromatography column  equilibrated in 20 mm freshly made DTT (Sigma) was added to MxiC(C184A/C233S/A247C/S290C) to reduce the SH of the cysteines before concentration and application on the gel filtration column. The fractions containing His-MxiC(C184A/C233S/A247C/S290C) (∼2.12 mg/ml) were pooled, and the spin label MTSL  was added at 2-fold molar excess (∼121 μm). This means one molecule of MTSL was added per cysteine in the sample. After incubation for overnight at 4 °C in the dark, the sample was concentrated to ∼1.842 mm (∼70 mg/ml) as determined by the A280 using Amicon Ultra-4 spin concentrators .1 mQ cavity. The samples were thawed on ice, and 20 μl were transferred into a 1.5-mm outer diameter glass capillary. A 14-mT field sweep was performed, with 0.15 mT modulation amplitude, 7.96 milliwatt incident microwave power, and ∼9.38 GHz frequency. As the measured signal is the first derivative, the resulting curve has to be integrated to obtain the absorbance spectrum. Double integration yields the spin concentration as horizontal asymptote. The area under the absorbance spectrum is proportional to the spin concentration. The correlation factor was experimentally determined with a solution of known concentration of tempol in water.To determine the labeling efficiency of His-MxiC(Cys) with MTSL, continuous wave EPR spectra were detected at room temperature on an E500 Elexsys Bruker spectrometer equipped with a super high Q-band DEER experiments were performed as described in Polyhach et al. (Q-band DEER experiments are described in Bordignon and Polyhach (V(t) = V(0)) are background corrected to obtain the DEER form factor (F(t) = F(0)) by division by the background function (which is mainly due to intermolecular interactions). The form factor oscillates around a horizontal line at (1 − Δ) (Δ is the modulation depth) after background correction. The distance distribution is extracted from the form factor using DeerAnalysis2013 , and contained a final concentration of ∼75 μm MxiC and 10-fold more of the other proteins (∼750 μm).To analyze the interaction between MxiC and other proteins, we performed continuous wave X-band EPR spectroscopy at room temperature to detect eventual changes in the dynamic properties of the two spin labels on MxiC upon complex formation. Each EPR sample was prepared in gel filtration buffer (20 mA. D. R., E. B., R. B. S., I. M. A., and A. J. B. conceived and designed the experiments; A. D. R., E. B., S. M., D. K. S., X. L., M. P., I. M., R. B. S., and A. J. B. performed the experiments; A. D. R., E. B., I. M. A., R. B. S., and A. J. B. analyzed the data; X. L. and M. P. generated reagents and materials; and A. D. R., E. B., I. M. A., R. B. S., and A. J. B. contributed to the writing of the manuscript. All authors approved the final version of the manuscript.