To examine the outcome of cervical lateral mass screw fixation focusing on analysis of the risk factors for screw-related complications.Ninety-four patients who underwent posterior cervical fixation with a total of 457 lateral mass screws were included in the study. The lateral mass screws were placed using a modified Magerl method. Computed tomographic (CT) images were taken in the early postoperative period in all patients, and the screw trajectory angle was measured on both axial and sagittal plane images.In the postoperative CT analysis for the screw trajectory, 56.5 % of the screws were directed within the acceptable range . As intraoperative screw-associated complications, 9.6 % of the screws were found to contact with or breach the vertebral artery foramen. In this group, the screw trajectory angle on axial plane was significantly lower than in the group without contact. Facet violation was observed in 13 screws (2.8 %). This complication was associated with a significantly lower trajectory angles in the sagittal plane, predominantly at C6 level (69.2 %). In the patient chart review, no serious neurovascular injuries were documented.In the analysis of potential risk factors for violation of the VA foramen as well as FV during screw insertion, the former incidence was significantly related to the screw trajectory angle  in the axial plane, while the latter incidence was related to a poor screw trajectory angle in the sagittal plane. Posterior cervical fixation is a commonly selected procedure in the surgical management of the unstable cervical spine caused by trauma, and other morbidities such as degenerative disorders, neoplasms, rheumatoid arthritis, and destructive spondyloarthopthy. For fixation, the application of wire between the spinal processes was the first technique described in the literature . SubsequAmong those techniques, the pedicle screw is predominantly used in Japan. Previous biomechanical experiments performed for this fixation method have shown its superior strength compared to other techniques , and favAnother option for posterior cervical fixation is the use of a screw applied to the lateral mass as an internal fixation device . Roy-Camille et al.  initiallHowever, there are complications in the use of lateral mass screwing, and VA, facet violation (FV), nerve root injuries, and lateral mass fracture are listed as potential intraoperative complications associated with screw insertion . Among tIn this study, the screw trajectory angle was evaluated on CT images taken in the early postoperative period. We hypothesized that there is a correlation between the screw trajectory angle and the potential risk for screw-related complications. The purpose of the present study was to review our clinical experience with this fixation technique, focusing on the analysis of risk factors for complications associated with cervical lateral mass screwing.The design of this study is a retrospective clinical review of our patient population who underwent cervical lateral mass screw fixation. One hundred and seven patients underwent this procedure at our institute from 2000 to 2010. Among these patients, CT images and clinical records in the early postoperative period were available in 94 patients (49 men and 45 women) and this patient population constituted the basis of the study. Screw placement and trajectory were assessed on the CT images taken within 3 weeks, while clinical findings indicating screw-related complications were reviewed in the patient’s chart within 1 month after surgery. The screws inserted at C7 were excluded from the analysis, since the pedicle screw was the device of choice at this level. In addition, C1 and C2 levels were not included in the analysis since lateral mass screw fixation was not applied to these levels in our practice during the study period. In total, 457 lateral mass screws were used and subject to analysis. The screws were inserted using the modified Magerl method. The average age at surgery was 56.8 year (range 15–86 years). Detailed information of the patient demographics such as body weight, height, and BMI is presented in Table During surgery, the patient is placed in the prone position after endotracheal intubation. Alignment of the cervical spine is maintained at neutral during the procedure using the three-pin skull fixation. We only use fluoroscopy to confirm the alignment of the cervical spine preoperatively. A standard midline posterior approach to the cervical spine is used. Posterior elements are fully exposed, extending to the lateral edges of the lateral mass and the facet joint at each fusion level. The facet joints to be fused are decorticated, while care is taken to protect the facet joint above and below the instrumented levels. The entry point was located 1 mm medial to the mid-point of the lateral mass. An awl was used to create the starting hole. The angle of screw trajectory was directed approximately 30° laterally and superiorly , which is a modification of Magerl’s proposal. Since the superolateral quadrant is regarded as the “safe zone” , this rePatients are instructed to wear a semi-rigid collar or a soft collar for at least 8 weeks after surgery except for patients with cerebral palsy. For patients with cerebral palsy, we apply a Halo-vest for at least 8 weeks followed by additional use of a semi-rigid collar for an additional 4 weeks.For all the included patients, CT examinations were performed within 3 weeks after the surgery. The screw trajectory angle was measured on both axial and sagittal planes following the method described by Seybold et al. . Among tThe spatial relationship between the screw and the VA foramen was evaluated on axial plane CT images. Based on the location of the screw tip in relation to the edge of the VA foramen, the screws subjected to the analysis were divided into two groups as follows. When the screws were shown to contact with or violate the VA foramen these screws were classified as contacting and allocated to the contact group. By contrast, the screws without any contact with the edge of the foramen were classified as not contacting and allocated to the non-contact group. The presence of FV was also assessed on CT images. Based on the location of the screw in relation to the facet joint, screws were divided into two groups (non-FV and FV groups). In addition, occurrence of intraopertive lateral mass fracture was assessed on the postoperative CT images.Regarding occurrence of intraoperative VA injury, the operative record was reviewed for description of profuse arterial bleeding. Description of any symptoms and signs indicating neurovascular injuries such as sensory or motor deficit, and visual impairment was reviewed in the patient chart. The data collection based on the chart review was limited to the descriptions during the initial month after the index surgery.To evaluate the potential risk of these complications, the length and trajectory angle of each screw were measured on the CT images and compared between the groups with and without the complications.P < 0.05).All numerical results were presented as mean ± SD. Differences between the groups were compared using the unpaired t-test and Fisher`s exact probability test. Differences between each of the fixation levels were analyzed with one-way ANOVA followed by Fisher`s PLSD test. A difference was accepted as significant if the probability was less than 5 % (P = 0.8081).A total of 457 lateral mass screws comprising 89 at C3, 140 at C4, 138 at C5, and 90 screws at C6 level. The mean screw length at each level was 16.2 ± 1.7 mm at C3, 16.4 ± 1.5 mm at C4, 16.3 ± 1.7 mm at C5 and 16.2 ± 1.7 mm at C6. There was no significant difference in screw length among the levels . When the axial trajectory angle in the contact group was compared among the levels, no difference was detected. In the comparison of the sagittal projection angle and screw length between the contact and non-contact groups, no significant difference was observed.Analysis of the axial image at each level revealed contact of the screw with the edge of the VA foramen in 8.5 % 39 of 457) of the screws. Moreover, 5 screws (1.1 %) were observed to violate the edge of the foramen 9 of 457 . When the sagittal projection angle in the contact group was compared among the levels, no difference was detected. In the comparison of the axial projection angle and screw length between the FV  and non-FV groups , no difference was demonstrated. With regard to the incidence of this complication among cervical levels, 69.2 % of the facet violation was detected at the C6 level with a significantly higher incidence compared to the other levels (P < 0.0001). No significant correlation between the occurrences of violation of the VA foramen and the FV was detected.In the analysis of the CT images, FV were identified for 13 screws Fig. . The scrIntraoperative lateral mass fractures were identified in 18 of the 471 lateral masses (3.8 %). When this complication was encountered during surgery, the screw was reinserted with a different trajectory angle in four lateral masses. In the remaining cases, screw reinsertion at the same level was deemed impossible and the corresponding site was skipped for screwing. Therefore, the relationship between the occurrence of this complication and the screw trajectory was not analyzed in this group.No description indicating intraoperative injury to neurovascular structures was identified in the review of the patients’ chart. For early postoperative complications, surgical site infection and postoperative C5 root palsy occurred in 1 case (1.7 %) and 5 cases (5.3 %), respectively. Revision of the lateral mass screw was not required in any case.In the present study, CT examination during the early postoperative period enabled accurate analysis of direction and depth of the screw on both axial and sagittal planes. Consequently, several findings of interest were demonstrated showing the relationship between the screw trajectory and screw-related complications.In our clinical experiences, the precision of lateral mass screwing during fluoroscopic images was not high, because the rate of acceptable trajectory angle  was only 56.5 % (258/457 screws). Heller et al.  assessedThere remains a potential risk of VA injury in this technique, and two conclusions of note can be drawn. First, although the actual incidence of major vascular injury is very low (no cases in this series), close contact or even violation of the VA foramen by the screw can be present with considerable incidence (9.6 % in this series). This situation raises the possibility of vascular injury. Second, the low axial screw trajectory  was strongly correlated with this potential complication. In a cadaveric study, Seybold et al.  reportedFV is another screw-related complication reported in the literature, and this complication was encountered in 13 screws (2.8 %) in this series. Heller et al.  reportedIn the review of patient charts, no serious complications such as neurovascular injuries, persistent postoperative palsy, or deep infection necessitating screw removal were documented. These results correspond to the majority of previous studies –22. AmonThe strength of this study is the analysis of CT images obtained in the early postoperative period. Therefore, accurate assessment of the direction and location of screws as well as detection of intraoperative screw-related complications could be made. By contrast, the limitation of this study is that only the screw trajectory and length are analyzed to find the relationship between the rates of screw-related complications, while other factors such as morphologic characteristics also can influence the incidence. In addition, regarding the analysis of vascular complication, the collection of clinical data was solely based on the description of the operative record and the clinical findings in the patient’s chart, and special diagnostic modalities such as angiography and color-coded duplex sonography were not adopted. It has been also reported that the majority of intraoperative VA injuries are asymptomatic, and clinical manifestation can be delayed by several weeks in some cases . TherefoAlthough the present study showed the safety of our screwing procedure was acceptable, there were a considerable number of screws inserted with less optimal placement and trajectory. Whether use of intraoperative navigation or fluoroscopic control is able to improve the surgical consistency and reduce the incidence of the screw-related complication has yet to be examined. Moreover, complications such as FV and lateral mass fracture can affect the mechanical properties of the fixation, and thus further investigation for the subsequent clinical outcome is still required to substantially evaluate the safety and efficacy of our screwing procedure.The present study indicates that lateral mass screw fixation without intraoperative fluoroscopic images can be performed without serious complications. In the analysis of potential risk factors for violation of the VA foramen as well as FV during screw insertion, the former incidence was significantly related to the screw trajectory angle  in the axial plane, while the latter incidence was related to a poor screw trajectory angle in the sagittal plane. Moreover, the incidence of FV was highest at the C6 level. An understanding of these findings may help reduce the complication rate associated with cervical lateral mass screwing.


Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573. Insulin and insulin-like growth factor (IGF)-1 mediate their metabolic and mitogenic effects on target tissues through activation of the IGF-1/insulin receptor. The activated receptors function as tyrosine kinases that phosphorylate proteins including the insulin receptor substrate proteins (IRS)-1 and -2 14-3-3 proteins are highly conserved and expressed in a wide range of eukaryotes. The high level of functional conservation of the 14-3-3 proteins is indicated by the finding that the isoforms from yeast, plant and mammals are functionally interchangeable The abundance of 14-3-3 proteins and their broad range of interaction partners explain why they are key regulators of various intracellular processes, such as neuronal development, cell cycle control, cell growth control, apoptosis and control of gene transcription. Recently, IRS-2 was identified by a differential 14-3-3 affinity capture approach as novel 14-3-3 binding protein regulated by IGF-1/insulin signaling To identify the serine/threonine residues of IRS-2 that are involved in the IGF-1/insulin signaling cascade GFP-IRS2 encoding mouse IRS-2 was expressed transiently in HEK293 cells and cells were either left unstimulated or stimulated with IGF-1 for 30 min. Following purification, SDS-PAGE and tryptic digestion of IRS-2 protein bands liquid chromatography mass spectrometry (LC-MS) was applied to identify phosphorylated peptides. Mass spectrometric fragmentation pattern were used to localize the phosphorylation sites. The sequence coverage was 92% and a total of 24 phosphorylated serine/threonine residues were identified. According to IRS-2 mouse numbering phosphorylations were detected at the residues Ser-66, Ser-303, Ser-305, Ser-347, Ser-385, Ser-388, Thr-401, Thr-517, Ser-556, Ser-573, Ser-675, Ser-722, Ser-727, Ser-728, Ser-762, Ser-907, Ser-968, Ser-977, Ser-999, Ser-1089, Ser-1151, Ser-1165, Ser-1190 and Ser-1266 XpS/pTXP with the sequences surrounding the identified phosphorylated residues it became apparent that potential 14-3-3 binding motifs frequently appeared in an amino acid area between the PTB (phosphotyrosine binding) and the KRLB (kinase regulatory loop binding) domain on IRS-2 stretching from amino acid position 300–600. In -2 forms . The ove-2 forms . A stron-2 forms . This frHaving localized the 14-3-3 binding region to amino acids 301–600 we focused on the phosphorylated serine/threonine residues that were identified by mass spectrometry and corresponded to a 14-3-3 binding motif as shown in Aligning of IRS-2 protein sequences from mammalian and amphibian species showed that serine 573 and the flanking motif is highly conserved . Sequenc573) conformed to a potential consensus site for phosphorylation by Akt/PKB . To test whether Akt/PKB was involved in phosphorylation of serine 573, Flp-In HEK293 cells stably expressing GFP-IRS2 were incubated with an Akt/PKB inhibitor (Akti-1/2) before IGF-1 stimulation. The compound Akti-1/2 reduced the phosphorylation of Thr-308 of Akt/PKB and subsequently the IGF-1-stimulated phosphorylation of serine 573  cells were treated with IGF-1 or insulin for 30 min. Insulin stimulation led to a strong interaction of 14-3-3 with IRS-2 and increased Akt/PKB phosphorylation at Thr-308 . StimulaTo investigate if the phosphorylation of serine 573 had an influence on downstream signaling of the IGF-1/insulin signaling cascade, the phosphorylation of Thr-308 and Ser-473 of Akt/PKB in HEK293 cells transfected transiently with GFP-IRS2 or GFP-IRS2-S573A was assessed after incubation with IGF-1 for 5, 15, 30, 60, 120 and 240 min. IRS-2 wild type expressing cells showed induction of phosphorylation of Thr-308 and Ser-473 after 5 min of IGF-1 stimulation and this stimulation was decreased after 120 min . In contThe results of the overlay assay using alanine mutants of the phosphorylation sites between amino acids 301–600, identified in the mass spectrometric approach, did not indicate a second site relevant for the 14-3-3 binding to IRS-2 . HoweverIRS-1 and -2 as intracellular signaling nodes contain numerous serine and threonine residues that can be modified by protein kinases and protein phosphatases. For IRS-1 this has been shown extensively Less is known about individual serine/threonine phosphorylation sites on IRS-2, the interaction of 14-3-3 with IRS-2 and the influence on cellular metabolism. The IRS-2 interaction with 14-3-3 proteins has been documented in large-scale proteomic studies, but only as one of many interaction partners without any further characterization in vivo. In addition, we present here a pattern of 24 phospho-sites of IRS-2 after IGF-1 stimulation and the IGF-1/insulin-regulated 14-3-3 binding region on IRS-2 involving the novel IRS-2 serine phosphorylation site 573. We showed that upon insulin or IGF-1 stimulation 14-3-3 associated with IRS-2 in transiently transfected HEK293 cells, in Flp-In HEK293 cells that stably expressed IRS-2 and in Fao cells, which expressed IRS-2 endogenously. In addition, we could provide evidence that the IGF-1/insulin-dependent activation of the PI 3-kinase-Akt/PKB pathway was necessary for the IGF-1/insulin-stimulated phosphorylation of serine 573 and the 14-3-3 binding of IRS-2. Our data suggest that serine 573 potentially is involved in the negative regulation of IGF-1/insulin signaling, since the 573 alanine mutant led to increased Akt/PKB signaling. In mice liver 14-3-3 associated with IRS-2 upon insulin stimulation and refeeding, thus indicating the physiological occurrence of this interaction.This interaction was verified here in different cell culture models and in the liver of mice 573SLTT) do not completely conform to a 14-3-3 binding motif with the lack of proline at position +2 relative to the phosphorylated serine being the most prominent feature to be different. However, a proline in positin +2 is found in only 50% of the target sites as has been reported by Johnson et al We used the IRS-2 mouse sequence to identify novel phosphorylation sites on IRS-2 and the mass spectrometry approach revealed 24 phosphorylated serine/threonine residues. These sites seem to be conserved in human IRS-2 since all sites could also be found in the human IRS-2 protein sequence. In addition, certain sites could be found in the corresponding human and mouse protein sequences of IRS-1. Phosphorylation of serine 675 and serine 907 after insulin stimulation has already been investigated and described by our group We focused on the characterization of serine 573 since a physiological importance of serine 573 is suggested by the fact that sequence alignments of different mammalian species showed complete sequence accordance, and even an amphibian sequence showed partial accordance. We could observe phosphorylation of serine 573 of mouse IRS-2 with insulin and IGF-1 and also phosphorylation of the corresponding residue serine 577 in human IRS-2. A time course of serine 573 phosphorylation in Fao cells showed peak phosphorylation levels after 60, 90 and 120 min of insulin treatment. Moreover, mutation of this site to alanine displayed clear effects on IGF-1/insulin signal transduction.IRS-1 and IRS-2 display a similar architecture and partially overlapping functions, therefore the IRS-1 sequence was screened for a homologue position corresponding to serine 573 of IRS-2. Giraud et al. described the homologue serine 522 in IRS-1 The data of the overlay assay indicated that only one phosphorylated residue on IRS-2 was sufficient to trigger IGF-1/insulin-dependent 14-3-3 interaction with IRS-2. This would be in line with several other studies which also identified single phosphorylated residues as 14-3-3 interaction site How can the persuasive result of the overlay assay be explained? A possibility is partial renaturation of the IRS-2 protein on the nitrocellulose membrane. The renaturation process probably was effective enough to enable binding of 14-3-3 to the phospho-serine 573 motif, whereas the second binding site might not have been easily accessible and full renaturation of the IRS-2 protein would have been required. This may have led to abrogation of 14-3-3 binding in the S573A mutant in the overlay assay, despite the presence of a second binding site.The 14-3-3 protein family shows high overall sequence conservation, in the central binding groove the residues are strictly conserved How could the interaction of IRS-2 with 14-3-3 proteins influence downstream insulin signaling? The rigid structure of the 14-3-3 proteins led to the hypothesis that 14-3-3 proteins provide themselves as an anvil whereon the target protein can be restructured/reshaped in vivo opens several novel perspectives in the (patho)physiological regulation of the biological function of IRS-2 including its role in metabolic disorders such as insulin resistance and type 2 diabetes.In summary, we demonstrate the identification of 24 phosphorylation sites including novel sites on IRS-2 and mapped the IGF-1/insulin-dependent 14-3-3 binding region. Serine 573 as novel phosphorylation site was shown to be part of IGF-1/insulin-dependent interaction between 14-3-3 and IRS-2 and its phosphorylation may have an inhibitory role in IGF-1/insulin signaling. The evidence of the interaction between 14-3-3 proteins and the signal transducer IRS-2 The animal experiments were conducted in accordance with the national guidelines of laboratory animal care and were approved by the local governmental commission for animal research .Flp-In HEK293 cells were from Invitrogen , Fao and HEK293 cells from The European Collection of Cell Cultures . Insulin, IGF-1 and phosphatase inhibitors  were obtained from Sigma , wortmannin and Akt/PKB inhibitor (Akti-1/2) from Calbiochem . IRS-2 polyclonal protein antibody (06–506) was from Millipore , Akt/PKB protein antibody (610861) from BD Transduction Laboratories , GFP (green fluorescent protein) polyclonal protein antibody (8334), 14-3-3 (C-17) (732) and 14-3-3 K-19 (629) from Santa Cruz  and phosphospecific Thr-308-Akt/PKB (9275) as well as Ser-473-Akt/PKB (9271) from Cell Signaling . Recombinant human 14-3-3ζ-HRP was from R&D Systems . Trypsin gold, mass spectrometry grade was from Promega , 4–12% Bis-Tris mini gels and Colloidal Blue Staining Kit were from Invitrogen . Protease Inhibitor Cocktail Tablets were from Roche and the Protein Assay was from BioRad . Protein A Sepharose, Sepharose 4B and GST (glutathione S-transferase) antibody (27-4577-01) were from GE Healthcare Europe . GFP-Trap® was obtained from Chromotek .2-atmosphere with 95% humidity at 37°C. HEK293 and Flp-In HEK293 cells were cultivated in DMEM with 4.5 g/l glucose, supplemented with 10% FBS, 1% glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Fao cells were cultivated in RPMI 1640 supplemented with 10% FBS, 1% glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Flp-In HEK293 and HEK293 cells were transfected either transiently with polyethylenimine or stably with the Calcium Phosphate method All cell lines were kept in a 5% CO2, 1 mM EDTA, 10% glycerol, 1% Triton-X-100, set to pH 7.5, containing phosphatase inhibitors . Lysates were cleared by centrifugation for 5 min at 4°C with 16000 g. IRS-2 was immunoprecipitated with 2 µg IRS-2 antibody coupled to Protein A Sepharose for 4 hours at 4°C. 14-3-3 was immunoprecipitated by mixing lysates with 2 µg 14-3-3 antibody for 3 hours and adding 30 µl Protein G Sepharose for another hour. The Sepharose pellet was washed two times with lysis buffer and resuspended in 5x Laemmli . Proteins were separated on either 7.5% or 5–15% SDS gels followed by transfer onto nitrocellulose membranes using semi-dry Western blot. Western blotting and enhanced chemiluminescence was described previously Cells were lysed with lysis buffer consisting of 50 mM HEPES, 150 mM NaCl, 1.5 mM MgClwww.dnaseq.co.uk). The plasmid construct for human IRS-2 was from Calum Sutherland (University of Dundee).All plasmids to express GFP tagged proteins were generated at the University of Dundee (UK). All PCR and mutagenesis reactions were carried out using KOD Hot Start DNA polymerase (Novagen). The coding region for mouse IRS-2 (NM_001081212.1) was amplified from a pRK5 IRS-2 vector  using primers 5′-gaggatccatggctagcgcgcccctgcctg-3′ and 5′-ctgcggccgctcgagtcactctttcacgactgtggcttccttc-3′, cloned into vector pSC-B (Stratagene) and sequenced. IRS-2 was sub-cloned from this plasmid into pcDNA5/FRT/TO-GFP as a BamH1/Not1 insert to give vector pcDNA5/FRT/TO-GFP-IRS2 in which IRS-2 is tagged with GFP at the N-terminus. For truncated IRS-2 versions containing the IRS-2 sequence from amino acid 1–300 and 1–600 a stop codon was created after position 300  or 600  respectively. For the constructs 301–1321 and 601–1321 a BamHI restriction site was created prior positions 301 and 601 and the obtained inserts were ligated into pcDNA5/FRT/TO-GFP. Construct 301–600 was generated by creating a stop codon after position 600 and pcDNA5/FRT/TO-GFP-IRS2-301–1321 served as template. pcDNA5/FRT/TO-GFP-IRS2 served as template for the point mutation of serine 573 to alanine  and for the point mutation of serine 556 to alanine . pcDNA5/FRT/TO-GFP-IRS2-S556A served as template for the generation of double mutant S556A/S573A . DNA sequencing was performed by The Sequencing Service, College of Life Sciences, University of Dundee  and twice with no salt buffer , the pellet was resuspended in 5x Laemmli buffer. Proteins were separated on 5–15% gradient gels and transferred onto nitrocellulose membranes. For Far Western blot membranes were blocked in 5% milk powder in TBS-T  for 1 hour at room temperature. Incubation of the membrane overnight at 4°C with recombinant human 14-3-3ζ-HRP was followed by washing with TBS-T and visualization using ECL. Incubation of membranes with 14-3-3ζ-HRP will be referred to as overlay assay.All centrifugation steps were carried out at 4°C for 2000 E. coli and protein expression was induced by 1 mM IPTG  for 5 hours. Bacterial cells were harvested at 4°C by centrifugation with 4500 g for 15 min and disrupted by applying 40 strokes with a probe-type sonicator. After adding Triton-X-100 to a final concentration of 1% the suspension was centrifuged at 4500 g for 20 min at 4°C. GST-14-3-3 proteins were coupled to 50% slurry of glutathione Sepharose 4B. The amount of GST-14-3-3 was determined by SDS-PAGE followed by Coomassie staining. Lysates were incubated with 2 µg GST-14-3-3 for 2 hours in lysis buffer at 4°C. Beads were washed three times with lysis buffer, resuspended in 5x Laemmli and denaturated at 95°C for 5 min.GST-14-3-3β and GST-14-3-3ε constructs were a kind gift from Angelika Hausser (University of Stuttgart). The GST-14-3-3 plasmids were propagated in m-maleimidobenzoic acid N-hydroxysuccinimide ester) according to the method of Harlow and Lane The peptide CLRKRTYS*LTTPAR  was used to generate a phosphospecific antibody against phosphorylated residue 573 of mouse IRS-2. The phosphopeptide was conjugated to KLH (keyhole limpet haemocyanin) and BSA using MBS  containing a Protease Inhibitor Cocktail tablet. Protein determination was followed by immunoprecipitation of GFP proteins from 10 mg of total protein using 15 µl GFP Trap® and separation on precast 4–12% Bis-Tris gels. Staining overnight using the Colloidal Blue Staining Kit followed cutting of IRS-2 bands and transfer into 1.5 ml reaction tubes. Gel pieces were washed sequentially with ultrapure H18 guard column (0.1×20 mm) equilibrated in 0.1% formic acid/water at 5 µl/min and then separated on a 0.075×150 mm PepMap C18 column, equilibrated in 0.1% formic acid/water . Peptides were eluted with a 100 min discontinuous gradient of acetonitrile/0.1% formic acid at a flow rate of 300 nl/min. The column outlet was connected to a Thermo Fisher Nanospray 1 source fitted with a New Objective FS360 20-10 uncoated emitter and a voltage of 1.3 kV was applied to the emitter. The orbitrap was set to analyse the survey scans (m/z 350–2000) at 60.000 resolution and top 5 ions in each duty cycle (minimum ion intensity of 50000 cps), were selected for MSMS in the LTQ linear ion trap with multistage activation. Ions were excluded for 30 s after 2 occurrences. The raw files were converted into mascot generic files using Raw2msm . The MSMS spectra (mgf file) were searched against SwissProt database using the Mascot search engine (Matrix Science) run on an in-house server using the following criteria: peptide tolerance = 10 ppm; trypsin as the enzyme; carboxyamidomethylation of cysteine as a fixed modification with oxidation of methionine and phosphorylation of serine, threonine and tyrosine as a variable modification. Any MSMS spectra that could be assigned to a phosphopeptide was inspected manually using QualBrowser software. IRS-2 tryptic digests were also analysed by LC-MS with precursor of 79 scanning on a 4000 Q-Trap system  as described previously IRS-2 tryptic digests were analysed by LC-MS (liquid chromatography mass spectrometry) on a LTQ-orbitrap classic mass spectrometer system  coupled to a Proxeon Easy-LC HPLC system. The peptide mixtures were loaded onto a nanoseparations Cg for 10 min at 4°C. All procedures were approved by the local Animal Care and Use committee.Male C57Bl/6 mice were from Charles River , maintained at a 12 hours dark/light cycle under standard chow. Liver was homogenized at 4°C in lysis buffer . Homogenates were cleared after 30 min of solubilization on ice by 3 centrifugation steps at 16 000 Data are presented as means ± SEM. Unpaired student’s t-test was employed and a result was considered significant if p<0.05.Table S1Phosphopeptides detected by mass spectrometry that are common to IRS-2 isolated from serum starved and IGF1 treated cells. GFP-IRS2 was expressed transiently in HEK293 cells and cells were either left unstimulated or stimulated with IGF-1 for 30 minutes. Using GFP-Trap® IRS-2 was purified from total cell lysate and after SDS-PAGE and Coomassie staining, the bands corresponding to IRS-2 were cut and peptides were digested using trypsin . A mass accuracy of 10 ppm was used in database searches and phosphosites were identified by MS/MS analysis and manual inspection of the spectra. The sequence coverage was 92%. The phosphorylated residues are indicated with pS or pT.(DOC)Click here for additional data file.Table S2Phosphopeptides detected by mass spectrometry that are unique to IRS-2 isolated from IGF-1-treated cells. Samples were prepared as in (DOC)Click here for additional data file.


The author hypothesized that multiple sclerosis (MS) is a humoral autoimmune disease, caused by faulty interplay between myelin-specific, dimeric IgE, specifically competing non-IgE antibodies and IgE-triggered degranulating mast cells. The principal fault was believed to be insufficient quantity of protective, specific non-IgE antibodies. Also conjectured was the possibility of an unexpected and adverse immune suppression caused by none-MS pharmaceuticals being consumed by patients for their MS or for other conditions. To test both hypotheses, a mimotopic, peptide antigen-based, serum immunoassay was developed to measure dimer-bound IgE excess among MS patients, wherein the IgE specifically complexes with two or more myelin surface epitopes at an interval of 40–100 Angstroms, a separation critical for mast cell degranulation and cell damaging effect. MS test sensitivity and specificity, when analyzing five previously untreated patients for dimeric IgE presence, was 100%. In direct comparison, twenty age- and gender-matched female and male control subjects were test negative. Analysis of 35 multiple sclerosis patients, who were concomitantly being treated with potentially immunosuppressive pharmaceuticals, appeared to show the substances' negative effect upon MS causation, progression, or specific immunoassay performance. Therefore, MS is likely an autoimmune disease caused by IgE-mediated mast cell degranulation possibly in conjunction with immunosuppressive agents. While it is commonly understood that multiple sclerosis (MS) is an autoimmune disease of multifactorial etiology, the exact mechanism of causation has not yet been elucidated. Consequently, disease-specific MS therapy has not advanced beyond the use of interferons and immunosuppressive agents, the application of which is now more than thirty-year old , 2 and oBased upon his early work  and the To test the hypothesis, a mimotopic, peptide antigen-based, serum immunoassay was developed to measure dimer-bound IgE excess among MS patients, wherein the IgE specifically complexes with two or more myelin surface epitopes at an interval of 40−100 Ångströms, a separation critical for mast cell degranulation  and tissBecause MS patients often consume numerous medications, some started before observed disease onset, questions arise about the medications' effect upon (1) disease origin, (2) serum antibody test function, and (3) hindrance of normal and protective, humoral immune processes. Epitope-specific IgE is but one isotype involved in the myelin inflammatory process as other investigators have also documented the presence of myelin-specific IgA, IgG, and IgM . ConcomiThe analytical method entailed quantification of the ratio of myelin epitope-specific IgE relative to the sum of the matching myelin-specific non-IgE isotypes. In order to simplify the process, the non-IgE antibody level was determined by measuring epitope-specific human kappa plus lambda chains and subtracting the matching epitope-specific IgE. With experience, it became obvious that the specific IgE subtraction was unnecessary as the IgE was exceedingly small in comparison to the matching non-IgE antibodies.Thereafter, the evolved MS test employed the formula: IgE/(kappa + lambda). Individual humoral epitopes were mimicked by single peptides that were 5 amino acids in length , the sizWhen analyzing surface pentameric structures of individual myelin proteins for potentially serving as humoral epitopes  and compThe Hopp and Woods hydrophilicity method for locating epitopic sites on linear protein sequences  was usedIn order to estimate the functional distance (in Ångströms) between epitopes on the surface of each myelin protein, as depicted on its Hopp and Woods plot, the following tasks were per formed. The average diameter of constituent amino acids was determined by (a) comparing the mass of each amino acid relative to the mass of alanine with its known diameter of 6.9 Ångströms ; (b) mulIndividual Hopp and Woods, Excel X-Y plots of each protein's amino acid sequence were modified so as to only depict the protein's hydrophilic surface, either extracellular or intracellular, as if flattened by trimming away all amino acids that were functionally hydrophobic, and likely to be located in the protein interior, but leaving 2 on each hydrophilic edge to reflect infolding toward the protein center Figures , 14, 15.The bridging distance between dimeric surface epitopes on individual myelin proteins was estimated by multiplying the intervening amino acids number by 10.6 Ångströms per amino acid.Because a protein surface is not flat but oscillates in depth, sera from an age- and gender-varied MS-negative control group were sequentially tested to measure dimer-point, myelin epitope-specific IgE/(kappa + lambda) values while reducing the estimated dimeric distances in 5 percent intervals to find a reduction percentage to use as a factorial adjustment which simulated staircase dips in normally rolling surface contours of proteins and thus afforded functionally negative test results for the controls . A 25% reduction of the linear distances attained in Step 3 between dimeric epitopes afforded test-negative results for all initially tested, negative-control serum samples by exhibiting no more than one dimer point as being IgE/(kappa + lambda) test positive.In validation, when testing positive control MS patient sera and employing the 25% reduction factor, dimer bridging, test-positive values were attained whose corresponding epitopes' separation ranged from between 40 to 100 Ångströms, thus indicating a likelihood of mast cell degranulation. Claudin 11 and oligodendrocyte myelin glycoprotein lacked IgE dimer bridging sites and were, therefore, not analyzed further. Pathological dimer bridging values and locations are depicted in Figures  A similar contour-mapping approach was not used for PLP because it has but one structurally unique epitope expressed on the myelin surface, ADARM Figures , 2, 3. HIn MOG has been shown to be differentially expressed in various isoforms. However, for the purpose of identifying the potential array of MOG humoral epitopes possible on all isoforms and the epitopes' utility in dimer formation, analysis of MOG Sanger Institute Isomer 1 Figures –9 provedMyelin basic protein, an oligodendrocyte intracellular protein also potentially accessible by myelin surface disruption, exhibits a more complex array of potentially tar getable epitopes and dimer conditions than PLP or MOG. All potential dimer sites are exhibited by a combination of myelin basic protein Isoform 1 Figures –12, Isof2 (amino-ADOOA-ADOOA). The epsilon amino group on peptide lysine residues was blocked with a -3-methylbutyl (ivDde) group, to prevent undesirable, lateral binding when attaching peptide constructs to test plate wells via their free amide groups.As depicted in Each construct was dissolved in pH 7.2 phosphate buffered saline (PBS) immobilization buffer , at an assay optimum concentration .μL of each peptide construct solution was applied in quadruplicate to 96-well, amide-binding, maleic anhydride-activated, white 96- well plates . Four wells were left blank for plate background determination. The plates were covered with acetate plate sealers  and the construct solutions incubated at 21−26 degrees C for 18−24 hrs.100 μL of HSA background blocking solution  was applied per well. The construct/blocking solutions were incubated at 21–26 degrees C for 18–24 hrs and plates aspirated and dried.120 μL of a 2% solution of hydrazine monohydrate  in DMSO  was applied per well and incubated at 21–26 degrees C for 10 minutes. The hydrazine solution was aspirated and the procedure repeated two additional times. 250 μL of phosphate buffered saline with 0.05% Tween-20  was applied per well. Plates were incubated at 21–26 degrees C for 30 minutes, aspirated, washed three additional times, aspirated, and dried.200 After drying at 21−26 degrees C for 18−24 hrs in a clean, covered container, test plates were sealed with acetate plate sealers and stored at room temperature until needed. Individual plates were used for both specific IgE and specific (kappa + lambda) assays.specific IgE immunoassay entailed use of 100 uL/well of neat subject serum that had been spiked with 1 mg/mL aminated hydrophilic linker solution to neutralize potential, antilinker antibodies (50 μL linker solution per 12 mL serum). Plates were sealed and incubated for 2 hours at 21–26 degrees C and then washed four times with PBST (250 μL/well followed by immediate aspiration). 100 μL of 4 μg/mL biotinylated, goat anti-human IgE was applied per well. ). After incubation for 2 hours at 21–26 degrees C, the plates were washed and 100 uL/well of 64 ng/mL streptavidin horseradish peroxidase (Thermo Scientific Product No. 21126 diluted in HSA conjugate diluent) applied. Test plates were incubated for 30 minutes at 21−26 degrees C and then washed. 100 uL/well of Thermo Scientific Chemiluminescence substrate (Product No. 37074) was applied and the plate(s) read 1−3 minutes after application using a Luminoskan Ascent Microplate Luminometer .The μL linker-spiked serum and 9.9 mL PBST and (b) spiking 11.950 HSA conjugate diluent with 48 μL of the 1/100 diluted serum. Plates were filled with 100 μL/well of 1/25 k diluted serum, sealed, and incubated for 2 hours at 21–26 degrees C. Equal volumes of Vector biotinylated, goat anti-human kappa antibody (BA-3060) plus biotinylated, goat anti-human lambda antibody (BA-3070) were mixed together to form a 50 : 50 biotinylated anti-(kappa + lambda) concentrate (500 μg/mL). 96 μL of the anti-(kappa + lambda) concentrate was mixed with 11.9 mL of HSA conjugate diluent and 100 μL of the resulting solution applied per well. After 2-hour incubation at 21–26 degrees C, plates were washed and 100 uL per well of 16 ng/mL streptavidin horseradish peroxidase solution applied. Test plates were incubated at 21–26 degrees C for 30 minutes, aspirated, and washed. 100 uL/well of Thermo Scientific chemiluminescence substrate was applied and the plates read at 1–3 minutes after application.The linker-spiked test serum sample used in the specific IgE assay was diluted 1/25,000 by (a) making a 1/100 dilution mixing together 100 Specific IgE and matching specific (kappa + lambda) signals were obtained by reading corresponding, matched test plates on the microplate luminometer. Average test values corresponding to individual mimotopic peptides were determined by discarding the highest and lowest of four values and averaging the remaining two. The same procedure was followed for the four background well values, together with calculation of twice the standard deviation of the two-point average. The blank well background was deemed to be its average value plus twice the standard deviation. The plate blank well value was subtracted from each peptide-coated well average to yield a net signal. Kappa + lambda values were multiplied by 25,000 in order to delineate the corresponding neat serum (undiluted) epitope-specific kappa + lambda antibody levels. IgE/(K+L) values were multiplied by 1,000,000 in order to bring each quotient to a positive number if attainable. Test results with negative values or values of less than 0.5 were assumed to be test negative. Disease-positive and disease-negative (control) sera were purchased from BioServe . Multiple sclerosis-positive samples were from patients who had not yet received MS-specific therapy, patients who were taking interferon and/or Copaxone (glatiramer acetate) with or without medication shown to be immunosuppressive (Tables n = 10) nor male (n = 10) age-varied, control serum samples were MS test positive by exhibiting IgE/(kappa + lambda) dimer-positive results. Being positive, the control sera would have been IgE/(kappa + lambda) positive when tested against the PLP ADARM peptide antigen , secondary progressive (SP), or primary progressive (PP) MS. Individual test scores were lower than scores for the untreated MS group. None was IgE dimer positive against MOG or MBP.One of twelve patients was PLP dimer test positive. None were positive against MOG or MBP. , a humanized monoclonal antibody whose therapeutic use results in a systemic increase in B lymphocytes and, ultimately, plasma cells and specific serum autoantibodies –54. WherWe have concluded that multiple sclerosis is a complex, humoral autoimmune disease caused by IgE dimer formation on the surface or immediate subsurface of CNS myelin that results in focal mast cell degranulation. The degranulating mast cells likely release proteolytic enzymes and possibly other factors that damage or destroy proximal neurons.Test results of MS patients taking medication shown to have an immunosuppressive effect suggest either interference with the IgE/competing antibody pathological process or with the MS test itself. This was inferred by the quantitative difference in MS test scores between the patients who were taking no medication and exhibited relatively high test scores, patients who were being treated with a single MS-specific pharmaceutical (beta interferon or Copaxone), but had relatively low positive test scores, and patients who were taking the described immunosuppressive agents, with or without interferon or Copaxone, and were only sporadically MS test positive.This suggests that the conventional, multiple therapy approach may potentially hinder therapeutic efficacy by altering normal immune function.As interferon and Copaxone are prescribed because of their MS-specific, immunosuppressive, effect and MS is likely an autoimmune disease, it is reasonable to expect some decrease in humoral immune function among specifically treated patients, as reflected by the low anti-PLP test scores exhibited in Also suggested is the possibility that (1) the ancillary medications, having only been in use since the early twentieth century, may have historically played an instigating or promoting role in MS causation and/or progression through disruption of homeostatic, immune system controls.The absence of MS test-positive results against MBP dimers may have been due to their deep, intracellular location and relative freedom from immune surveillance and pathological action. It may also indicate that (a) successful MBP humoral targeting first calls for myelin surface disruption via action against PLP and/or outer MOG in order to provide penetrating access for anti-MBP antibodies and/or lymphocytes; (b) MBP is an autoimmune target, but of lesser frequency or importance than the epitopes on the outer surface or immediate sub-surface of myelin.If MS proves to be an IgE dimer-driven, humoral autoimmune disease, as is suggested by the test data, treatment with mimotopic peptides homologous to those used in the diagnostic immunoassay might prove to be therapeutically beneficial by neutralizing anti-myelin IgE antibodies. The therapeutic peptides would need to be administered in such a way as to insure intravascular delivery of quantities sufficient to neutralize all epitope-specific dimeric IgE autoantibodies through antibody-to-peptide complexing or by neutralizing key epitope-specific IgE antibodies whose target epitopes are cornerstones of a number of pathological dimers. The elimination of one or two dimer-point-specific autoantibodies could prevent multi-site-initiated mast cell degranulation and halt the pathological autoimmune process.Cornerstone dimer-blocking peptides are highlighted in bold within the full array of potentially therapeutic peptides listed in the sixth column from the left of To be effective, therapeutic peptides would need to (a) possess an exact structural match with the specific myelin protein epitope; (b) be of sufficient length (5-6 amino acids) to easily fit and be avidly bound by a single autoantibody; (c) be relatively hydrophilic so as to be functionally soluble when injected or ingested; (d) if ingested, be aided in enteric absorption by pharmaceutical agents such as medium-chain fatty acid constructs , superpoEvidence is presented that multiple sclerosis is a humoral autoimmune disease caused by IgE dimer formation on the surface or immediate subsurface of CNS myelin that results in focal mast cell degranulation with release of tissue-damaging enzymes and/or other substances.


Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood.Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. C. burnetii by non-phagocytic cells. C. burnetii internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein’s ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of C. burnetii, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited C. burnetii internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of C. burnetii infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection.Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor C. burnetii internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen.Our results suggest that the SH3 domain of cortactin is implicated in  Phagocytosis is the process that cells have developed for the engulfment of particulate material such as apoptotic cells, cell debris and, also, inert particles. Furthermore, phagocytosis represents a crucial event that triggers host defense mechanisms against invading pathogens. Nevertheless, several pathogens have acquired different strategies to alter these mechanisms to survive and multiply within host cell, causing infectious diseases Dynamic remodeling of the actin cytoskeleton is not only intimately involved in phagocytosis Escherichia coliShigella, Neisseria, Chlamydia, Staphylococcus and Listeria. The phosphorylation status of cortactin has been proposed to differentially regulate the invasion of many microbial pathogens. Cortactin is also involved in actin-based motility of many pathogens during their intracellular trafficking Cortactin is a key regulator of the actin cytoskeleton, and it plays a crucial role in tumor cell invasion Cortactin possesses an N-terminal acidic domain (NTA) and F-actin-binding repeats that activate the Arp2/3 complex to initiate actin polymerization Coxiella burnetii, the causative agent of human Q fever, is an obligate intracellular bacterium found in a wide range of hosts, including livestock and humans. The primary route of infection in humans is inhalation of contaminated aerosols C. burnetii in their milk, urine and feces, and the bacteria are dispersed together with amniotic fluids and the placenta during birthing. These bacteria can survive for long periods in the environment, since they are highly resistant to heat, desiccation and common disinfectants.C. burnetii inhabits mainly monocytes/macrophages but can infect a wide variety of cultured cell lines in vitroC. burnetii internalization into HeLa cells, a non-professional phagocyte cell line. We investigated the role of the Arp2/3-activating DDW motif in the N-terminal acidic region and of the SH3 domain at the C-terminus of cortactin during C. burnetii internalization. We observed that overexpression of cortactin mutated in the SH3 domain inhibits uptake of the bacterium, suggesting that the SH3 domain is important for internalization. We also analyzed the role of cortactin phosphorylation in internalization. By overexpressing cortactin mutants that are non-phosphorylatable and that mimic phosphorylation, we show that cortactin favors C. burnetii internalization in a tyrosine dephosphorylation- and/or serine phosphorylation-dependent manner. Furthermore, pharmacological inhibition of Src and ERK kinases reduce C. burnetii uptake. Our results indicate that phosphorylation status of cortactin affect internalization of C. burnetii.In this report we describe the involvement of cortactin in Coxiella antibody against Nine Mile phase II, clone 4 (RSA439) was generously provided by Dr. Robert Heinzen . Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. . Rabbit monoclonal anti-phosphocortactin (Tyr421) antibody was purchased from Abcam , mouse monoclonal anti-actin Ab-5 antibody was purchased from BD , rabbit anti-phosphoSrc (Tyr416)  and anti-Src antibodies were generously provided by Arlinet Kierbel , and mouse monoclonal anti-phosphoERK1/2 (Tyr204) and rabbit polyclonal anti-ERK antibodies were purchased from Santa Cruz Biotechnology . The inhibitors PD98059 and SU6656 were from Invitrogen  and CalBiochem , respectively. Protease inhibitor cocktail was from Sigma .Dulbecco’s Modified Eagle's Medium (D-MEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco BRL/Life Technologies . Vectors encoding a fusion of green fluorescent protein (GFP) with cortactin WT (full-length cortactin) or GFP-cortactin 3F (cortactin mutated in the three tyrosine-phosphorylation sites recognized by Src) were kindly provided by S. Bourdoulous . Rabbit polyclonal anti-2.HeLa cells were grown in DMEM supplemented with 10% heat-inactivated FBS, 2.2 g/l sodium bicarbonate, 2 mM glutamine and 0.1% penicillin/streptomycin at 37°C under 5% COC. burnetii bacteria were provided by Ted Hackstadt  and handled in a biosafety level II facility. Non-confluent Vero cells were cultured in T25 flasks at 37°C under 5% CO2 in DMEM medium supplemented with 5% FBS, 0.22 g/l sodium bicarbonate and 20 mM Hepes, pH 7 (MfbH). Cultures were infected with C. burnetii phase II suspensions for 6 days at 37°C under 5% CO2. After freezing at −70°C, the flasks were thawed, and the cells scraped and passed 20 times through a 27-gauge needle connected to a syringe. Cell lysates were centrifuged at 800×g for 10 min at 4°C. The supernatants were centrifuged at 24,000×g for 30 min at 4°C, and pellets containing C. burnetii were resuspended in phosphate-buffered saline , aliquoted and frozen at −70°C.Clone 4 phase II Nine Mile strain of 5) were seeded on sterile glass coverslips placed in 24-well plates and grown overnight in MfbH medium. For infection, a 5-µl aliquot of C. burnetii suspension was added per well (Multiplicity of infection: ∼20). Cells were incubated for different lengths of time at 37°C under 5% CO2.Cells  and donkey anti-rabbit secondary antibody conjugated with Cy3 (1∶600) in PBS containing 0.5% BSA (nonpermeabilizing conditions to label extracellular bacteria). After washing, cells were incubated with the same rabbit antibody against C. burnetii (1∶1000) and a donkey anti-rabbit secondary antibody conjugated with Cy5 (1∶600) in PBS containing 0.5% BSA and 0.05% saponin . Coverslips were mounted with Mowiol and examined by confocal microscopy. The intracellular bacteria are expressed as a percentage of the total number of bacteria per cell.To determine the number of internalized bacteria a double cycle antibody staining protocol was used 2.Cells were transfected for 6 h with 2 µg/ml pGFP empty vector or pGFP plasmids expressing fusions of GFP with wild-type cortactin (WT) or one of the following mutants: single point mutants, W22A and W525K; double mutants, S405/418D (SD) and S405/418A (2A); and triple mutants, Y421/466/482D (3D) and Y421/466/482F (3F). Cell transfection was carried out using Lipofectamine™ 2000 , according to the manufacturer’s instructions. After 6 h of transfection, the cells were washed and incubated for 18 h in MfbH medium at 37°C under 5% CO2, 2 mM Na3VO4, 10 mM NaF, 0.5 mg/ml DTT, 2 mM EDTA) supplemented with a protease inhibitor cocktail and kept on ice for 20 min. Lysates were clarified by centrifugation at 2000×g for 15 min at 4°C. Clarified lysates were transferred to clean tubes, mixed with Laemmli buffer and boiled for 5 min. The samples were resolved by SDS-PAGE and the proteins transferred to nitrocellulose membranes using standard procedures. Membranes were blocked for 2 h at 4°C in Tween-Tris-buffered saline  supplemented with 5% BSA, then incubated overnight at 4°C with the appropriate primary antibodies. The membranes were washed three times with TTBS, then, incubated for 2 h at room temperature with appropriate peroxidase-conjugated secondary antibodies. Membranes were washed again with TTBS and developed using the ECL Western blotting system  according to the supplier’s recommendations. Blotting with anti-GAPDH or anti-actin antibody was carried out to provide loading controls. Band densitometry was carried out using ImageJ software .Hela cells were cultured on 60-mm dishes and infected as described above for different lengths of time. After the indicated infection periods, cells were washed with PBS, scraped into ice-cold lysis buffer . Images were processed using ImageJ software.Results were analyzed by the ANOVA test in conjunction with Tuckey and Dunnett tests.Cortactin is an F-actin regulatory protein that plays an important function in various cellular processes such as cell adhesion, motility and endocytosis. However, its role in phagocytosis has been poorly characterized. Interestingly, cortactin is recruited to the contact sites made by several pathogens with the host plasma membrane during infection C. burnetii internalization, we tested two cortactin mutants: W22A (20DDW22 motif mutated to 20DDA22), an NTA mutant that has lost its ability to bind and activate Arp2/3 C. burnetii at 37°C for 4 h, processed for indirect immunofluorescence and analyzed by fluorescence microscopy , cortactin contains a short motif called DDW that binds and activates the Arp2/3 complex copy see . We deciC. burnetii internalization by immunofluorescence we used the conventional double cycle antibody staining protocol for discriminate between extra- and intracellular bacteria , while ERK and PAK phosphorylate Ser405/Ser418 and Ser113, respectively C. burnetii at 37°C for 4 h, processed for indirect immunofluorescence and analyzed by fluorescence microscopy. As shown in C. burnetii internalization were similar in cells expressing either GFP (control) or GFP-cortactin WT, but significantly higher in cells expressing GFP-cortactin 3F. In contrast, when HeLa cells were transfected with a plasmid encoding pGFP-cortactin 3D , which mimics cortactin phosphorylated by Src, internalization of C. burnetii was similar to that in cells expressing GFP-cortactin WT  cannot be phosphorylated by ERK, while cortactin SD  mimics the effects of ERK phosphorylation. As shown in C. burnetii during infection.Since the cellular function of cortactin is also regulated by phosphorylation of its serine residues, we studied whether serine phosphorylation is important for C. burnetii uptake and that cortactin is a substrate of kinases of the Src and ERK families, we analyzed directly whether these kinases are involved in C. burnetii internalization. HeLa cells were treated during infection with Src kinase inhibitor SU6656 or with PD98059, an inhibitor of MEK kinases which are upstream activators of ERK kinases C. burnetii uptake was observed during infection of a macrophage cell line treated with the MEK kinase inhibitor , the activated form of the kinase. C. burnetii-host cell interaction.Considering that cortactin is phosphorylated on tyrosine residues and that it is a substrate of Src kinase, we reasoned that this enzyme may be activated during C. burnetii infection, lysates of infected HeLa cells were analyzed by SDS-PAGE and Western blotting using an antibody that specifically recognizes phosphorylated ERK. As shown in C. burnetii-host cell interaction.Cortactin can also be regulated by phosphorylation of serine residues and ERK is one kinase involved in this reaction. To investigate whether ERK is activated during Bacterial pathogens manipulate the host cell cytoskeleton to avoid phagocytosis, to invade and/or to become mobile in the host cell cytoplasm. They often interact with actin filaments by modulating the activity of different actin-interacting effectors in the host. One such effector is cortactin, an actin-binding protein that plays a crucial role in the regulation of actin dynamics. Cortactin has been implicated in the infection process of several microbial pathogens C. burnetti into non-phagocytic cells, whereas tyrosine phosphorylation of cortactin suppresses this internalization. We also show that Src and ERK kinases are activated during the initial stages of C. burnetti infection.The present study contributes to understanding the role of cortactin in bacterial pathogenesis. We provide evidence that the SH3 domain and serine phosphorylation of cortactin are involved in signal transduction pathways that support the internalization of avirulent C. burnetii internalization  is sufficient to stimulate actin assembly and bacterial uptake. Our results are similar to those observed in the formation of the pedestal-like structure during E. coli infection. Indeed, expression of the cortactin mutant W525K significantly reduced the number of pedestals induced by enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic E. coli (EHEC) in HeLa cells in vitroCortactin stimulates actin polymerization by binding, via its N-terminal domain, the Arp2/3 complex, or by binding, through its C-terminal SH3 domain, N-WASP, a well-known activator of Arp2/3 lization , suggestE. coli, entry of C. burnetii into HeLa cells requires serine phosphorylation of cortactin. Our conclusion is also supported by the observations that ERK1/2 kinases were transiently activated early during HeLa cell infection and then later deactivated , which inactivates c-Src and leads to cortactin dephosphorylation by an unknown mechanism C. burnetii internalization occurs by a mechanism similar to that of H. pylori. To our knowledge, C. burnetii, H. pylori and EHEC are the only three pathogens known to induce dephosphorylation of cortactin during host cell infection. Recently, H. pylori has been shown to induce cortactin phosphorylation on serines in a CagA-independent manner, and this form of cortactin stimulates actin rearrangement and cell elongation C. burnetii internalization, which suggests that serine phosphorylation of cortactin is necessary for C. burnetii entry.We show here that during d 60 min . In addinfection . This seC. burnetii exhibit intense membrane protrusions associated with major actin cytoskeleton reorganization, while infection with avirulent bacteria induced a few membrane folds without significantly affecting cell morphology C. burnetii result from a modest actin cytoskeleton rearrangement that facilitates bacterial uptake. Meconi and collaborators also showed that actin cytoskeleton reorganization is associated with tyrosine phosphorylation of the Src family kinases Hck and Lyn very early during infection of THP-1 cells with virulent, but not avirulent, C. burnetiiC. burnetii, but not avirulent bacteria, stimulate the tyrosine phosphorylation of several proteins. Using a similar experimental strategy, we observed a significant labeling of proteins with masses around 85 kDa at 1 h of HeLa infection with avirulent C. burnetii (data not shown). Cortactin migrates as a doublet of 80 and 85 kDa in SDS-PAGE THP-1 monocytes infected with virulent C. burnetii uptake, while tyrosine phosphorylation suppresses this uptake. Our results suggest that a complex series of events occurs during C. burnetii internalization into non-phagocytic cells. Early after infection, Src and ERK kinases may phosphorylate unknown substrates, perhaps other kinases such as Abl that in turn phosphorylate tyrosine residues in cortactin. This may regulate an early step in internalization. At a later stage, tyrosine dephosphorylation and serine phosphorylation of cortactin take place, which regulates a later step of internalization. In this way, tyrosine phosphatases and serine kinases from the host cell and/or C. burnetti regulate the phosphorylation status of cortactin to favor C. burnetti entry into the host cell. Thus, the results reported here indicate that dynamic phosphorylation of cortactin is important for C. burnetii internalization during infection.In conclusion, our results indicate that serine phosphorylation of cortactin and its SH3 domain are involved in a signal transduction mechanism that favors Figure S1ERK kinase is involved in C. burnetii internalization. (A) RAW macrophages were incubated for 1 h at room temperature with 0.05% DMSO (control) or 15 µM PD 98059 (MEK-ERK inhibitor). Then the cells were infected for 2 h with C. burnetii in the presence of the inhibitor. Cells were fixed and processed for indirect immunofluorescence using a specific anti-C. burnetii antibody , percentage of the total number of bacteria.body see . Bars, 1(TIF)Click here for additional data file.Figure S2Tyrosine phosphorylation of cortactin and ERK proteins during C. burnetii infection. Lysates of RAW macrophages infected with C. burnetii for different lengths of time were analyzed by SDS-PAGE and Western blot using antibodies against phosphoTyr421-cortactin (P-cortactin), phospho-ERK (P-ERK) or GAPDH. 0 min: control RAW macrophages incubated in the absence of C. burnetii. Data were analyzed with ImageJ software. The ratio between phosphorylated ERK and GAPDH (A), and phosphorylated cortactin and GAPDH (B) levels are shown. The results are representative of two independent experiments. (RU), relative units.(TIF)Click here for additional data file.


The relatively common experimental visual search task of finding a red X amongst red O’s and green X’s (conjunction search) presents the visual system with a binding problem. Illusory conjunctions (ICs) of features across objects must be avoided and only features present in the same object bound together. Correct binding into unique objects by the visual system may be promoted, and ICs minimized, by inhibiting the locations of distractors possessing non-target features (e.g., Treisman and Sato,  Some of the most compelling cases in the neuropsychology of vision are patients who, following brain damage, experience selective loss of particular stimulus qualities. Patients with specific and restricted cortical damage may present with selective deficits for color  for targets defined by conjunctions of features came to light in the late 1980s . Treisman and Sato, There is a further key difference between FIT as proposed by Treisman and Sato  and the In the next section we review the experimental approaches used to address whether there is suppressive rejection of distractors in general and also specifically in relation to the theories discussed above.There have been a number of approaches to testing whether target selection can take place by distractor suppression, and, if it can, how this process might operate. We briefly consider four types of study examining: (i) manipulations of distractor heterogeneity, (ii) trial-by-trial variations in stimulus properties, (iii) effects of stimulus foreknowledge, and (iv) probe-dot detection.Treisman and Sato , FriedmaStudies employing this method have generated data that support roles for both excitatory and inhibitory mechanisms in search. Treisman and Sato  argued tDriver et al.  also useOn the other hand, similar studies have found no requirement for an inhibitory process. Friedman-Hill and Wolfe  examinedMcLeod et al.  asked paClearly the evidence from studies manipulating distractor heterogeneity is not straightforward. It is difficult to unambiguously attribute changes in performance to attentional tuning (inhibitory or otherwise) in this task. As a consequence of manipulating the number of features present in the displays the grouping structure of the display also changes , compared to when the features on the two trials were unrelated. Thus RTs were facilitated when features repeated and impaired when the target took the previous distractor features, consistent with a role for both excitatory and inhibitory processes.Trial-by-trial priming effects have also been used to make arguments about inhibitory and excitatory guidance in search. Here the logic is that any inhibitory or excitatory effects will carry-over in time to the next trial. Koshino  varied wLamy et al.  also exaOne interpretative issue with these studies of carry-over effects, is whether they directly reflect distractor inhibition, or rather the costs that might be involved when features from a past trial have to be re-bound to define the target  of inhibitory and excitatory guidance in search is to look at whether providing fore knowledge of either target or distractor features can lead to costs and benefits to search.Shih and Sperling  and MoorIn these tasks it is possible to observe both benefits for valid cues and costs for invalid cues, and at least logically these two effects are dissociable. Take the situation where the target appears 80% of the time in red and 20% of the time in green. Compared to the situation where target color is equiprobable, there can be benefits when the cue is valid, consistent with increased activation of items with likely features. There may also be costs on invalid trials, and these costs are at least consistent with inhibition of items with unlikely features. Relative to the case where the target was equally likely to be one of two possible colors, both Shih and Sperling, and Moore and Egeth reported benefits for validly cued color targets, and costs for trials where the initial target color cue was invalid . The presence of both benefits and costs is consistent with both excitatory and inhibitory processes. In particular Shih and Sperling  say the relative strength of a competing stimulus even though its absolute activation level remains constant. Thus the processing costs seen in these studies could be traced to increased competition from highly activated items in the cued color. In order to circumvent this problem what is needed is a measure of the relative loss of competitive strength for distractors that is independent of the search task.However, the results from manipulations of target foreknowledge are inconclusive. In particular, attentional selection may be understood as the outcome of a competition between different stimuli  but also of features carried by the distractors that are irrelevant to the search task. For example, in the studies of Braithwaite, Humphreys and colleagues . IOR refers to the reduction in processing efficiency that follows the withdrawal of spatial attention from a previously attended location  in search.per se, probe detection can be examined as a function of search activity. In other approaches where the search response itself provides the index of attentional priority it becomes difficult to vary the degree of engagement with the search task without also varying the nature of the search stimuli. Additionally, probe detection provides a quite direct and relatively unambiguous measure of attentional priority that by-passes issues related to rebinding of features common to trial-by-trial approaches to measuring distractor activation and inhibition.As noted above, most attempts to examine excitatory and inhibitory contributions to search have manipulated the nature of the search task itself. Here, the experimental observation changes the very nature of the task under scrutiny. The method for measuring inhibition is an integral part of the search task, and is measured with some response to the search display. By changing the nature of the search task the experimenter may also inadvertently change display-wide grouping or bottom-up salience. We argue that the probe-dot task provides a tool that allows the experimenter to measure components of search, without disrupting or changing the nature of the search task under scrutiny. Since the probe task is a tool that is independent of search In addition to providing evidence for the existence of feature inhibition in search, results from the probe-dot task provide important constraints on the nature of this inhibition. Note that in this task the probe does not necessarily share any features with the distractor on which it is presented apart from location. Thus the probe itself would not be represented in the same feature maps as the search distractors. Thus any disadvantage for probe processing is unlikely to be rooted in inhibition in the feature maps themselves but rather from the coding of priority at the level of a general masters alience map.Our review so far indicates that there is evidence for the parallel suppression of distractors under conditions in which targets can be efficiently segmented from distractors, with the inhibition across groups of distractors with a common feature not shared by the target. This result is consistent with ideas put forward by both Treisman and Sato , in theiincreased rather than decreased . Thus when particular features are used to signal inhibition, increases in activation in mid-level feature-specific areas may occur. This might reflect direction of an inhibitory signal itself or the initial allocation of attention to the distractor in order to subsequently inhibit it  signals the to-be ignored feature, and setting of appropriate inhibitory weights to translate feature activation into reduced priority at the master-map level. Subsequently, interactions between feature-specific regions and downstream areas in the parietal cortex and precuneus create a spatial template that is used to coordinate location specific deactivation in V1. There are also other candidate structures that could implement a master salience map. Notably, the temporo-parietal junction has been highlighted as an important neural structure for bottom-up attentional capture (see Corbetta and Shulman, One way to reconcile the idea that feature inhibition is realized by reductions in V1 activation is to view V1 activity, as revealed by the BOLD signal, as reflecting a salience representation (e.g., Li, Feature inhibition has been hypothesized to play an important role in search, guiding attention away from distractors and preventing ICs. Here we show by exploring the literature, that there is a theoretical niche for such a mechanism. Probe-dot studies are highlighted as being well suited to providing behavioral evidence for this mechanism. Although there is good evidence for inhibitory guidance of attention, there is also good evidence supporting the proposal that the multiple features of objects may not always be independent targets for inhibition, inhibition of one feature of an object may inadvertently lead to the inhibition of other features of the same object. Such feature non-independence of suppressive mechanisms in search is consistent with the AET account (Duncan and Humphreys, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases  These effectors are finely tuned and regulated by post-translational modifications, including phosphorylation, which in turn is able to control subcellular localization and activity. Intensity and duration of the phosphorylation depend on the equilibrium between the activities of kinases and phosphatases, as well as on the crosstalk and connectivity established between pathways. All together, these parameters ensure appropriate and specific cell decisions.Xenopus oocytes, where feedback loops and feedforward loops elaborate physical properties like ultrasensitivity, bistability and irreversibility [Among the signaling cascades of phosphorylated molecules, which are dedicated to conveying information and integrating extracellular signals, the Mitogen Activated Protein Kinase (MAPK) signaling network stands out ,2. Each sibility –7. Spatisibility .During the past two decades, new tools have emerged concomitantly to the discovery of GFP. Among these tools, genetically encoded enzyme activity reporters based on Förster Resonance Energy Transfer (FRET) imaging have become more and more attractive due to their ability to sense and report the level of several analytes such as second messengers, ions or protein activity in living cells or tissues . FRET isKinase activity biosensors are constituted by two fluorophores adapted for FRET experiments. Those fluorophores are flanking a specific peptide substrate of ERK and a domain recognizing and binding this peptide substrate when phosphorylated. The latter recognition allows a conformational change of the biosensor and thus a FRET signal.vice versa [Upon FRET several properties of light are modified and can be measured. The emission intensity of the donor fluorophore decreases, while emission intensity of the acceptor increases. In this way FRET can thus be evaluated by dividing the YFP signal by the CFP signal. An increase of this YFP/CFP ratio thus corresponds to an increase of kinase activity and ce versa . The lifThe purpose of optimizing such tools directly relies on kinase activity behavior. In fact, in some biological processes, kinase activity changes are too low to be detected with the existing biosensors. This depends directly on an intrinsic property of the biosensor called the dynamic range, which corresponds to the maximal difference of “FRET value” between two conditions: when there is no kinase activity and thus no phosphorylation of the biosensor and when kinases are fully activated and thus biosensors are folded and give a strong signal. Dynamic range corresponds thus to the ability of the biosensor to give a measurable FRET signal even when kinase activity is low.MAPK Indicator Unit ERK2) [Xenopus ERK2 sequence, from which they were spaced by short linkers of two and three aminoacids. When the MAPK/ERK pathway cascade is recruited by an external signal, activated MEK binds to endogenous ERK as well as the ERK enclosed within the sensor in order to achieve phosphorylation. This binding lead to a modification of ERK conformation within Miu2, bringing closer the two fluorophores and therefore noticeably increasing the FRET signal. However, Miu2 expression acts as an overexpression of ERK2, which is a disadvantage because ERK2 overexpression disturbs cellular processes like in the case of stimulation of proliferation in human hepatocellular carcinoma study [per se ERK activity. This last issue was resolved in 2007 with the engineering of Erkus [Regarding MAPK/ERK signaling pathway, the first FRET-biosensor designed was named Miu2 (for it ERK2) . This bima study . Moreoveof Erkus , whose pof Erkus .In 2008, another ERK biosensor named Extracellular-regulated Kinase Activity Reporter (EKAR) was published by Harvey and collaborators , taking i.e., orientation factor, distance between fluorophores) and (ii) from a photophysical point of view, on the fluorophores' properties . Two fluorescence properties, modified by FRET, are measured: fluorescence emission is monitored by ratiometric measurements while fluorescence lifetime is measured in Fluorescence Lifetime Imaging Microscopy-FRET (FLiM-FRET). Here we report new ERK biosensors, namely EKAR-Cep-CpV, EKAR-TVV and EKAREV-TVV, modified from the two last generation of biosensors EKAR and EKAREV [The dynamic range of the preexisting MAPK biosensors to be optimized is directly associated with FRET efficiency , which, d EKAREV ,16. Thos2.2.1.prK5 cEKAR was amplified by PCR using the sense-oligonucleotide 5′-AGCGGATCCATATGGCGGACGAGGAGAAGC-3′ and the antisense-nucleotide 5′-CATGAGCTCGATATCCCGGGCCCGCGG-3′ containing, respectively, one BamHI and one SacI restriction site. The resulting fragment was then subcloned into the pCS2 AKAR4 vector using BamHI/SacI enzymes.Three different constructs were produced based on EKAR and EAKREV sequences ,16, taki2.2.TEPACVV [TEPACVV is surrounded by two unique restriction enzymes EcoRV and NheI. Because those restriction sites were already present in the coding sequence of EKAR, we proceed to a multisites-directed mutagenesis to delete those sites by introducing two silent mutations in EKAR with two primers: 5′-GGAGGAGGAGGAGCCAGCGGCGG AGGTGG-3′ and 5′-GCGGGCCCGGGACATCATGGTGAGCAAGG-3′. The molecular recognition element sequence of prK5 cEKAR was then amplified by PCR using the sense-oligonucleotide 5′-CGAT ATCTCCGGATCCATATGGCGGACGAG-3′ and the antisense-nucleotide 5′-CGCTAGCGATGTCC CGGGCCCGCGGG-3′ containing respectively EcoRV and NheI. The resulting fragment was then sub-cloned into the pCDNA3 TEPACVV vector.This was generated using the same kind of strategy: sub-cloning the sequence of the molecular recognition element of EKAR  between TEPACVV . The who2.3.TEPACVV. The same procedure was used to obtain the negative control sensor. We amplified the mutated (Threonine to Alanine within the substrate) EKAREV and sub-cloned this fragment into the pCDNA3 TEPACVV.As the FRET pair used in the second construction gives us good results, we tried to optimize it with an improved linker named EV-linker contained in another MAPK biosensor EKAREV . The maj2.4.Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum and antibiotics (100 units/mL of penicillin and 100 μg/mL of streptomycin) at 37 °C with 5% CO2. Cells were seeded on 32 mm coverslips in six-well plates and grown to reach 50% of confluency. Cells were then transfected with 1 μg of DNA per well using FuGENE HD transfection reagent . Twenty four hours later, cells were starved in DMEM with 0.5% FCS overnight. Prior to images acquisition, DMEM was removed and cells were placed in L-15 buffered medium. Epidermal Growth Factor  was resuspended in PBS and used at 100 ng/mL final concentration. MEK inhibitor U0126 dihydrocholride  was resuspended in DMSO and used at the final concentration of 50 μM.2.5.Cells on coverslips were placed on a thermostatted (37 °C) inverted Leica AF6000 videomicroscope  with 63×, 1.3 numerical aperture (NA) glycerin-immersion objective. Fluorescence excitation was performed with a 427 ± 10 nm bandpass filter through a double band dichroic mirror (440/520 nm). To avoid displacements between donor and acceptor's fluorescence measurements, we used fast detection filter wheel (respectively with 472 ± 30 nm and 542 ± 27 nm bandpass filters). Images were acquired every two minutes. After 6 min, drugs were added to the cell medium by gentle pipetting.p-values were obtained using Student t-test.YFP/CFP ratio for cell by cell data analysis was realized with the ImageJ software as described in . Curves 2.6.p-values were obtained using Student t-test.Frequency domain FLiM experiments were performed using the LIFA system  assembled on a wide field Nikon Eclipse TE2000 inverted microscope  in a 37 °C thermostatted chamber and with a 60×, 1.4 numerical aperture (NA) oil immersion objective and appropriate CFP filter cube . The modulation frequency was set to 40 MHz and MCP to 800 V. We used a solution of 7-(diethylamino)coumarin-3-carboxylic acid (Sigma) diluted in methanol as lifetime reference (4 ns). 36 phases shifted intensity images were recorded every two minutes, pre-diluted EGF and U0126, according to the experimental designs, were manually added into the culture medium. Lifetime values were extracted with LiFliM (Lambert Instruments). For each cell, lifetime variation was obtained by subtracting the average of the baseline values (time points 0 to 6 min) from each time point. Histograms represent the variation before (time points 0 to 6 min) and after (10–20 min) EGF induction. In both cases the average and standard deviation is calculated and represented. 3.EKAR-Cer-CpV, including the Cerulean/cpVenus FRET pair . The difference between the basal ratio values corresponding to starved cells and the ratio values following EGF stimulation corresponded to the dynamic range of the biosensors. In order to compare the results among themselves, each value obtained over time for every single cell, was normalized on the average of baseline values . WW is a phospho-aminoacid binding domain. The substrate corresponds to a part of the Cdc25C sequence, coding for the amino acid sequence PDVRTPVGK specifically recognized and phosphorylated by ERK on the Threonine 48 residue. Dotted red boxes highlight the changes bring to the original constructions. EKAR-Cer/CpV and EKAR-TVV derive respectively from EKAR [rom EKAR  (A) and rom EKAR  (B).p value ≤0.01 and three symbols represent a p value ≤0.001 when compared to EKAR (circles) or EKAREV (stars).p value ≤0.001 when compared to EKAR or EKAREV.The YFP/CFP ratio was normalized on the average of the baseline values (time points 0 to 6 min) for each cell . Lifetime variation was obtained by subtracting the average of the baseline values (time points 0 to 6 min) from each time point for each cell . For each time point either the average YFP/CFP ratio or the average delta lifetime of n cells and the standard deviation were calculated and represented. Histograms  were calculated by subtracting the same averages of baseline values from the average of plateau values after EGF stimulation (points at time 12 to 20 min). p-Values were obtained using Student t-test.The ability of the biosensor to be dephosphorylated and return to its original conformation, when kinase activity decreases or stops, is a critical parameter. Since many kinases are regulated by negative feedback loops or more specifically if we take the sustained/transient activation of MAPK, a non-reversible probe would lead to wrong interpretation of physiological events, because we will be only able to detect the first activation step. To reverse the signal, the biosensor in its folded conformation has to allow the phosphatases to access to the phosphorylated site within the probe. We thus tested our best construction for each technique upon inhibition of the pathway by a chemical inhibitor of MEK, U0126  and 3. MEKAR-TVV revealed the highest dynamic range in FRET ratio study. Following an EGF stimulation cells expressing this probe, cells were exposed to the MEK inhibitor U0126. The CFP/YFP ratio returned to a basal level when MAPK were inhibited, which demonstrated that EKAR-TVV sensing was reversible. The variations were compared to the mutated biosensor . A repre4.Increasing biosensor sensitivity may rely on different strategies optimizing the combination of sensing domains (substrate and PAABD), linkers and fluorophores. Each optimization process for a biosensor is challenging because the global dimension and topology of each of them is different, and thus the structural requirements of each biosensor are unique. In some cases, a systematic approach, building all the combinations of the sensors key elements might be carried out . In othei.e., donor fluorescence lifetime for FLIM FRET experiments or both fluorophores brightness for ratiometric measurements. Thus, these properties can be used for a stepwise procedure of optimization of preexisting biosensors [Intensity and lifetime are two different properties of fluorescence. Intrinsic properties of fluorophores will therefore increase the dynamic range, osensors ,16, in cosensors . HoweverSeveral requirements necessary to achieve the best dynamic range are independent of the measured properties of light, like the relative orientation of fluorophores. Due to the flexible nature of EKAR, it is hard to predict this orientation in a structural point of view. Then, while we can narrow the combination of what we identified as the best FRET pairs  and