Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV–VAP tethering complex and the formation of ER-Inclusion MCS. Via a C-terminal region containing three CK2 phosphorylation motifs, IncV recruits CK2 to the inclusion leading to IncV hyperphosphorylation of the noncanonical FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.Membrane contact sites (MCS) are crucial for nonvesicular trafficking-based interorganelle communication. Endoplasmic reticulum (ER)–organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT motif-containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen  They provide physical platforms for the nonvesicular transfer of lipids and ions, and cell signaling events important for interorganelle communication and organelle positioning and dynamics . Since tsequence . In addisequence .Chlamydia trachomatis, the causative agent of the most commonly reported bacterial sexually transmitted infection. Upon invasion of the genital epithelium, C. trachomatis replicates within a membrane-bound vacuole called the inclusion , are inserted into the inclusion membrane and are therefore strategically positioned to mediate inclusion interactions with host cell organelles  is similar to the canonical sequence, whereas a second motif (262S1F2H3T4P5P6N7268) deviates significantly and was originally defined as a noncanonical FFAT  is essential for the IncV–VAP interaction during infection. However, it remains unclear whether additional determinants promote the assembly of this bacterial tether.Characterization of the protein composition of ER-Inclusion MCS led to the identification the Inc protein IncV, which constitutes a structural component that tethers the ER membrane to the inclusion membrane through interaction with VAP . The Inccal FFAT . SimilarChlamydia evolved a post-translocation phosphorylation strategy in order to accommodate proper secretion via the Type III secretion system, while achieving full mimicry of eukaryotic FFAT motifs.Here, we show that multiple layers of host cell kinase-mediated phosphorylation govern the assembly of the IncV–VAP tethering complex and ER-Inclusion MCS formation. IncV phosphorylation supports the IncV–VAP interaction through FFAT motifs displaying core domains immediately downstream of phosphorylation-mediated acidic tracts. Since the substitution for genetically encoded acidic tracts interfered with IncV translocation, we propose that C. trachomatis overexpressing IncV-3xFLAG displayed a doublet consisting of a 50 and 60 kDa band (Ct). By contrast, IncV-3xFLAG ectopically expressed in HEK293 cells had an apparent molecular weight that was shifted toward the 60 kDa band of the doublet . This result led us to hypothesize that IncV is post-translationally modified by a host factor.When subjected to anti-FLAG western blot analysis, we noticed that lysates of HEK293 eukaryotic cells infected with wild-type C. trachomatis expressing IncV-3xFLAG. Following the release of IncV-3xFLAG from the beads by FLAG peptide competition, the eluate was treated with lambda (λ) phosphatase or phosphatase buffer alone, and subsequently subjected to anti-FLAG western blot analysis -inducible promoter were analyzed by confocal immunofluorescence microscopy (We next focused on identifying the host cell kinase(s) responsible for phosphorylating IncV. All three subunits of Protein Kinase CK2 were identified as potential interacting partners of IncV in an Inc-human interactome . To detecroscopy . In the croscopy , −aTc. Hcroscopy , +aTc. Tcroscopy . Altoget167–363) or GST alone, purified from E. coli. To detect phosphorylation, we used ATPγS, which can be utilized by kinases to thiophosphorylate a substrate, followed by an alkylation reaction of the thiol group to generate an epitope that is detected using an antibody that recognizes thiophosphate esters  fused to GST  purified from E. coli  phosphatase dephosphorylation of IncV coupled with a GST-VAPMSP pull-down assay  and GST-IncV167–363 were expressed separately in E. coli and purified using amylose resin and glutathione Sepharose beads, respectively. GST-IncV167–363 was left attached to glutathione Sepharose beads and was phosphorylated by incubation with recombinant CK2 and ATP before being combined with purified MBP-VAPMSP. GST-IncV167–363 was pulled down and the samples were subjected to western blot using anti-MBP antibodies , or a pool of all four siRNA duplexes (pool), were infected with a previously characterized incV mutant strain of C. trachomatis  at 18 hours post infection (h pi), prior to the induction of IncVWT-3xFLAG expression at 20 h pi. This experimental setup allowed for CK2 inhibition, prior to IncVWT-3xFLAG synthesis, translocation, insertion into the inclusion membrane and exposure to the host cell cytosol. The cells were lysed 24 h pi and subjected to western blot analysis to determine the effect of CK2 inhibition on the apparent molecular weight of IncV. The apparent molecular weight of IncV decreased in a dose-dependent manner  or aTc only (CX-4945 washout) for an additional hour (25 h pi). Samples where CX-4945 was omitted and collected at 25 h pi (No CX-4945), and samples processed in the presence of CX-4945 and collected at 24 h pi prior to the wash (CX-4945) were used as controls. None of the treatment prevented IncV localization to the inclusion  contains seven CK2 recognition motifs (S/T-x-x-D/E/pS/pY) . HeLa cells expressing YFP-CK2β or YFP-VAP were infected with C. trachomatis incV mutant strains expressing IncVWT- or IncVS3A-3xFLAG. The cells were fixed at 24 h pi, immunostained with anti-FLAG antibody, and analyzed by confocal immunofluorescence microscopy. IncVWT- and IncVS3A-3xFLAG displayed similar inclusion localization  . Three olization . Howevernclusion . AltogetS3A failed to recruit VAP to the inclusion because of a lack of IncV phosphorylation, we assessed IncVS3A apparent molecular weight by western blot analysis of lysates from HeLa cells infected with a C. trachomatis incV mutant expressing IncVWT- or IncVS3A-3xFLAG. Compared to IncVWT-3xFLAG, which as previously observed ran as a doublet corresponding to both phosphorylated and unphosphorylated species of IncV   . Altoget345, S346, and S350 was sufficient to mediate the in vitro IncV–VAP interaction observed upon CK2 phosphorylation of IncV  and one canonical FFAT motif (286E1Y2M3D4A5L6E7292) (F263A or IncVY287A) and in combination (IncVF263A/Y287A), led to a partial and full reduction of the IncV–VAP interaction, respectively, indicating that both FFAT motifs cooperate for VAP binding (265) , or in combination with alanine mutation of the tyrosine residue in position two of the canonical FFAT (IncVT265A/Y287A). In parallel, a phosphomimetic mutation was generated by substituting both T265 to an aspartic acid residue and the proline residue at position 266 (P266) to an alanine residue, as described by T265D/P266A) or in combination with a mutation in the canonical FFAT (IncVT265D/P266A/Y287A). HeLa cells expressing YFP-VAP were infected with the incV mutant strain of C. trachomatis expressing IncVT265A-, IncVT265A/Y287A-, IncVT265D/P266A-, or IncVT265D/P266A/Y287A-3xFLAG. Cells infected with incV mutant strains expressing IncVWT-, IncVY287A-, or IncVF263A/Y287A-3xFLAG were included as controls. The cells were fixed at 24 h pi and immunostained with anti-FLAG antibody, followed by qualitative and quantitative analysis of confocal immunofluorescence microscopy images  . In agreL6E7292) , we had  binding . Recentl binding . The preng (265)  suggestsy images . Althougy images , the redy images , IncVWT ectively . Similarectively  indicati265 of the IncV noncanonical FFAT (IncVT265D/P266A) was sufficient to promote the IncV–VAP interaction in vitro. GST-IncVT265D/P266A failed to interact with MBP-VAPMSP in our vitro assay . The ability of IncVS/A-3xFLAG to recruit VAP to the inclusion was assessed. HeLa cells expressing YFP-VAP were infected with C. trachomatis incV mutant strains expressing either IncVWT-, IncVF263A/Y287A-, or IncVS/A-3xFLAG under an aTc-inducible promoter. The cells were fixed at 24 h pi and analyzed by confocal immunofluorescence microscopy  was sufficient to rescue the ability of the cytosolic domain of IncV expressed in E. coli to interact with the MSP domain of VAP in our VAP binding in vitro assay. As observed before, there was minimal binding of VAPMSP to IncVWT  that are part of CK2 recognition sites  that match the CK2 recognition motifs (S-x-x-D/E) located in a C-terminal domain of IncV are essential for CK2 recruitment to the inclusion, and IncV phosphorylation.Few kinases phosphorylating VAP-dependent tethers have been identified  and how 345, S346, and S350 was not sufficient to promote the IncV–VAP interaction in vitro. While it is possible that the phosphomimetic mutations failed to mimic phosphorylation, it is more likely that additional phosphorylation sites exist. Kinase-substrate recognition is a complex process that goes beyond the simple recognition of a consensus sequence and can involve docking sites away from the phosphorylation sites , and S278, S281, S282, and S284  can be phosphorylated upon priming the serine or tyrosine residue at the last position of the CK2 recognition motif (S/T-x-x-D/E/pS/pY). We therefore propose that alanine substitution of S345, S346, and S350 eliminates an essential docking site for subsequent CK2-mediated phosphorylation of distal residues in the cytosolic domain of IncV, including the serine tracts next to the FFAT motifs (see below). Further investigation of the IncV-dependent recruitment of CK2 to ER-Inclusion MCS could offer some insights into kinase targeting to cellular MCS. Moreover, since intracellular pathogens often mimic cellular processes, our study may have identified CK2 as a regulator of cellular MCS.However, while CK2 was required for ER-Inclusion MCS formation during infection, and sufficient for IncV–VAP interaction in vitro, the introduction of phosphomimetic mutations at Son sites . The cyt265 was detrimental to the IncV-dependent recruitment of VAP to the inclusion. Conversely, phosphomimetic mutation of T265, in the context of an intact canonical-FFAT, resulted in efficient VAP association with the inclusion. These results are similar to those obtained with the phospho-FFAT motif-containing proteins STARD3 and VPS13D  enhances the CERT–VAP interaction (259 and S261) are putative CK2 targets, so it would be interesting to determine if CK2 also plays a role in assembly of the ACBD5-VAP tether. Another example are six serine residues, spread over 21 residues upstream of the core FFAT motif of Miga, shown to facilitate the Miga–VAP interaction  was propagated in HeLa cells as previously described  was obtained from Ted Hackstadt  (Chlamydia strains are routinely tested for mycoplasma contamination.HeLa cells (ATCC CCL-2) and HEK293 cells (ATCC CRL-1573) were maintained in DMEM high glucose (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco) at 37°C and 5% COescribed . The incatories) . All celInserts were generated by PCR using the primers (IDT) and templates listed in K50L plasmid was constructed by cloning the VAPA ORF encoding the K50L mutation (generated my overlapping PCR), into pCMV-N1-YFP using AgeI and HindIII restriction sites.The IncV-3xFLAG construct cloned in the pCMV-IE-N2-3xFLAG vector was previously described . The YFPMSP plasmid was previously described  vector  from the aTc-inducible promoter were generated by overlapping PCR and cloned into KpnI and NotI of the p2TK2) vector . The Tetescribed .C. trachomatis or an incV mutant (incV::bla) were transformed using our previously described calcium-based Chlamydia transformation procedure .Cells were either directly lysed in 2× Laemmli buffer with 10 mM DTT(Dithiothreitol) or IncV was purified as described in the immunoprecipitation and protein purification sections then suspended in a final concentration of 1× Laemmli buffer with 10 mM DTT. Protein samples were separated using Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).2O. Membranes were incubated for 1 hr with shaking at room temperature in blocking buffer . Membranes were then incubated with primary and secondary (horseradish peroxidase [HRP]-conjugated) antibodies diluted in blocking buffer overnight at 4°C and 1 hr at room temperature, respectively, with shaking. ECL Standard western blotting detection reagents (Amersham) were used to detect HRP-conjugated secondary antibodies on a BioRad ChemiDoc imaging system. CK2β was detected using secondary antibodies conjugated to Alexa Fluor 800 on Li-Cor Odyssey imaging system.After SDS/PAGE, proteins were transferred onto nitrocellulose membranes . Prior to blocking, membranes were stained with Ponceau S in 5% acetic acid and washed in dHThe following antibodies were used for immunofluorescence microscopy (IF) and immunoblotting (WB): mouse monoclonal anti-FLAG , rabbit polyclonal anti-CK2β ; rabbit polyclonal anti-thiophosphate ester antibody , rabbit polyclonal anti-MBP , rabbit polyclonal anti-GAPDH , rabbit polyclonal anti-mCherry , rabbit polyclonal anti-actin , HRP-conjugated goat anti-rabbit IgG , HRP-conjugated goat anti-mouse IgG , Alexa Fluor 514-, 800-, or Pacific Blue-conjugated goat anti-mouse IgG .C. trachomatis at a multiplicity of infection (MOI) of 5. 8 h pi, media containing 2 ng/ml aTc was added to the infected cells for 16 hr. Twenty-four hours postinfection, culture media was removed from the cells and 500 μl of lysis buffer , 1% Triton X-100, protease inhibitor mixture EDTA-free [Roche]) was added per well. Cells were lysed for 20 min at 4°C with rotation. Lysates were centrifuged at 16,000 × g for 10 min at 4°C to pellet nuclei and unlysed cells. Cleared lysates were incubated with 10 μl of anti-FLAG M2 affinity beads (Sigma) for 2 hr at 4°C with rotation. The beads were washed with lysis buffer three times. Proteins were eluted with 50 μl of 100 μg/ml 3xFLAG peptide (Sigma) in 1× Tris-buffered saline (TBS). For cells transfected with pCMV-IE-N2-IncV-3xFLAG, cells were not infected, and the remainder of the protocol remained the same starting with removal of media and lysing.800,000 HEK293 cells were seeded into one well of a 6-well plate  and infected the following day with 2, 2.5 μl of 10× PMP buffer (NEB), and 400 units of lambda (λ) phosphatase (NEB) for 24 hr at 4°C. The assay was halted by adding 5 μl of 6× Laemmli buffer with 10 mM DTT. Samples were boiled and 10 μl of sample was then used in SDS–PAGE.Immunoprecipitation was performed as described above with the following changes: The beads were washed with 1× TBS three times and proteins were eluted with 55 μl of 100 μg/ml 3xFLAG peptide (Sigma) in 1× TBS. 20 μl of eluate was combined with 2.5 μl of 10 mM MnClK50L the following day. Twenty-four hours post-transfection, cells were infected with the indicated strain of C. trachomatis at a MOI of 1. Twenty-four hours postinfection, media containing 20 ng/ml aTc  was added for 4 hr to induce expression of IncV-3xFLAG.HeLa cells were seeded onto glass coverslips and transfected with YFP-CK2 (α or β), CFP-VAP, YFP-VAP, or YFP-VAPC. trachomatis were fixed 24 hr postinfection with 4% paraformaldehyde in 1× PBS for 20 min at room temperature then washed with 1× PBS three times. The coverslips were sequentially incubated with primary and secondary antibodies in 0.1% Triton X-100 in 1× PBS for 1 hr at room temperature. For coverslips stained with anti-CK2β, antibodies were diluted in 0.5% Triton X-100% and 5% bovine serum albumin (BSA) in 1× PBS. Coverslips were washed with 1× PBS three times then mounted with glycerol containing DABCO and Tris pH 8.0. Confocal images were obtained using an Andor iXon ULTRA 888BV EMCCD camera and a Yokogawa CSU-W1 Confocal Scanner Unit attached to a Leica DMi8 microscope. 1 μm thick Z slices covering the entirety of the cell were captured. Image analysis was performed using the Imaris software. All the micrographs within a given figure panels are at the same scale.HeLa cells seeded on glass coverslips and infected with Quantification was performed using the Imaris imaging software. A step-by-step illustration of the quantification method is presented in t-tests or one-way ANOVA with multiple comparisons was performed.Each experiment was performed in triplicate with at least 20–30 inclusions analyzed per condition per replicate. Unless specified, data from three independent replicates are combined into a single graph. Each point on the graph represents a single inclusion with the average value and SEM shown. Student’s MSP, GST-IncV167-363, GST-IncV167-363 S/D, or MBP-VAPMSP was induced for 2 hr by the addition of isopropyl-β-ᴅ-thiogalactopyranoside  to a 10 ml culture of E. coli BL21-λDE3 at OD 0.8. Bacterial pellets were stored at −80°C. Frozen pellets were thawed and resuspended in 800 μl sonication buffer ). The samples were sonicated using five 5-s pulses at 40% power then centrifuged at 13,000 × g for 10 min at 4°C. 40 μ of glutathione Sepharose beads (GE) for GST-tagged constructs and 40 μl of Amylose resin for MBP-tagged constructs were washed three times with sonication buffer then added to the cleared lysate and incubated for 2 hr at 4°C with rotation. The beads were washed three times in TBS.Expression of GST, GST-VAP2O. The membrane was then probed with anti-thiophosphate ester antibodies to detect phosphorylated proteins which were detected with HRP-conjugated secondary antibodies.Protein-bound glutathione Sepharose beads were resuspended in 1× NEBuffer for Protein Kinases supplemented with 1 mM ATPγS and 10 units of CK2 (NEB) and incubated at 30°C for 45 min. P-nitrobenzyl mesylate (PNBM) was added to the kinase reaction at a final concentration of 2.4 mM for 2 hr at room temperature in the dark. The PNBM alkylation reaction was quenched by adding an equal volume of 2× Laemmli buffer. Proteins were separated using SDS–PAGE on a 12% acrylamide gel then transferred to a nitrocellulose membrane. The membrane was stained with Ponceau S in 5% acetic acid to detect total protein then washed in dH167-363, GST-IncV167-363 S/D, and MBP-VAPMSP were purified as described in protein purification. MBP-VAPMSP was eluted from amylose resin using 100 μl 1× TBS supplemented with 10 mM maltose monohydrate. GST, GST-IncV167-363, or GST-IncV167-363 S/D attached to glutathione beads were washed three times in sonication buffer. 500 μl of sonication buffer containing 1.25 μg MBP-VAPMSP was added to each tube with GST beads and binding was allowed to occur overnight at 4°C with rotation. Following overnight binding, beads were washed three times in 1× TBS. After the final wash, all liquid was removed from the beads which were then suspended in 20 μl 2× Laemmli buffer. The entire sample was separated by SDS–PAGE, proteins transferred to a nitrocellulose membrane which was stained with Ponceau S to detect the GST construct then probed with anti-MBP to detect MBP-VAPMSP.First, GST, GST-IncVFirst, the phosphatase assay was performed with the following changes: 1,000,000 HEK293 cells stably transfected with pCMV-IE-N2-IncV-3xFLAG were seeded per 6 well. Six wells were lysed in 500 μl lysis buffer each and lysates from two wells were combined. 10 μl of anti-FLAG beads were added per 1000 μl cleared lysate for 2 hr at 4°C with rotation. All beads were combined after the first wash, and proteins were eluted in 150 μl elution buffer (130 μl eluate collected).MSP were purified as described in protein purification. Per phosphatase assay tube: 1.5 μg of GST or GST-VAPMSP attached to beads  was suspended in 500 μl lysis buffer then added to tubes containing the IncV-3xFLAG-containing eluate (±phosphatase treatment). Binding was allowed to occur overnight at 4°C with rotation.Next, GST and GST-VAPTo confirm that IncV dephosphorylation was successful, a set of control tubes were incubated with beads alone (no GST construct) in lysis buffer to mimic experimental conditions.Twenty-four hours after binding, beads were washed three times in 1× TBS. After the final wash, all liquid was removed from the beads which were then suspended in 20 μl 2× Laemmli buffer. The entire sample was separated by SDS–PAGE, proteins transferred to a nitrocellulose membrane which was stained with Ponceau S to detect the GST construct then probed with anti-FLAG to detect IncV-3xFLAG.167-363, and MBP-VAPMSP were purified as described in protein purification. MBP-VAPMSP was eluted from amylose resin using 100 μl 1× TBS supplemented with 10 mM maltose monohydrate. 1.5 μg of GST or GST-IncV167-363 attached to glutathione beads  or beads alone was suspended in 1× NEBuffer for Protein Kinases with 200 μM ATP (Thermo) and 100 units of CK2 (NEB) at 30°C for 45 min. Beads were washed three times in sonication buffer. 1.25 μg MBP-VAPMSP suspended in 500 μl sonication buffer was added to each tube with beads and binding was allowed to occur overnight at 4°C with rotation. Twenty-four hours after binding, beads were washed three times in 1× TBS. After the final wash, all liquid was removed from the beads which were then suspended in 20 μl 2× Laemmli buffer. The entire sample was separated by SDS–PAGE, proteins transferred to a nitrocellulose membrane which was stained with Ponceau S to detect the GST construct then probed with anti-MBP to detect MBP-VAPMSP.First, GST, GST-IncVImmunoblots and Ponceau S staining were quantified using the ImageJ software (NIH). The immunoblot band intensity was normalized to the Ponceau S band intensity and the fold change determined relative to wild-type or untreated conditions.2 for 3 days. The total volume for one 96 well was 120 μl. The CSNK2B target sequence for each individual siRNA duplex was: A, CAACCAGAGUGACCUGAUU; B, GACAAGCUCUAGACAUGAU; C, CAGCCGAGAUGCUUUAUGG; D, GCUCUACGGUUUCAAGAUC. The efficacy of the knock down was quantified using the ImageJ software (NIH). The CK2 band intensity was normalized to the GAPDH band intensity and the knockdown efficacy was determined relative to the mock condition.CK2 was depleted from cells using a pool of four siRNA duplexes or each duplex individually that was transfected with Dhamafect 1 transfection reagents. On day 0, one volume of 200 nM siRNA in siRNA buffer was incubated with one volume of 5 μl/ml of Dharmafect 1 transfection reagent in DMEM high glucose in a well for 20 min at room temperature. Two volumes of DMEM High Glucose supplemented with 20% fetal bovine serum and 200,000 HeLa cells per ml were added to the well. Cells were incubated at 37°C with 5% COCK2 was inactivated using the CK2-specific inhibitor CX-4945  or GO289 , as described in the result sections.C. trachomatis expressing IncVWT or IncVS3A-3xFLAG under the control of the aTc-inducible promoter. Twenty hours postinfection cells were incubated in the presence of 20 ng/ml of aTc. Twenty-four hours postinfection, the samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 60 min at RT. After fixation, cells were scraped off and pelleted, as described previously  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Chlamydia inclusion membrane contact sites" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Dominique Soldati-Favre as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Lena Pernas (Reviewer #2).Thank you for submitting your article "Phospho-regulation accommodates Type III secretion and assembly of a tether of ER-The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Although they find the work interesting, the reviewers raise caveats that could invalidate the interpretations of some results. Additional experiments are needed to clarify these. In particular, while the title and abstract of the paper put a great emphasis on membrane contact sites, these sites are, per se, not shown anywhere in the paper, and the effects of IncV phosphorylation on their establishment are inferred indirectly. Another thing requiring clarification is an incongruity with the proposed model; IncV phosphorylated by CK2 in vitro appears to bind VAP efficiently  despite the fact that CK2 is unlikely to phosphorylate the Threonine residue of the phosphoFFAT, and therefore, that this residue is likely unphosphorylated in this assay. There is therefore a discrepancy with the results of Figure 1, indicating that phosphorylation of the phosphoFFAT is necessary for VAP binding. The same can be said for the phosphomimetic mutant of figure 6D: VAP appears to bind robustly despite the threonine not being phosphorylated.Here are important points to address:C. trachomatis inclusion in the absence of IncV phosphorylation.1) The authors state in the title that "Phospho-regulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion contact sites." However, nowhere in the manuscript do they actually analyze contact sites (only VAP recruitment by IF). As done in a previous study published by the authors , electron microscopy (EM) in this study is required to depict the lack of ER-contact sites to the 2) The discrepancy pointed above likely stems from the fact that different assays are used. How would a Threonine to alanine mutant bind VAP in the GST pull down assay? What about a phosphomimetic (see below)?3) As it stands, the only data supporting a phosphoFFAT are based on a single threonine to alanine mutation. This is insufficient to clearly demonstrate that this is a phosphoFFAT, as a T to A mutation could mess up the protein in various ways, unrelated to phosphorylation. It is therefore important to better characterize the phosphoFFAT motif both in vitro and in vivo. Can a phosphomimetic be generated? phosphomimetic FFAT requires, in addition to mutate the T to D, to mutate the prolines to alanines to allow flexibility in the chain and allow the aspartate to work as a phosphomimic . Can this rescue VAP recruitment? Alternatively, can the phosphorylation be demonstrated by phosphoproteomics in vivo?4) The model implies that Phosphorylation of the C-terminal S residues is necessary to trigger the phosphorylation of the phsophoFFAT T-residue. There is however no evidence for that. It is equally plausible that S-residue and T-residue phosphorylation contribute independently to strengthen the interaction with VAP . Can phosphoproteomics be used to assess if S-residue mutation or CK2 inhibition affects T phosphorylation? Can a phosphoFFAT phosphomimetic (see above) bypass the need for CK2 phosphorylation? Otherwise, it is equally plausible that the phosphoFFAT is constitutively phosphorylated, and that phosphorylation of the C-terminal S-residues are only necessary to phosphorlylate the S-track around the FFAT motifs. If both models cannot be distinguished, then both should be presented.5) Using CK2 inhibitors, siRNA, and IncV mutants, the authors arrive at a model where CK2 is recruited to IncV at inclusions , phosphorylates IncV , and this then enables an interaction between IncV and VAP  causing the creation of an MCS. Demonstrating this order of event in vivo as well would strengthen the model. Could the authors provide a time-lapse, or other sort of kinetic analysis of CK2 and VAP recruitment to the bacterial vacuole to support their model that CK2 phosphorylation enables ER tethering (or at least to show that VAP is not recruited prior to CK2)?6) Related to the point above, the association of CK2 to the inclusion in the presence of CX4945 should be analysed qualitatively and quantitively. The model predicts that CK2 should colocalize with IncV at inclusions whether or not it is active. IncV should bind its recognition sequences on IncV, and unless there is a positive feedback loop (where more phosphorylation causes more recruitment of the kinase), you should be able to observe CK2 at inclusions in the absence of VAP recruitment, showing that subsequent association of VAP to the inclusion depends on CK2-dependent phosphorylation of IncV.7) The siRNA-mediated knockdown of CK2 was incomplete. If CK2 is not critical for host cell survival, a CRISPR/Casß gene knock-out of CK2α and CK2ß might provide the result that the siRNA approach did not achieve-the authors could then simply assess IncV phosphorylation in 293 cells in the absence of infection.Chlamydia inclusion membrane contact sites" for further consideration by eLife. Your revised article has been evaluated by Dominique Soldati-Favre (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Phospho-regulation accommodates Type III secretion and assembly of a tether of ER-You can see that a third reviewer has been brought into the process, who was not able to see and comment on your initial work. This reviewer has a number of concerns, especially concerning the fact that all of the study was performed using overexpressed IncV rather than endogenously expressed from Chlamydia. Although it would be unfair to ask you to address the concerns in full at this stage, we would like to encourage you to look into this matter. In particular, as was already pointed out in the first round of review, it is regrettable that phosphoproteomics proved challenging at nailing an important point of the paper. The reviewer suggests, as an alternative, to utilise anti-phosphoserine antibodies that can be used to probe immunoprecipitated endogenous IncV or to decorate endogenous IncV at the MCS in immunofluorescence.In addition, reviewer #1 has a remaining unaddressed concern about the function of the phosphoFFAT. Your pull-down assays show that IncV phosphorylated in vitro by CK2 binds robustly to VAP, despite a presumed absence of phosphorylation of the phosphoFFAT . The reviewer proposes alternative interpretations of the data and suggests performing the pull down with phosphomimic and phosphor-null mutants of IncV (phosphorylated in vitro by CK2) to settle these alternatives.Reviewer #1 (Recommendations for the authors):This revised version brings new and exciting data, consolidating the story.The effect of a phospho mutant on the establishment of contact sites is convincing. The partial bypass of CK2 by the FFAT phosphomimic is a compelling evidence that the phosphoFFAT is not constitutively phosphorylated, but requires CK2 phosphorylation on other residues to become phosphorylated. The reorganisation of the manuscript, and keeping the phosphoFFAT to the end is also facilitating the reading.Further clarification below would help the understanding of this complex story.– Asked how mutations in the phosphoFFAT motif would influence VAP binding in the GST pull down assay, the author states in their response to the reviewers that "IncVWT does not bind to VAP in this assay". This is demonstrably wrong, as shown in Figure 2D, where IncVWT binds robustly to VAP, provided it was phosphorylated by CK2 beforehand. Since CK2 is unlikely to be responsible for phosphoFFAT phosphorylation, and that the phosphoFFAT is likely unphosphorylated in this assay, then it means that phosphoFFAT phosphorylation is not necessary for VAP binding. There could be several explanations for this surprising result.a. One possibility is that the binding observed here is suboptimal, because only wrought by the canonical FFAT. In this case a phosphomimic FFAT should increase binding, while a phosphonull FFAT should behave quite exactly like the WT (i.e. this assay should be impervious to the F263A and T265A mutations).b. Another possibility is that, in contradiction to expectations, CK2 does indeed phosphorylate the FFAT motif, in which case a phosphonull mutation in FFAT should abrogate binding (while a phosphomimic should have no effect).c. A third possibility is that the phosphoFFAT is necessary for something else than the direct binding of VAP. For instance, it could be necessary in vivo for recruitment or activation of CK2, a step that may be bypassable in vitro given the high amount of CK2.These should be clarified. The same concern applies for Figure 7D. Here the S/D mutations in the serine track adjacent to the FFATs is sufficient for VAP binding even though the phosphoFFAT motif is clearly non-phosphorylated here. While, here, possibility b. is not existing, a. and c. remain to be clarified. Again, a phosphomimic FFAT should increase binding, while a phosphonull FFAT should behave quite exactly like the WT.– In the same vein, Figure 5 – supplement figure 4 shows that phosphomimics mutants of the three C-terminal serines do not bind VAP. This is in stark contrast with figure 2D, where CK2-mediated phosphorylation leads to robust VAP binding. This indicates either of two things. Either the phosphomimics mutation do not actually mimic phosphorylation, or CK2 phosphorylates more than the three target sites at the C-terminus. The second explanation is likely favoured by the authors, but this is not explicitly stated. What are the additional residues that could be phosphorylated by CK2? There are arguments against the idea the CK2 phosphorylates the phosphoFFAT. Could CK2 phosphorylate the serine track adjacent to the FFATs?– Experiment with the VapK50L are not entirely conclusive. Indeed, if the mutant was not able to bind to phosphoFFAT, then this mutant should bind to the T265A mutant as much  as to WT IncV. This is not the case. Therefore, at face value, the VapK50L appears more like a mutant of general reduced activity (for instance less expressed) than a phosphoFFAT-specific mutant. A positive control would be to test ER-recruitement of a FFAT protein (non-phosphoFFAT), which should bind this mutant as well as a wild-type.– There is a problem in the assembly of fig6. In panel B, A picture that represented a double mutant in the previous version, now represents a single mutant. Moreover, the double mutant shows robust VAP recruitment.Reviewer #3 (Recommendations for the authors):C. trachomatis inclusion and the endoplasmic reticulum (ER) of the host cell. They also analysed a phosphomimietic mutant.In this revised manuscript the authors have performed additional experiments to address the fair comments raised by the previous two referees. This predominantly involved electron microscopy to further establish the link between phospho-IncV and the formation of membrane contact sites (MCS) that form between the Strengths:As a new referee, there are some more general comments on the above and also the content of the original manuscript. Firstly, it should be recognised that the authors have performed a considerable amount of work, including the generation of various genetic tools, to study the role of the virulence factor IncV in the formation of ER-inclusion MCS. Understanding mimicry by bacterial effectors in an important topic, of interest to those studying microbial pathogenesis and also to the cell biology community who study related canonical mechanisms. Indeed, it is often the case that such cellular microbiology experiments illuminate new aspects of fundamental cellular mechanisms as is potentially the case here when studying FFAT motifs and their relationship to VAT and ER-MCS / other MCS in uninfected cells.E. coli translocated intimin receptor (Tir). Reporting another transmembrane effector, this time at the intracellular interface, that becomes phosphorylated during host-pathogen interaction, has in itself significant novelty.Post-translational modifications of translocated bacterial effectors have rarely been reported, and in the case of phosphorylation, the archetypal example is the enteropathogenic That said, there are striking similarities between IncV FFAT and phospho-FFAT motifs recently identified in eukaryotic cells . While this on the one hand enhances the mimicry story, it dents the novelty of phospho-FFAT per se. It seems the kinases involved in the phosphorylation of phospho-FFAT in cells are yet to be resolved.Weaknesses:While this reviewer appreciates the authors attention to detail in trying to study the full biochemical pathway leading to IncV phosphorylation, some intricacies of this story are less well developed and the model in its current form remains speculative. There is no doubt that studying Chlamydia is challenging, but from both the bacterial and host angle there are conclusions drawn from combinations of in vitro and cell culture based approaches, often in combination with overexpression of the virulence factor and/or the putative host targets, which compounds interpretation. I expect as a result, some of the findings correlate with the model, while others do not. Rather than presenting all the current data, it might be preferable to streamline the story focusing on the phosphorylation of IncV to establish firm groundwork for future detailed studies of the mechanism.To this reviewer, the firmest aspects of the work are that:- IncV is phosphorylated (Thr/Ser)- IncV can recruit CK2 via C-terminal Ser residues- IncV phosphyorylation relates to VAP recruitment and the formation of ER-MCSC. trachomatis) has been previously shown to enhance MCS formation, and mislocalise or over titrate IncV around the inclusion membrane and one wonders whether this affects the phenotypes. Similarly VAP and CK2 are overexpressed as a fusion protein in most of the experiments, and endogenous CK2 visualised only when IncV-FLAG is over-expressed. Although these approaches implicate IncV, VAP and CK2 in a common event, without true endogenous data details of the mechanistic pathway cannot be inferred.A major limitation is the lack of information on the post-translational modifications of endogenous IncV. Which residues are phosphorylated during infection when the protein is not overexpressed? Over-expression , the authors speculate on the action of CK2 and host kinases responsible for IncV phosphorylation. This seems quite subjective, especially given the promiscuity and constitutive activation of CK2 (>300 cellular substrates). Their in vitro data support a view that CK2 can bind IncV, but not that this is necessarily the relevant kinase responsible for the phosphorylaton of key FFAT residues in IncV during infection. Why would this be a static recruitment? Did the authors perform any broader kinase screens or use additional kinase inhibitors to verify in a less directed manner the relevance of CK2 and/or implicate other host kinase families?Also relating to CK2, what is the effect of CK2 knockdown on other substrates and cellular processes? How much CK2 is required in the cell to sustain essential functions in apoptosis and cell proliferation for example? Assuming chemical inhibition also inhibits these processes do they have any pleiotropic effects during infection? What is the effect of CX-4945 and GO289 on known CK2 substrates? How were these inhibitors titrated?Caution should be taken in the interpretation of in vitro kinase assays. Just because CK2 can phosphorylate IncV in vitro does not in itself mean this is the relevant kinase during infection.What is the effect of mutating IncV T265 during infection? Is there an influence on infectivity or ER-inclusion MCS?Thank you very much for your letter. We would first like to apologise for the complicated path your manuscript had to go through with the addition of a third reviewer at the revision stage. This third reviewer was involved in reviewing your 1st manuscript but could not manage to complete the evaluation for reasons beyond their control. To avoid any further delay, we therefore had to complete the first round of review without the unique expertise of this reviewer. But because of this expertise, we decided to bring them back at the 2nd round, aware that it created an awkward situation. Because of that, we agreed to not push forward most of the 3rd reviewer's concern and to focus only on one, i.e. that all evidence herein, and in your previous manuscript come from over-expression of the bacterial effector and/or the host targets CK2/VAP.The model that you are putting forward sets a strong precedent and a bold claim, and surely you would want it to reflect the behaviour of endogenous proteins and not an artefact of protein overexpression.The reviewer would like to see a simple experiment using wild-type Chlamydia in non-manipulated cells demonstrating that these modifications occur. The reviewer suggests to use an IncV antibody to probe WT chlamydia infections, which, I realise, might not be available, or to purify phosphoproteins and probe if endogenous IncV is in the lot. The concern that overexpression can generate artefacts by triggering non-natural interactions and artificially expanding contact sites is a valid one. Irrespective of the unfortunate circumstances that led this concern to come to light belatedly, this is a point that you should be keen to address unless you had good grounds to dismiss it.Concerning the point of reviewer #1, we beg to differ that phospho-FFAT is a minor part of your study. Phospho-FFAT is mentioned in your abstract, in three main figures, and is central to your model in figure 8. Right now, your data show a major discrepancy with this model.The motif that you identify does appear to be necessary for VAP recruitment to inclusions, as a T>A mutation decreases VAP fluorescence there , and this effect appears to be downstream of the C-terminal serine phosphorylation, since a T>D mutation partially rescues the S3A mutation. This is consistent with the idea that this motif could act as a phosphoFFAT, through which VAP binds IncV.However, there are also discrepant data; this motif does not need to be phosphorylated to allow IncV-VAP binding . Therefore, while this motif appears to be doing something, it is unclear that it acts as a phosphoFFAT. Indeed, the S/D mutations appear to bypass the need for T265 phosphorylation , which, following the same logic as above, would seem to indicate that the serine track phosphorylation is downstream of T265 phosphorylation, and that this motif doesn't act as a phosphoFFAT but instead as a primer for serine tract phosphorylation.So, an important unaddressed point remains; is it really a phosphoFFAT? Is it fair to call it a phosphoFFAT? Or is this motif doing something entirely different?Without evidence that this phosphorylated motif binds VAP directly (hence in vitro), it seems premature to call it a phosphoFFAT. And as you concede, the VAPk50l mutant does little to lift the ambiguity.We would like to renew our apologies for the way the review process has gone and assure you that this has been beyond our control.Ultimately, authors and editors want to publish solid manuscripts that stand the test of time. Therefore, we would like to invite you to consider the above concerns and address them the best you can. We are open to further discussions if you think we made an incorrect evaluation of your data, or if other circumstances prevent you from doing requested experiments. Essential revisions:Although they find the work interesting, the reviewers raise caveats that could invalidate the interpretations of some results. Additional experiments are needed to clarify these. In particular, while the title and abstract of the paper put a great emphasis on membrane contact sites, these sites are, per se, not shown anywhere in the paper, and the effects of IncV phosphorylation on their establishment are inferred indirectly. Another thing requiring clarification is an incongruity with the proposed model; IncV phosphorylated by CK2 in vitro appears to bind VAP efficiently  despite the fact that CK2 is unlikely to phosphorylate the Threonine residue of the phosphoFFAT, and therefore, that this residue is likely unphosphorylated in this assay. There is therefore a discrepancy with the results of Figure 1, indicating that phosphorylation of the phosphoFFAT is necessary for VAP binding. The same can be said for the phosphomimetic mutant of figure 6D: VAP appears to bind robustly despite the threonine not being phosphorylated.Here are important points to address:C. trachomatis inclusion in the absence of IncV phosphorylation.1) The authors state in the title that "Phospho-regulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion contact sites." However, nowhere in the manuscript do they actually analyze contact sites (only VAP recruitment by IF). As done in a previous study published by the authors , electron microscopy (EM) in this study is required to depict the lack of ER-contact sites to the WT or with IncVS3A, by electron microscopy. IncVS3A was chosen because mutation of the corresponding serine residues prevents CK2 association with the inclusion, IncV phosphorylation and VAP recruitment to the inclusion . IncVWT was used as a positive control based on our previous published data showing robust MCS formation under these conditions . Quantification of the proportion of inclusion membrane associated with the ER confirmed a reduction in ER-Inclusion MCS upon IncVS3A expression, compared to IncVWT. We note that some levels of ER-Inclusion MCS is expected with IncVS3A because of redundant tethering mechanism(s) . The corresponding data is presented in Figure 5G-H and Lines 360-375.To directly demonstrate that the defect in VAP recruitment observed when IncV phosphorylation is prevented correlates with a defect in ER-Inclusion MCS formation, we performed ultra-structural analysis of cells infected with an IncV mutant complemented with IncV2) The discrepancy pointed above likely stems from the fact that different assays are used. How would a Threonine to alanine mutant bind VAP in the GST pull down assay? What about a phosphomimetic (see below)?T625A mutant in the GST pull down assay. IncVWT does not bind to VAP in this assay, so we did not expect that a loss of function of the phospho-FFAT to lead to VAP binding. Please see point (3) for our response regarding the phosphomimetic mutant.We did not test the IncV3) As it stands, the only data supporting a phosphoFFAT are based on a single threonine to alanine mutation. This is insufficient to clearly demonstrate that this is a phosphoFFAT, as a T to A mutation could mess up the protein in various ways, unrelated to phosphorylation. It is therefore important to better characterize the phosphoFFAT motif both in vitro and in vivo. Can a phosphomimetic be generated? phosphomimetic FFAT requires, in addition to mutate the T to D, to mutate the prolines to alanines to allow flexibility in the chain and allow the aspartate to work as a phosphomimic . Can this rescue VAP recruitment? Alternatively, can the phosphorylation be demonstrated by phosphoproteomics in vivo?We conducted the following experiments to further characterize the phospho-FFAT.T265D/P266A) as explained in Di Mattia et al.  and tested the ability of the phosphoFFAT phosphomimetic mutant to interact with VAP in vitro and during infection. IncVT265D/P266A was not sufficient to rescue VAP binding in vitro . However, IncVT265D/P266A was as efficient as IncVWT to recruit VAP to the inclusion .1- We generated a phosphomimetic mutation  . VAPK50L recruitment to IncVWT-positive inclusions was intermediate, due to the contribution of IncV canonical FFAT, which was confirmed by the lack of VAPK50L recruitment to IncVY287A-positive inclusions .2- We tested the recruitment of VAPCollectively, these results are in line with the characterization of the phospho-FFAT of STARD3 and VPS13D  and support that the non-canonical FFAT of IncV is a phospho-FFAT that contributes to VAP binding. The corresponding data is presented in Figure 6 – supplemental figure 2 and Figure 6B-E (Lines 388-448) and discussed in the Discussion section (Lines 550-571).Performing phosphoproteomics has been one of our main goals with this project. Unfortunately, preparing samples to analyze IncV phosphorylation status by phosphoproteomics has been technically challenging and unfruitful so far. This was acknowledged on lines 577-580 of the Discussion section.4) The model implies that Phosphorylation of the C-terminal S residues is necessary to trigger the phosphorylation of the phsophoFFAT T-residue. There is however no evidence for that. It is equally plausible that S-residue and T-residue phosphorylation contribute independently to strengthen the interaction with VAP . Can phosphoproteomics be used to assess if S-residue mutation or CK2 inhibition affects T phosphorylation? Can a phosphoFFAT phosphomimetic (see above) bypass the need for CK2 phosphorylation? Otherwise, it is equally plausible that the phosphoFFAT is constitutively phosphorylated, and that phosphorylation of the C-terminal S-residues are only necessary to phosphorlylate the S-track around the FFAT motifs. If both models cannot be distinguished, then both should be presented.Please see our response above, in point 3, regarding the technical challenges associated with a phophoproteomics approach.T265D/P266A/S3A-positive inclusions, which supports an indirect role for CK2 in the phosphorylation of IncV phospho-FFAT, despite the fact that T265 is not a direct CK2 target. The corresponding data is presented in Figure 6F-G (Lines 449-458) and discussed in the Discussion section (Lines 572-577 ).To addressed if a phospho-FFAT phosphomimetic can bypass the need for CK2, we generated a phopho-FFAT phosphomimetic mutant in the background of the S3A mutation (The S3A mutation prevents CK2 mediated phosphorylation of IncV). Our results indicate an intermediate VAP recruitment to IncV5) Using CK2 inhibitors, siRNA, and IncV mutants, the authors arrive at a model where CK2 is recruited to IncV at inclusions , phosphorylates IncV , and this then enables an interaction between IncV and VAP  causing the creation of an MCS. Demonstrating this order of event in vivo as well would strengthen the model. Could the authors provide a time-lapse, or other sort of kinetic analysis of CK2 and VAP recruitment to the bacterial vacuole to support their model that CK2 phosphorylation enables ER tethering (or at least to show that VAP is not recruited prior to CK2)?Analysis of the sequential recruitment of CK2 and VAP to the inclusion, using time-lapse video microscopy, proved to be technically challenging. Instead, we took advantage of the fact that the kinase activity of CK2 is not required for CK2 recruitment to the inclusion (please see point 6 below). In our experimental setup, we first allowed for CK2 association to the inclusion in the presence of the CK2 inhibitor, which prevents VAP recruitment, and then washed the inhibitor away and assayed for VAP recruitment. Under these conditions, VAP association with the inclusion was observed 1 hour after removal of the CK2 inhibitor, supporting the model that CK2 is recruited first and that phosphorylation enables VAP-dependent ER tethering. The corresponding data is presented in Figure 4C-E (Lines 294-314).6) Related to the point above, the association of CK2 to the inclusion in the presence of CX4945 should be analysed qualitatively and quantitively. The model predicts that CK2 should colocalize with IncV at inclusions whether or not it is active. IncV should bind its recognition sequences on IncV, and unless there is a positive feedback loop (where more phosphorylation causes more recruitment of the kinase), you should be able to observe CK2 at inclusions in the absence of VAP recruitment, showing that subsequent association of VAP to the inclusion depends on CK2-dependent phosphorylation of IncV.In Figure 4A-B (Lines 284-293), we provide qualitative and quantitative evidence that the kinase activity of CK2 is not required for CK2 association with the inclusion, supporting the model that CK2 recruitment precede VAP and that VAP is only recruited once CK2 phosphorylates IncV The latter is further supported by our answer to point 5 above.7) The siRNA-mediated knockdown of CK2 was incomplete. If CK2 is not critical for host cell survival, a CRISPR/Casß gene knock-out of CK2α and CK2ß might provide the result that the siRNA approach did not achieve-the authors could then simply assess IncV phosphorylation in 293 cells in the absence of infection.-/- cell line reported so far is a CK2-/- C2C12 myotube cell line  and the lack of CK2 was associated with phosphoproteomics perturbations that could potentially confound our results. We also note that the myotube nature of the C2C12 cell line would not be compatible with Chlamydia infection.When considering our options for genetic targeting of CK2, we favored silencing over a knockout, because ck2 is an essential gene , but also because we did notice that ck2 silencing was affecting the growth of the cells to some extent. To our knowledge, the only CK2To strengthen our results and rule out any potential off target effect of the CK2 inhibitor CX-4945, we complemented our approach by using an independent CK2 inhibitor, GO289 . As observed with CX-4945, GO289 prevented IncV phosphorylation and VAP recruitment to the inclusion. The corresponding data is presented in Figure 3 – supplemental figure 3 (Lines 273-277).ReferencesBorgo, C., Cesaro, L., Hirota, T., Kuwata, K., D'Amore, C., Ruppert, T., Blatnik, R., Salvi, M., & Pinna, L. A. . Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach. Eur J Med Chem, 214, 113217. https://doi.org/10.1016/j.ejmech.2021.113217Buchou, T., Vernet, M., Blond, O., Jensen, H. H., Pointu, H., Olsen, B. B., Cochet, C., Issinger, O. G., & Boldyreff, B. . Disruption of the regulatory β subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Mol Cell Biol, 23(3), 908-915. https://doi.org/10.1128/MCB.23.3.908-915.2003Di Mattia, T., Martinet, A., Ikhlef, S., McEwen, A. G., Nomine, Y., Wendling, C., Poussin-Courmontagne, P., Voilquin, L., Eberling, P., Ruffenach, F., Cavarelli, J., Slee, J., Levine, T. P., Drin, G., Tomasetto, C., & Alpy, F. . FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts. EMBO J, 39(23), e104369. https://doi.org/10.15252/embj.2019104369Franchin, C., Borgo, C., Cesaro, L., Zaramella, S., Vilardell, J., Salvi, M., Arrigoni, G., & Pinna, L. A. . Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells. Cell Mol Life Sci, 75(11), 2011-2026. https://doi.org/10.1007/s00018-017-2705-8Guillen-Samander, A., Leonzino, M., Hanna, M. G., Tang, N., Shen, H., & De Camilli, P. . VPS13D bridges the ER to mitochondria and peroxisomes via Miro. J Cell Biol, 220(5). https://doi.org/10.1083/jcb.202010004Stanhope, R., Flora, E., Bayne, C., & Derre, I. . IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole. Proc Natl Acad Sci U S A, 114(45), 12039-12044. https://doi.org/10.1073/pnas.1709060114Zhang, R., Ou, H. Y., & Zhang, C. T. . DEG: a database of essential genes. Nucleic Acids Res, 32(Database issue), D271-272. https://doi.org/10.1093/nar/gkh024You can see that a third reviewer has been brought into the process, who was not able to see and comment on your initial work. This reviewer has a number of concerns, especially concerning the fact that all of the study was performed using overexpressed IncV rather than endogenously expressed from Chlamydia. Although it would be unfair to ask you to address the concerns in full at this stage, we would like to encourage you to look into this matter. In particular, as was already pointed out in the first round of review, it is regrettable that phosphoproteomics proved challenging at nailing an important point of the paper. The reviewer suggests, as an alternative, to utilise anti-phosphoserine antibodies that can be used to probe immunoprecipitated endogenous IncV or to decorate endogenous IncV at the MCS in immunofluorescence.In addition, reviewer #1 has a remaining unaddressed concern about the function of the phosphoFFAT. Your pull-down assays show that IncV phosphorylated in vitro by CK2 binds robustly to VAP, despite a presumed absence of phosphorylation of the phosphoFFAT . The reviewer proposes alternative interpretations of the data and suggests performing the pull down with phosphomimic and phosphor-null mutants of IncV (phosphorylated in vitro by CK2) to settle these alternatives.Reviewer #1 (Recommendations for the authors):This revised version brings new and exciting data, consolidating the story.The effect of a phospho mutant on the establishment of contact sites is convincing. The partial bypass of CK2 by the FFAT phosphomimic is a compelling evidence that the phosphoFFAT is not constitutively phosphorylated, but requires CK2 phosphorylation on other residues to become phosphorylated. The reorganisation of the manuscript, and keeping the phosphoFFAT to the end is also facilitating the reading.Further clarification below would help the understanding of this complex story.– Asked how mutations in the phosphoFFAT motif would influence VAP binding in the GST pull down assay, the author states in their response to the reviewers that "IncVWT does not bind to VAP in this assay". This is demonstrably wrong, as shown in Figure 2D, where IncVWT binds robustly to VAP, provided it was phosphorylated by CK2 beforehand. Since CK2 is unlikely to be responsible for phosphoFFAT phosphorylation, and that the phosphoFFAT is likely unphosphorylated in this assay, then it means that phosphoFFAT phosphorylation is not necessary for VAP binding. There could be several explanations for this surprising result.a. One possibility is that the binding observed here is suboptimal, because only wrought by the canonical FFAT. In this case a phosphomimic FFAT should increase binding, while a phosphonull FFAT should behave quite exactly like the WT (i.e. this assay should be impervious to the F263A and T265A mutations).b. Another possibility is that, in contradiction to expectations, CK2 does indeed phosphorylate the FFAT motif, in which case a phosphonull mutation in FFAT should abrogate binding (while a phosphomimic should have no effect).c. A third possibility is that the phosphoFFAT is necessary for something else than the direct binding of VAP. For instance, it could be necessary in vivo for recruitment or activation of CK2, a step that may be bypassable in vitro given the high amount of CK2.These should be clarified. The same concern applies for Figure 7D. Here the S/D mutations in the serine track adjacent to the FFATs is sufficient for VAP binding even though the phosphoFFAT motif is clearly non-phosphorylated here. While, here, possibility b. is not existing, a. and c. remain to be clarified. Again, a phosphomimic FFAT should increase binding, while a phosphonull FFAT should behave quite exactly like the WT.As stated in our letter, the phospho-FFAT constitutes a minor aspect of our revised manuscript. As such, and based on what is discussed below, we do not believe that additional experiments will significantly change the impact and overall message of our study.K50L mutant (Please see below).In response to the initial review, we added Figure 6, a multi-panel figure dedicated to the phospho-FFAT which includes a significant amount of data to characterize the phospho-FFAT during infection, using established tools to study phospho-FFAT motifs such as loss- and gain-of-function mutations that affect the phospho-FFAT/VAP interface. Our results, for which we have discussed the limitations, are consistent with phosphorylation of the phospho-FFAT motif during infection. Our conclusions were not challenged by the reviewers. However, in light of reviewer 1 comment’s, we agree to further discuss the potential limitations of the results obtained with the VAPReviewer 1 noted some incongruity in our model regarding the role of the phospho-FFAT in the IncV-VAP interaction, because, in vitro, using purified proteins, IncV interacts with VAP only when IncV is phosphorylated by CK2, despite the fact that the phospho-FFAT is not a CK2 target and is therefore presumably not phosphorylated in this particular experiment . Reviewer 1 concluded that this result indicates that “phospho-FFAT phosphorylation is not necessary for VAP binding”, suggested additional experiments to explain this result, and proposed three possible outcomes with the following explanations: (a) suboptimal binding, (b) CK2 phosphorylation of the phospho-FFAT, and (c) phospho-FFAT involved in something else.We realize that we may have partially misunderstood points 2 and 3 of the original review. More specifically, in point 2 we took “How would a threonine to alanine mutant bind VAP in the GST pull down assay” literally, and did not understand that by “GST pull down assay”, the reviewer most likely meant “GST pull down assay after in vitro phosphorylation by CK2”. We would like to sincerely apologize to reviewer 1 for this misunderstanding and convey that we never intended to dismiss his/her/their comment.With regards to further revising our manuscript by performing additional experiments to clarify the role of the phospho-FFAT in VAP binding in vitro, regardless of the outcome of these experiments, the conclusions of our studies will remain unchanged and the manuscript will not be substantially improved, especially because the data presented in figure 6BC and 6FG are consistent with phospho-FFAT phosphorylation during infection, which in our opinion is more relevant to the overall model. We do however agree that the discussion will benefit from presenting the three possible explanations proposed by reviewer 1 when discussing the phosphoFFAT in the context of the result of Figure 2D. See lines 532-541 of revised manuscript.Similarly, discussing the two possibilities to explain the results with the S/D mutation presented in Figure 7D, seems more appropriate than performing experiments at this stage of the review process since the concern was not raised in the initial review, and here again the outcome will not significantly elevate our study. See lines 541-545 of revised manuscript.We also note that while the model presented in Figure 8 is derived from the data presented in the manuscript, this model is not set in stone and is mostly intended as a framework to guide future studies. The following has been added to the first paragraph of the discussion after describing the model presented in Figure 8: “This model constitutes a framework to guide futures studies to further dissect the complex phosphorylation events leading to the assembly of the IncV-VAP tether. Below we discuss our results…”. See lines 477-479 of revised manuscript.– In the same vein, Figure 5 – supplement figure 4 shows that phosphomimics mutants of the three C-terminal serines do not bind VAP. This is in stark contrast with figure 2D, where CK2-mediated phosphorylation leads to robust VAP binding. This indicates either of two things. Either the phosphomimics mutation do not actually mimic phosphorylation, or CK2 phosphorylates more than the three target sites at the C-terminus. The second explanation is likely favoured by the authors, but this is not explicitly stated. What are the additional residues that could be phosphorylated by CK2? There are arguments against the idea the CK2 phosphorylates the phosphoFFAT. Could CK2 phosphorylate the serine track adjacent to the FFATs?345, S346, and S350 alone is not sufficient to promote VAP binding in vitro, suggesting that additional IncV phosphorylation sites are required to promote optimal interaction between IncV and VAP.” Based on the reviewer's comment we have revised our statement to “While we cannot exclude that the phosphomimetic mutations failed to mimic phosphorylation, these results indicate that although critical for CK2 recruitment, IncV hyper-phosphorylation status, and assembly of the IncV-VAP tether at the ER-inclusion MCS, phosphorylation of S345, S346, and S350 alone is not sufficient to promote VAP binding in vitro, suggesting that additional IncV phosphorylation sites are required to promote optimal interaction between IncV and VAP.” See lines 333-334 of revised manuscript.In the result section of the revised manuscript, after presenting the data of Figure 5 – supplement figure 4, we stated “These results indicated that, although critical for CK2 recruitment, IncV hyper-phosphorylation status, and assembly of the IncV-VAP tether at the ER-inclusion MCS, phosphorylation of SWe have also amended the discussion as follow:345, S346, and S350 was not sufficient to promote the IncV-VAP interaction in vitro. While it is possible that the phosphomimetic mutations failed to mimic phosphorylation, it is more likely that additional phosphorylation sites exist. Kinase-substrate recognition is a complex process that goes beyond the simple recognition of a consensus sequence and can involve docking sites away from the phosphorylation sites . The cytosolic domain of IncV contains a large number of additional potential CK2 recognition sites. Among those, two serine residues, S253 and S283, located in the serine tracts upstream of the phospho-FFAT and the canonical FFAT respectively, are direct CK2 target. Additionally, serine residues S257, S258, and S259 (serine tract upstream of the phospho-FFAT), and S278, S281, S282, and S284  can be phosphorylated upon priming the serine or tyrosine residues at the last position of the CK2 recognition motif (S/T-x-x-D/E/pS/pY). We therefore propose that alanine substitution of S345, S346, and S350 eliminates an essential docking site for subsequent CK2-mediated phosphorylation of distal residues in the cytosolic domain of IncV, including the serine tracts next to the FFAT motifs (see below). Further investigation of the IncV-dependent recruitment of CK2 to ER-Inclusion MCS could offer some insights into kinase targeting to cellular MCS. Moreover, since intracellular pathogens often mimic cellular processes, our study may have identified CK2 as a regulator of cellular MCS.” See lines 488-501 of revised manuscript.“However, while CK2 was required for ER-Inclusion MCS formation during infection, and sufficient for IncV-VAP interaction in vitro, the introduction of phosphomimetic mutations at S– Experiment with the VapK50L are not entirely conclusive. Indeed, if the mutant was not able to bind to phosphoFFAT, then this mutant should bind to the T265A mutant as much  as to WT IncV. This is not the case. Therefore, at face value, the VapK50L appears more like a mutant of general reduced activity (for instance less expressed) than a phosphoFFAT-specific mutant. A positive control would be to test ER-recruitement of a FFAT protein (non-phosphoFFAT), which should bind this mutant as well as a wild-type.K50L mutant was chosen for its specificity to phospho-FFAT motifs, based on the data by Di Mattia et al. who showed decreased binding to phospho-FFAT, but not to canonical FFAT motifs by performing the positive control suggested above.The VAPK50L mutant was not less expressed than the WT .In our assay, the VAPK50L to IncV phosphoFFAT motifs and/or an overall reduced activity towards the canonical FFAT. We have amended the text accordingly. See lines 398-400 (results) and 517-521 (discussion) of revised manuscript.As opposed to what was shown by Di Mattia et al., our data could be consistent with low residual binding of VAP– There is a problem in the assembly of fig6. In panel B, A picture that represented a double mutant in the previous version, now represents a single mutant. Moreover, the double mutant shows robust VAP recruitment.st, we realized that panels had been improperly positioned during the revision of the phospho-FFAT figure  and immediately notified the staff, who uploaded a corrected Figure 6. We apologize for this oversight.On April 21Reviewer #3 (Recommendations for the authors):C. trachomatis inclusion and the endoplasmic reticulum (ER) of the host cell. They also analysed a phosphomimietic mutant.In this revised manuscript the authors have performed additional experiments to address the fair comments raised by the previous two referees. This predominantly involved electron microscopy to further establish the link between phospho-IncV and the formation of membrane contact sites (MCS) that form between the Strengths:As a new referee, there are some more general comments on the above and also the content of the original manuscript. Firstly, it should be recognised that the authors have performed a considerable amount of work, including the generation of various genetic tools, to study the role of the virulence factor IncV in the formation of ER-inclusion MCS. Understanding mimicry by bacterial effectors in an important topic, of interest to those studying microbial pathogenesis and also to the cell biology community who study related canonical mechanisms. Indeed, it is often the case that such cellular microbiology experiments illuminate new aspects of fundamental cellular mechanisms as is potentially the case here when studying FFAT motifs and their relationship to VAT and ER-MCS / other MCS in uninfected cells.E. coli translocated intimin receptor (Tir). Reporting another transmembrane effector, this time at the intracellular interface, that becomes phosphorylated during host-pathogen interaction, has in itself significant novelty.Post-translational modifications of translocated bacterial effectors have rarely been reported, and in the case of phosphorylation, the archetypal example is the enteropathogenic That said, there are striking similarities between IncV FFAT and phospho-FFAT motifs recently identified in eukaryotic cells . While this on the one hand enhances the mimicry story, it dents the novelty of phospho-FFAT per se. It seems the kinases involved in the phosphorylation of phospho-FFAT in cells are yet to be resolved.Weaknesses:While this reviewer appreciates the authors attention to detail in trying to study the full biochemical pathway leading to IncV phosphorylation, some intricacies of this story are less well developed and the model in its current form remains speculative. There is no doubt that studying Chlamydia is challenging, but from both the bacterial and host angle there are conclusions drawn from combinations of in vitro and cell culture based approaches, often in combination with overexpression of the virulence factor and/or the putative host targets, which compounds interpretation. I expect as a result, some of the findings correlate with the model, while others do not. Rather than presenting all the current data, it might be preferable to streamline the story focusing on the phosphorylation of IncV to establish firm groundwork for future detailed studies of the mechanism.To this reviewer, the firmest aspects of the work are that:- IncV is phosphorylated (Thr/Ser)- IncV can recruit CK2 via C-terminal Ser residues- IncV phosphyorylation relates to VAP recruitment and the formation of ER-MCSC. trachomatis) has been previously shown to enhance MCS formation, and mislocalise or over titrate IncV around the inclusion membrane and one wonders whether this affects the phenotypes. Similarly VAP and CK2 are overexpressed as a fusion protein in most of the experiments, and endogenous CK2 visualised only when IncV-FLAG is over-expressed. Although these approaches implicate IncV, VAP and CK2 in a common event, without true endogenous data details of the mechanistic pathway cannot be inferred.A major limitation is the lack of information on the post-translational modifications of endogenous IncV. Which residues are phosphorylated during infection when the protein is not overexpressed? Over-expression , the authors speculate on the action of CK2 and host kinases responsible for IncV phosphorylation. This seems quite subjective, especially given the promiscuity and constitutive activation of CK2 (>300 cellular substrates). Their in vitro data support a view that CK2 can bind IncV, but not that this is necessarily the relevant kinase responsible for the phosphorylaton of key FFAT residues in IncV during infection. Why would this be a static recruitment? Did the authors perform any broader kinase screens or use additional kinase inhibitors to verify in a less directed manner the relevance of CK2 and/or implicate other host kinase families?Also relating to CK2, what is the effect of CK2 knockdown on other substrates and cellular processes? How much CK2 is required in the cell to sustain essential functions in apoptosis and cell proliferation for example? Assuming chemical inhibition also inhibits these processes do they have any pleiotropic effects during infection? What is the effect of CX-4945 and GO289 on known CK2 substrates? How were these inhibitors titrated?Caution should be taken in the interpretation of in vitro kinase assays. Just because CK2 can phosphorylate IncV in vitro does not in itself mean this is the relevant kinase during infection.What is the effect of mutating IncV T265 during infection? Is there an influence on infectivity or ER-inclusion MCS?As stated in our letter and acknowledged in the decision letter, we believe that it would be unfair to ask us to experimentally address reviewer 3 concerns at this stage.However, we have acknowledged in the revised manuscript that the studies were performed under IncV overexpression by adding “Although this model was in part inferred from overexpression studies, it constitutes a framework to guide futures studies to further dissect the complex phosphorylation events leading to the assembly of the IncV-VAP tether”. See lines 476-479 of revised manuscript.The model that you are putting forward sets a strong precedent and a bold claim, and surely you would want it to reflect the behaviour of endogenous proteins and not an artefact of protein overexpression.The reviewer would like to see a simple experiment using wild-type Chlamydia in non-manipulated cells demonstrating that these modifications occur. The reviewer suggests to use an IncV antibody to probe WT chlamydia infections, which, I realise, might not be available, or to purify phosphoproteins and probe if endogenous IncV is in the lot. The concern that overexpression can generate artefacts by triggering non-natural interactions and artificially expanding contact sites is a valid one. Irrespective of the unfortunate circumstances that led this concern to come to light belatedly, this is a point that you should be keen to address unless you had good grounds to dismiss it.Concerning the point of reviewer #1, we beg to differ that phospho-FFAT is a minor part of your study. Phospho-FFAT is mentioned in your abstract, in three main figures, and is central to your model in figure 8. Right now, your data show a major discrepancy with this model.The motif that you identify does appear to be necessary for VAP recruitment to inclusions, as a T>A mutation decreases VAP fluorescence there , and this effect appears to be downstream of the C-terminal serine phosphorylation, since a T>D mutation partially rescues the S3A mutation. This is consistent with the idea that this motif could act as a phosphoFFAT, through which VAP binds IncV.However, there are also discrepant data; this motif does not need to be phosphorylated to allow IncV-VAP binding . Therefore, while this motif appears to be doing something, it is unclear that it acts as a phosphoFFAT. Indeed, the S/D mutations appear to bypass the need for T265 phosphorylation , which, following the same logic as above, would seem to indicate that the serine track phosphorylation is downstream of T265 phosphorylation, and that this motif doesn't act as a phosphoFFAT but instead as a primer for serine tract phosphorylation.So, an important unaddressed point remains; is it really a phosphoFFAT? Is it fair to call it a phosphoFFAT? Or is this motif doing something entirely different?Without evidence that this phosphorylated motif binds VAP directly (hence in vitro), it seems premature to call it a phosphoFFAT. And as you concede, the VAPk50l mutant does little to lift the ambiguity.We would like to renew our apologies for the way the review process has gone and assure you that this has been beyond our control.Ultimately, authors and editors want to publish solid manuscripts that stand the test of time. Therefore, we would like to invite you to consider the above concerns and address them the best you can. We are open to further discussions if you think we made an incorrect evaluation of your data, or if other circumstances prevent you from doing requested experiments.We have addressed the comments as follow.– The IncV overexpression conditions is now acknowledged in the discussion (Lines 472-274).– We have toned down our conclusions regarding the phospho-FFAT. We refer to this motif as the non-canonical FFAT instead of a phospho-FFAT, and discuss the role of T265 in VAP recruitment to the inclusion either as part of a phospho-FFAT or as an independent mechanism.We have reorganized Figure 6. Because the results were inconclusive, panels 6DE (VAPK50L) have been moved to a new Figure 6 – supplement figure 3; the source data have been edited accordingly. The phospho-FFAT has also been removed from our model in Figure 8 and the related text has been modified accordingly. (Results lines 340-414 and Discussion Lines 505-563)


PMP22 gene, this condition is often underdiagnosed or misdiagnosed due to the heterogeneity of clinical and electrophysiological presentation. This case series describes the clinical and electrodiagnostic (EDX) features of three patients presenting with clinically heterogenous phenotypes of HNPP. A retrospective analysis of patients referred to our facility for EDX studies identified three patients with genetically confirmed HNPP. The clinical presentations were unique in each patient, and the referring physicians did not consider HNPP in their differential diagnosis. The patients underwent comprehensive clinical and EDX evaluations, which led to the diagnosis of HNPP with subsequent confirmation by genetic studies. The reasons for referral for EDX studies were foot drop in one patient, carpal tunnel syndrome (CTS) in another, and wrist drop in the third patient. Patient #1 was initially diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) at another facility and developed a foot drop while recovering from a cervical discectomy and fusion. A positive family history of neuropathy led to the suspicion of hereditary neuropathy, and further studies confirmed a PMP22 gene deletion. Patient #2 gave a typical history suggestive of CTS. EDX studies showed focal slowing of conduction at potential areas of entrapment/compression. A history of foot drop in the patient and many other family members led to the confirmation of HNPP by genetic testing. US studies aided in confirming CTS by documenting a significant drop in the diameter of the median nerve within the carpal tunnel. The third patient developed radial nerve palsy after using axillary crutches. The possibility of HNPP was considered due to the pattern of nerve conduction slowing and a family history of foot drop. Physicians should be cognizant of the varied presentation of HNPP and the potential for misdiagnosis and underdiagnosis of this condition. In each case, elicitation of a detailed family history led to the suspicion of HNPP. A high index of suspicion is necessary for patients with multiple episodes of compressive neuropathies, mono/multi mononeuropathies, unexplained polyneuropathy, and a family history of neuropathy. The EDX pattern in patients presenting with acute focal neuropathies, consisting of a global increase in distal motor latencies, moderately decreased motor nerve conduction velocities, and focal slowing of conduction in potential sites of entrapment, should trigger genetic testing for HNPP.Hereditary neuropathy with liability to pressure palsies (HNPP) is characterized by the acute onset of focal sensory and/or motor deficits when the peripheral nerves are stressed by a mechanical force. Caused by a deletion in the  Peripheral myelin protein-22 (PMP22) is mainly expressed in compact myelin of the peripheral nervous system and is an integral membrane glycoprotein of internodal myelin ,2. CondiWhen patients present with foot drop, wrist drop, or other mononeuropathies, it is important to look for a history of similar episodes and similar occurrences in other family members. Electrodiagnostic (EDX) studies play a valuable role in diagnosing HNPP as they often demonstrate increased distal motor latencies, prolonged F-wave latencies, and focal slowing of conduction velocities at sites of compression and are normal in other nerve segments ,4,5. SenWe present three cases of HNPP with heterogeneous clinical and electrophysiological findings. The physical examination, as well as the EDX and US findings, are discussed. The differential diagnosis and management of HNPP are also highlighted. Case oneHistory and Physical Examination2) presented with a three-week history of right foot drop that began while he was recovering from an anterior cervical discectomy and fusion from C4 through C7. The patient had a nine-year history of polyneuropathy. He had a previous episode of foot drop on the right side two years earlier, which improved after 2-3 months. The patient was initially diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and received intravenous immunoglobulin (IVIG) treatment without benefit. His mother and maternal grandfather had been diagnosed with polyneuropathy, although the cause was not determined.A 47-year-old male  of the Lower ExtremitiesCompound muscle action potentials (CMAP) could not be recorded over the extensor digitorum brevis (EDB) on stimulation of the peroneal nerve bilaterally. The tibial nerves showed decreased motor conduction velocity. No sensory nerve action potentials (SNAP) could be recorded on stimulation of the plantar, superficial peroneal, or sural nerves. Needle EMG revealed fibrillations and positive waves in the right tibialis anterior and peroneus longus with no motor units; the short head of the biceps was normal. The left tibialis anterior and peroneus longus did not show denervation changes. These findings suggested an acute right peroneal nerve neuropathy at the level of the popliteal fossa as well as severe sensorimotor polyneuropathy involving the lower extremities.EMG/NCV of the Upper ExtremitiesIn view of the findings suggestive of motor sensory polyneuropathy in the lower extremities, the upper extremities were also studied. Both median nerves showed marked prolongation of transcarpal motor latency with significant slowing of motor conduction velocity in the forearms Table .The amplitude of CMAP over the abductor pollicis brevis (APB) was decreased. Both ulnar nerves demonstrated significant slowing of motor conduction velocity across the wrist, forearm, and elbow. No SNAP of the median, ulnar, or superficial radial nerves could be recorded bilaterally. Needle EMG showed decreased motor unit recruitment and increased polyphasic motor units diffusely in the upper extremity muscles. Diagnosis and Family History of HNPPPMP22 gene deletion.This patient was diagnosed and received treatment for CIDP, although without much benefit. The occurrence of two episodes of foot drop, EDX findings suggestive of motor sensory polyneuropathy, peroneal nerve palsy, and family history of polyneuropathy raised the possibility of HNPP. The diagnosis was subsequently confirmed by the documentation of a Follow-UpFive years after the initial EDX studies, the patient continued to have significant weakness of the distal muscles of both upper and lower extremities with bilateral wrist drop and foot drop. Deep tendon reflexes were hypoactive in the upper extremities. Pinprick, light touch, and position sense were impaired. Three years later, the upper and lower extremities were increasingly weak, with worsening wrist drop and balance. Case twoHistory, Family History, and Physical Examination2) presented with a several-year history of numbness and aching of the hands with nocturnal exacerbations of symptoms. She was a cake decorator and spent a considerable time painting. She was diagnosed with CTS and wore a wrist splint with minimal benefit. Her history was significant for the occurrence of foot drop after childbirth which improved after several months. Several family members, including her father and grandfather, experienced foot drop, and some had foot deformities. Her daughter was later confirmed to have HNPP by genetic testing.A 51-year-old woman  with a linear array transducer of 8-18 MHz, according to our lab protocol . The US PMP22 gene deletion. While the EDX and the US findings confirmed right median nerve entrapment at the carpal tunnel, there was still the question of an underlying neuropathy, HNPP. The patient subsequently had genetic testing that confirmed a Surgical InterventionOne month following the neurodiagnostic evaluation, the patient underwent a right carpal tunnel release (CTR) and a right cubital tunnel release simultaneously. Case ThreeHistory, Physical Examination, and Radiological Imaging2) presented with a one-week history of sudden onset of right wrist drop. He was using crutches after undergoing surgery for a middle cruciate ligament tear at the knee. On exam, significant weakness of all muscles innervated by the right radial nerve, including the triceps, was noted , extensor carpi radialis longus (ECRL), and extensor indicis (EI); a single motor unit was recorded in the triceps. Family HistoryThe patient’s maternal aunt accompanied him to the neurodiagnostic evaluation. She disclosed that she had experienced episodes of wrist and foot drop. EDX studies on the aunt revealed prolonged distal motor latency and low motor conduction velocity of the right median nerve forearm segment; sensory potentials showed prolonged latency and decreased amplitude. There was also a slowing of motor conduction of the right ulnar nerve across the elbow. The clinical EDX findings and family history suggested the possibility of HNPP, which may have been predisposed to radial nerve palsy due to pressure from the crutches. The underlying pathology was most likely a conduction block in the right radial nerve at or proximal to the innervation of the triceps. A genetic test for PMP22 deletion was subsequently performed, which confirmed HNPP. Follow-UpThe patient underwent occupational therapy with electrical stimulation to the affected muscles and continued to wear the wrist brace. Evaluation five weeks later demonstrated greatly improved muscle strength in his right arm, although the triceps and wrist extensors were still weak. There was an increase in motor unit recruitment in the muscles innervated by the right radial nerve compared to the previous study, indicating significant improvement. HNPP is often underdiagnosed or misdiagnosed due to the heterogeneity of the clinical and electrophysiological presentation . RecurreIn addition to the common phenotype of episodic painless focal neuropathies, Mouton and colleagues described five other clinical presentations: recurrent short-term positional, progressive mononeuropathy, CMT-like polyneuropathy, chronic sensory polyneuropathy, and CIDP-like, recurrent subacute polyneuropathy . Taioli Each of the cases in our case series illustrates phenotypical heterogeneity of HNPP, which may have caused the referring physician to miss the diagnosis. Patient #1 illustrates the situation where the patient was initially diagnosed and treated with IVIG for CIDP without benefit. This problem has been previously reported in the literature. Beydoun and colleagues reported two patients who were initially diagnosed and treated for an acquired demyelinating disorder or alternative inherited demyelinating disorder . Upon rePatient #2 in our case series presented another unique problem: how to conclusively diagnose CTS in patients with HNPP. EDX studies may not be conclusive in patients with HNPP who show slowing of sensory and motor conduction in median nerves, findings similar to entrapment at the carpal tunnel. US studies may also not provide the answer, as an increase in CSA of nerves at potential entrapment sites is typically seen in HNPP ,17. In tUS imaging plays an important role in differentiating between HNPP and other demyelinating polyneuropathies, compression neuropathies, and nerve injuries through pattern recognition  ,19. HighPatient #3 presented with radial nerve palsy after the use of crutches. The only reason to suspect HNPP was the history of foot drop in several family members and the presence of more diffuse nerve conduction abnormalities.Physicians should be alert to the varied presentation of HNPP and the potential for misdiagnosis and underdiagnosis of this condition. A high index of suspicion is warranted in patients with multiple episodes of compressive neuropathies, mono/multiple mononeuropathies, and unexplained polyneuropathy. Our cases also highlight the importance of taking a detailed family history for neuropathy. The EDX pattern in patients presenting with acute focal neuropathies, consisting of a combination of a global increase in distal motor latencies coupled with moderately decreased motor nerve conduction velocities and focal slowing of conduction in potential sites of entrapment, should trigger genetic testing for HNPP.


Indeed, we find nonvolcanic S-isotopic anomalies (Δ33S ≠ 0‰) in background Antarctic ice-core sulfate during GE/SN periods, thereby confirming our hypothesis. This suggests that ice-core-Δ33S records can serve as a proxy for past ozone-layer-depletion events.Changes in the cosmic-ray background of the Earth can impact the ozone layer. High-energy cosmic events [e.g. supernova (SN)] or rapid changes in the Earth's magnetic field [e.g. geomagnetic Excursion (GE)] can lead to a cascade of cosmic rays. Ensuing chemical reactions can then cause thinning/destruction of the ozone layer—leading to enhanced penetration of harmful ultraviolet (UV) radiation toward the Earth's surface. However, observational evidence for such UV “windows” is still lacking. Here, we conduct a pilot study and investigate this notion during two well-known events: the multiple SN event (≈10 kBP) and the Laschamp GE event (≈41 kBP). We hypothesize that ice-core-Δ Significance StatementThe ozone layer in the upper troposphere–lower stratosphere (UTLS) region absorbs ultraviolet (UV) radiation, thereby limiting the penetration of harmful UV rays into the Earth's atmosphere. However, in the event of destruction or thinning of the ozone layer, this harmful UV radiation could penetrate deeper into the troposphere, causing detrimental effects on humans. Here, we provide observational evidence of changes in the cosmic-ray background of the Earth during past events such as magnetic field collapses and Supernovas, having affected the ozone layer. As such, future events of a similar kind could potentially lead to UV-induced environmental effects.14C), beryllium-10 (10Be), and chlorine-36 (36Cl), are powerful proxies often used to investigate changes in the cosmic-ray background   in tropospheric sulfate aerosols due to the penetration of UV rays toward Earth's surface  of aerosol sulfate trapped in ice cores. The ice-core sulfate isotope records have been used as an unambiguous tracer to fingerprint past volcanic eruptions     and as sSO, CS2) . This regerprint —e.g. of anism(s) , 47–51. 33S signals in ice-core sulfate were observed during both the Laschamp GE event (≈41 kBP) and the multiple SN event (≈10 kBP), respectively  values found in ice-core sulfate records    , Serua eruption (Δ33S = 0.16‰) detected in ice cores from Dome C, Antarctica .33S when examined in ice-core sulfate records, as reported previously  (Evidence of tropospheric origin can be obtained further by investigating points with lower sulfate concentrations but higher Δ14 ± 2‰) . However14 ± 2‰) . Remarka14 ± 2‰)   than the rest of the EPs (7 to 12). The overlap of isotopic anomalies in these signals with EPs 4, 5, and 6, as seen in Figure Given the different kinds of events studied and the study's exploratory nature, a range of sampling resolutions was needed to understand the likelihood of detecting any peculiar signals. To further probe this aspect, we investigated another event with a potential for affecting the cosmic-ray background of Earth—the multiple SN event Figure . EPs 7 tΔ33S dynamics contrast the well-established notion of SO2 oxidation kinetics imprinted in ice-core sulfate, they align with our ozone depletion hypothesis during the GE/SN events    . Howevers study) . Taking s period . The coriments)  . The resiments)  . A blip Figure   , this blFigure   . Here, w33S records for the corresponding periods suggest that past GE/SN events had impacted the ozone layer simultaneously, creating a so-called UV “window” in the Earth's atmosphere. This led to the traceable enhanced UV-induced tropospheric photochemical imprints during these periods, thereby corroborating our hypothesis.Taken together, these fluctuations in the radionuclide and ice-core Δ33S values) was not witnessed in the Antarctic ice-core records. This can be attributed to different aspects, which could influence the detection of such signals:Despite the likely existence of a UV “window” during the Laschamp GE event (≈41 kBP) and the multiple SN event (≈10 kBP), a continuous photochemical imprint (nonzero Δ2 + OH reaction), which is not associated with any isotopic anomaly (The transport and dilution/mixing processes could play a significant role in this regard. The magnitude of S-MIF detected in ice-core sulfate would be governed by sulfate formed from competing processes (photolysis vs. photoexcitation) , 44, 51. anomaly .The role of site-specific characteristics, e.g. the regional pattern of snow accumulation, a central variable when investigating the evolution of polar ice sheets and their sensitivities to changes in the atmosphere/climate, needs to be considered. We speculate that the difference in accumulation rates is likely to affect the frequency of detection of the episodic events between different ice-core records from Northern Hemispherical polar sites (e.g. in Greenland) having a higher snow accumulation rate [nearly an order higher in some regions ] vs. mosThe influence of field morphology and the potency of the UV “window” are critical factors. In the ∼2000 y of the Laschamp GE, the Earth's magnetic field  featured several changes , 64 that33S values in the period before the SN event (≈11.2 kBP). This analysis did not reveal any S-MIF anomaly  site in Antarctica, as detailed elsewhere . High-re G site i32S, 33S, and 34S and to increase the sensitivity to the signal    are given as an average of at least five independent measurements on the Neptune for a given sample. This analytical error  is an improvement from previous MC-ICPMS-based studies  as sampl32S, 33S, 34S) are reported relative to Vienna-Canyon Diablo Troilite (V-CDT). The bracketing standard was calibrated against the international standard IAEA-S1 assuming δ34S = −0.3‰ and δ33S = −0.061‰ (and thus Δ33S = 0.094‰). Further quality control was conducted using a secondary sulfur isotope reference material “SMIF-2” which yielded a δ34S value of 10.06‰ ± 0.14‰ and Δ33S of 9.46‰ ± 0.14‰ (n = 5) in agreement with the reported Δ33S = 9.54‰ ± 0.09‰ from five different laboratories worldwide , in agreement with previously published values (34S and Δ33S were ensured during this investigation.The triple sulfur isotopes (orldwide . A seawad values . The sead values , 66. Propgac170_Supplemental_FileClick here for additional data file.