Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and future zoonotic sarbecoviruses. We describe the isolation and characterization of a human monoclonal antibody, designated S2K146, that broadly neutralizes viruses belonging to SARS-CoV– and SARS-CoV-2–related sarbecovirus clades, which use angiotensin-converting enzyme 2 (ACE2) as an entry receptor. Structural and functional studies show that most of the virus residues that directly bind S2K146 are also involved in binding to ACE2. This allows the antibody to potently inhibit receptor attachment. S2K146 protects against SARS-CoV-2 Beta variant challenge in hamsters, and viral passaging experiments reveal a high barrier for emergence of escape mutants, making it a good candidate for clinical development. The conserved ACE2-binding residues present a site of vulnerability that might be leveraged for developing vaccines eliciting broad sarbecovirus immunity. et al. describe a monoclonal antibody that neutralizes a broad range of sarbecoviruses, including both the 2003 SARS-CoV and SARS-CoV-2. This antibody also binds in the RBM, but targets residues that are more conserved because they are involved in ACE2 binding. The antibody protects against the SARS-CoV-2 Beta variant, and none of the individual mutations in the Omicron variant affected antibody binding. —VVNeutralizing antibodies are a key defense against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many neutralizing antibodies target the region of the viral spike protein that is involved in binding to the human ACE2 receptor, known as the receptor binding motif (RBM). This region of the protein is divergent between SARS-CoV-2 variants, leading to failure of existing monoclonal antibody treatments and evasion of antibodies elicited by previous infection or vaccination. Park  An antibody neutralizes across sarbecoviruses that use ACE2 to enter cells by binding viral residues that interact with ACE2. The zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic causing more than 266 million infections and more than 5.2 million fatalities as of December 2021. Continued SARS-CoV-2 evolution leads to the emergence of variants of concern (VOCs) that are characterized by higher transmissibility, immune evasion, and/or disease severity. For pandemic preparedness, we need pan-sarbecovirus countermeasures, such as vaccines and therapeutics that are effective against all SARS-CoV-2 variants and divergent zoonotic sarbecoviruses  and two additional domains designated C and D (The coronavirus spike (S) glycoprotein promotes viral entry into host cells and is the main target of neutralizing antibodies elicited by infection or vaccination ] memory B cells from one symptomatic COVID-19 convalescent individual  35 days after symptoms onset. We identified one mAb, designated S2K146 , which did not compete with S309 (site IV)  and yeas50) of 108 and 16 ng/ml, respectively (fig. S2B). Moreover, S2K146 potently neutralized VSV pseudotypes harboring SARS-CoV-2 S glycoproteins from VOCs including Alpha, Beta, Gamma, Delta plus (AY.1/AY.2), Epsilon, and Lambda (fig. S2B). S2K146 also weakly neutralized VSV pseudotyped with BtKY72 S (clade 3) harboring the K493Y/T498W mutations (SARS-CoV-2 numbering) (fig. S2C), which enable human ACE2–mediated entry (50 = 10 ng/ml) and SARS-CoV-2 VOCs  with a potency approaching that observed with the ultrapotent S2E12 mAb  in a side-by-side comparison  pseudotyping platform. S2K146 efficiently blocked SARS-CoV and SARS-CoV-2 S-mediated entry into cells with median inhibitory concentration (ICmparison .H (variable region of immunoglobulin heavy chain) identity: 94.4%; VL (variable region of immunoglobulin light chain) identity: 98.9%] (fig. S2D). Except for WIV-1, S2K146 and S2K146 UCA showed no major cross-reactivity differences with a panel of RBDs representative of clade 1 sarbecoviruses, as determined by ELISA (fig. S2E). Nevertheless, biolayer interferometry revealed that S2K146 bound to prefusion-stabilized SARS-CoV and SARS-CoV-2 S ectodomain trimers with enhanced avidities compared with S2K146 UCA (fig. S2F). Accordingly, S2K146 UCA showed a marked loss in neutralizing activity against both SARS-CoV S and SARS-CoV-2 S VSV pseudotypes . Alignment with the UCA amino acid sequence reveals that S2K146 harbors seven and two somatic hypermutations in the heavy- and light-chain complementarity determining regions (CDR), respectively [Vudotypes . Our resTo understand the sarbecovirus cross-reactivity of the RBM-specific S2K146 mAb, we determined a cryo–electron microscopy (cryo-EM) structure of the S2K146 Fab fragment in complex with the SARS-CoV-2 S ectodomain trimer at 3.2-Å resolution , where human ACE2 binding first arose during sarbecovirus evolution  of a yeast-displayed RBD mutant library covering all possible single residue substitutions in the Wuhan-Hu-1 RBD background . Only onTo explore whether our escape map was consistent with in vitro viral evolution under mAb pressure, a replication competent VSV–SARS-CoV-2 S Wuhan-Hu-1/D614G chimera , in lineTo evaluate the fitness of the Y489H mutant, we carried out a competition assay in which replicating VSV chimeras harboring the Wuhan-Hu-1/D614G S with or without the Y489H substitution were mixed at equal titers and passaged together without mAb. Because of the fitness cost associated with the mutation, which dampens ~4.5-fold the 1:1 ACE2-binding affinity to the SARS-CoV-2 RBD (fig. S8B), the Y489H S chimera was outcompeted by the Wuhan-Hu-1/D614G S chimera after only four rounds of passaging . Accordi1 subunit from cell surface–expressed full-length SARS-CoV-2 S, similar to some other RBD-specific mAbs . However, S2K146 also did not activate FcγRIIa and triggered FcγRIIIa only weakly when target cells expressed an uncleavable prefusion-stabilized SARS-CoV-2 S protein . The greater efficiency of S2E12 for activating FcγRIIIa, relative to S2K146, might be explained by the different angles of approach at which these two mAbs bind to the RBD .The efficient S2K146-induced SNext, we evaluated the therapeutic activity of S2K146 against challenge with the SARS-CoV-2 Beta VOC in a Syrian hamster model of infection . Viral rThe SARS-CoV-2 RBD accounts for most serum neutralizing activity in both COVID-19 convalescent  has received emergency use authorizations in several countries around the world for the early treatment of mild-to-moderate COVID-19 in adults and some pediatric patients who test positive for SARS-CoV-2 by direct viral testing and who are at high risk for progression to severe COVID-19, including hospitalization or death (20220107-1Click here for additional data file.


Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 ( Clinical outcome for invasive lobular breast cancer (ILC) patients with metastatic disease is dismal once tumors stop responding to estrogen receptor antagonists . ILC hasIn non-cycling G0/G1 cells the retinoblastoma (Rb) tumor suppressor is hypo-phosphorylated and interacts with E2F factors to control repression of its target genes. Upon a mitotic stimulus or engagement of integrin-dependent anchorage, Rb is inactivated through cyclin-CDK-dependent phosphorylation, which disrupts its binding to the E2Fs (reviewed in: ). Rb-depId2 expression has been linked to tumor initiation, growth, dissemination, and chemotherapy responses –20. IncrHere, we demonstrate that Id2 represents a key factor that promotes sustained cell cycle quiescence in human and mouse models of ILC. Our data provides a causal link from E-cadherin loss to a direct and p120/Kaiso-dependent transcriptional upregulation of Id2, which is exacerbated in the absence of cell-matrix engagement. Functionally, Id2 promotes a low proliferative indolent state in lobular breast cancer cells through inhibition of CDK4/6-Rb-dependent cell cycle progression, enabling anchorage independence and subsequent metastatic dissemination.ILC cells are resistant to anchorage-independent programmed cell death (anoikis), a hallmark that underlies their metastatic capacity in vivo Fig. B, C.Fig.Because cell cycle arrest in G0/G1 is mediated through sequestering and inhibition of E2F transcription factors by hypo-phosphorylated Rb, we assessed the phosphorylation status of Rb under anchorage-independent conditions. In accordance with the observed anchorage-independent cell cycle arrest, Rb phosphorylation shifts from the hyper-phosphorylated (inactive) form under adherent conditions to the hypo-phosphorylated (active) in anchorage-independent suspended cells Fig. . MoreoveNEG mILC cell lines cultured under anchorage-independent conditions and performed genome-wide mRNA sequencing as described previously [Id2 is upregulated in suspended mILC cells  cell line versus two isogenic MCF7 E-cadherin knockout (∆CDH1) clones  in SUM44PE, an established ILC cell line, and generated two SUM44PE::iCDH1 cell lines. . Apoptosis was verified by blotting for cleaved Caspase-3 , we performed Id2 knock-downs in mILC using two independent shRNAs  in mouse Id2 [Id2-specific PCR confirms that Id2 is a Kaiso target gene in mouse and human E-cadherin deficient breast cancer cells (mILC-1 and MDA-MB231)  [∆versus mILC-1) and isogenic human MCF7 cells (WT versus ∆CDH1) models, we observe that loss of E-cadherin increases Id2 reporter activity when transferring cells to anchorage independent conditions  leads to an approximate 3-fold decrease in Id2 protein levels  Fig. . We nextpro1036)  upstreamion Fig. . We also-1A Fig. , indicatels Fig. . TogetheBecause Id2 can modulate cell cycle responses through the Rb pocket proteins , 16, we We next confirmed the impact of Id2 or Rb knockdown  Rb to dampen cell cycle progression. Further upregulation of Id2 under anchorage-independent conditions blocks cell cycle progression, which prevents accumulation of DNA damage and fosters anoikis resistance.CDH1  ILC models to assess anchorage independent Id2 expression in 3D. We compared KCL320, a classical E-cadherin negative ILC PDO model to P008, an E-cadherin positive ILC PDO model that contains a S180Y mutation in DH1 Fig. . The ext008 Fig. . We next008 Fig.  and obseres Fig. . Howeverels Fig. . In shorp = 0.003). Analysis of the publicly available TCGA database reveals that Id2 mRNA expression is indeed elevated in ILC when compared to IDC-NST  . These clinical findings concur with our in vitro data that Id2 predominantly localizes in the cytoplasm of ILC cells.We next analyzed a cohort of 148 invasive breast cancer samples (Supplementary Table 01) Fig. . As expeIn conclusion, loss of E-cadherin induces a p120/Kaiso-dependent upregulation of Id2, a factor that is essential for anchorage independence and binds hypo-phosphorylated Rb in the cytosol, leading to CDK4/6-dependent cell cycle repression in ILC Fig. .POS and ERNEG models of human and mouse origin, it also indicates that this mechanism operates independent of estrogen receptor function.It is well-established that luminal ER expressing breast cancers tend to relapse with a longer latency than non-luminal tumors . Interesi.e. anchorage-dependent conditions [INK4A activity [High Id2 levels are associated with terminally differentiated mammary cells, coinciding with β-casein expression in the mammary gland at day 12 of pregnancy . In linenditions , 42. In nditions . The Rb-nditions , and connditions . Howevernditions , it is aactivity .Id2 transcriptional repression, a phenomenon that is enhanced in the absence of anchorage. Although our data fully support the reported binding of Id2 to hypo-phosphorylated Rb, we hypothesize that in anchorage-independent carcinoma cells, formation of the Id2-Rb complex prevents CDK4/6-dependent Rb phosphorylation by retaining or accumulating Rb in its hypo-phosphorylated active form. Supporting this is the fact that loss of Id2 leads to an increase in Rb phosphorylation and the number of cycling cells in ILC cells in suspension cultures. Second, low to intermediate grade ILC is strongly associated with overexpression of Cyclin D1 [Our studies demonstrate that loss of E-cadherin leads to a p120/Kaiso-dependent relief of yclin D1 –48, a reyclin D1 , and theyclin D1 . Also, oyclin D1 , point tNEG and HER2POS ILC patients for clinical CDK4/6 breast cancer intervention trials.In closing, our study provides novel mechanistic insight and a conceptual rationale for the control over indolent proliferation of ILC, cellular quiescence and cycle re-entry upon cell anchorage. We propose that inactivation of E-cadherin, a lobular histology, and cytosolic Id2, should be considered as criteria to include ER∆-3 and mILC1  have been generated from mammary tumors that developed in K14cre;Cdh1F/F;Trp53F/F female mice and cultured as described previously [Wcre;Cdh1F/F;Trp53F/Ffemale mice [CDH1 and Trp53∆::∆Cdh1 cells using CRISPR-Cas9 editing has been described previously [CDH1 is described in detail in the supplementary methods.Mouse cell lines Trp53ale mice . 293T, Male mice . GeneratThe following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 , polyclonal rabbit Id2 C-20 , polyclonal rabbit Rb M-153 , rabbit monoclonal phospho-Rb Ser 780 , monoclonal rabbit phospho-Rb Ser 807/811 , monoclonal mouse E-cadherin ; purified mouse p120-Catenin , polyclonal rabbit GFP , and polyclonal goat Akt1 C-20 . The following primary antibodies were used for Immunofluorescence (IF): monoclonal rabbit phospho-Rb Ser 807/811 , polyclonal rabbit Cleaved Caspase-3 (Asp175) , monoclonal TRITC-conjugated mouse E-cadherin , monoclonal mouse phospho-Histone H2A.X (Ser139) , monoclonal mouse E-cadherin ; purified mouse p120-Catenin . immunohistochemistry (IHC) antibodies: Monoclonal mouse Id2 , Monoclonal rabbit phospho-Rb Ser 807/811 .Lentiviral and inducible p120-specific shRNA constructs (p120-iKD) have been described previously . SequencHEK 293T cells were cultured and transfected with 100 ng pGL4-Id2pro-1036 and 5 ng pRL-Renilla in combination with either 400 ng empty vector (pcDNA3.1) or pC2-eGFP-p120-1A or pcDNA3.1-Kaiso using XtremeGene9 (Roche) Reporter activity was measured using the Dual-Luciferase Reporter kit (Promega) on a Lumat LB9507 Luminometer (Berthold Technologies) according to manufacturer’s instructions. Cell lines transduced with the Id2-mCherry reporter viruses were cultured in anchorage-independent conditions for the indicated time periods and fluorescence was quantified by flowcytometry using a FACS Celesta (BD Biosciences). Data was processed using the FACSdiva software (Version 8.0) and analyzed in Prism (Version 8.0).ChIP procedures and PCR were done as previously described . The folProduction of lentiviral particles and transduction was performed as previously described .IHC was carried out on 4 µm thick tissue sections as described previously . Detailet tests assuming non-gaussian distributions. Unless stated differently, a minimum of three independent experiments, with three biological replicates, were considered. Error bars show either standard deviation (SD) or standard error of the mean (SEM), as stated in the figure legend. Western blots were quantified using ImageJ, converting pixel intensity to white/black scales and normalized to loading controls. Statistical test on the clinical tissue micro array of invasive breast cancer was performed using IBM SPSS Statistics version 27.0.0.0. Associations between categorical variables were calculated using the 369 Pearson’s Chi-square test. P-values <0.05 were considered statistically significant. For the bioinformatics analysis, Wilcoxon Rank tests were performed. Violin plots were generated using GraphPad Prism 8.Statistical tests were performed in Microsoft Excel and GraphPad Prism 8 using Student Additional experiments procedures regarding anoikis resistance and FACS analysis, RNA sequencing, co-immunoprecipitations, RT-qPCR, colony formation assays, lung colonization assays, immunofluorescence microscopy, and analysis of human expression data are available in the supplementary information –67.Collated Supplemental Information


A 70-year-old man reported a 2-year history of progressive gait imbalance and cognitive decline . A physical examination revealed spastic ataxia without peripheral involvement. The patient worked with car battery solutions for 40 years, until 13 years ago.


Scientific Reports 10.1038/s41598-021-89943-5, published online 19 May 2021Correction to: The original version of this Article contained an error in the Acknowledgments section.“This work was supported by National Science Centre (Poland) Grant OPUS (UMO-2019/33/B/NZ7/01077) awarded to Dr. Maciej Serda.”now reads:“This work was supported by National Science Centre (Poland) Grant OPUS (UMO-2019/35/B/NZ7/02459) awarded to Dr. Maciej Serda.”The original Article has been corrected.


Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease.Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition.Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3+ cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis.Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease. Despite the approval of several vaccines against SARS-CoV-2, there is a high medical need for further therapeutic options to facilitate convalescence from the infection and prevent a severe course of the disease. Early treatment strategies for COVID-19 patients include immunomodulatory approaches such as dexamethasone, antiviral substances such as remdesivir, antimalarial drugs, and monoclonal antibodies or convalescent plasma . The app+ memory T cell exhaustion , Nucleocapsid (N), and Membrane (M) proteins of SARS-CoV-2 are detectable . Particuhaustion . Reducedhaustion . In patihaustion . The adohaustion .The concept of adoptive T cell therapy has been developed for viral infections in immunosuppressed patients after allogeneic stem cell (HSCT) and solid organ transplantation (SOT). Refractory viral infections due to Cytomegalovirus (CMV), Adenovirus (AdV), and Epstein-Barr virus (EBV) after HSCT represent life-threatening conditions due to the insufficient endogenous T cell response of the recipient and lack of effective treatment options. However, the concept of adoptive T cell therapy not only applies to patients with transplantation history but also to those generally immunocompromised due to a variety of underlying diseases. In detail, progressive multifocal leukoencephalopathy (PML), caused by reactivation of JC polyomavirus (JCV), occurs in immunocompromised patients, and is associated with high morbidity and mortality mainly because there are no targeted therapies available . Hence, The repertoire of SARS-CoV-2-specific T cells in COVID-19 patients convalescent from mild disease covers a broad range of viral antigens including antigens from different VOCs. In contrast, in the majority of hospitalized patients reduced functionally active antiviral T cells are observed . AdoptivThe study was approved by the Internal Review Board of Hannover Medical School . All 198 donors  aged 18–69 years (mean 41 years) were recruited from MHH between October 2020 and July 2021. Convalescent COVID-19 patients with confirmed history of SARS-CoV-2 infection were sampled up to 61 weeks after symptom onset and at least 2 weeks after negative PCR.7 cells/ml, seeded in 24-well plates and rested overnight. For retrospective analyses, PBMCs were frozen and stored at −70°C until thawing and overnight resting prior to ELISpot assay. Rested PBMCs were cultured in anti-IFN-γ pre-coated ELISpot plates  for 16–18 h at a density of 2.5 × 105 cells/well in presence of PepTivator SARS-CoV-2 membrane (M), nucleoprotein (N) or Select  or PepMix SARS-CoV-2 Spike (S) protein from wild type, Delta or Omicron VOC  at a final concentration of 1 µg of each peptide/ml). PepTivator SARS-CoV-2 Select consists of 88 peptides with a length of 9–22 amino acids originating from structural and non-structural proteins. The peptides are restricted to Major Histocompatibility (MHC) class I (n = 63 peptides), covering n = 19 frequent Human Leukocyte Antigen (HLA) alleles, and MHC class II (n = 25), covering n = 6 frequent HLA alleles. Cells stimulated with staphylococcal enterotoxin B  served as positive controls, and PBMCs incubated in media alone as negative controls (NC). IFN-γ secretion was detected using streptavidin-alkaline phosphatase  and revealed by 5–13 bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium . Spots were counted using AID ELISPOT 8.0 on an AID iSpot spectrum reader system . Means of duplicate well readings were calculated and expressed as the number of spots per well (spw). The threshold for a response was set at 2xNC+1 or ≥3.Virus-specific T lymphocytes were detected by Interferon-gamma (IFN-γ) Enzyme-Linked Immunospot (ELISpot) Assay as previously described . BrieflyThe SARS-CoV-2 T Cell Analysis Kit (Whole Blood) (Miltenyi Biotec) was performed according to the manufacturer’s instructions. In brief, heparinized whole blood was filled into 48 well plates (1 ml/well) and stimulated with PepTivator SARS-CoV-2 Select (1 µg of each peptide/ml) for 8 h. Unstimulated samples served as negative control and samples stimulated with CytoStim (Miltenyi Biotec) served as positive control. Cytokine secretion was blocked by supplementation with Brefeldin A (Miltenyi Biotec). After a total of 8 h of stimulation, erythrocytes were lysed , samples were fixed, and permeabilized followed by staining with the following antibodies: anti-CD3 Allophycocyanin (APC), anti-CD4 Vio Bright B515, anti-CD8 VioGreen, anti-IFN-γ Phycoerythrin (PE), anti-Tumor Necrosis Factor alpha (TNF-α) PE-Vio 770, anti-CD20 VioBlue, and anti-CD154 APC-Vio 770 . Samples were acquired on a MACSQuant Analyzer 10 (Miltenyi Biotec).7 isolated PBMCs were stimulated with PepTivator SARS-CoV-2 Select (Miltenyi Biotec) at a final concentration of 1 µg of each peptide/ml peptide pool. Unstimulated PBMCs served as negative controls. Activated IFN-γ-secreting T cells were specifically captured during the magnetic enrichment process using anti-IFN-γ-PE antibodies and paramagnetic anti-PE microbeads. Aliquots of the respective cell fractions collected before (pre drug substance (preDS)) and after enrichment (DS) were used for analysis of IFN-γ+ T cell subsets by multicolor flow cytometry. Dead cells were excluded from analysis by 7-amino-actinomycin D (7-AAD) staining . The percentage of viable IFN-γ+ cells was determined by staining the cells with anti-CD45 APC-H7, anti-CD3 Fluorescein Isothiocyanate (FITC), anti-CD8 APC, and anti-CD4 Alexa Fluor 700 (AF700) mAbs . At least 10,000 events were acquired in the viable CD45+ leukocyte gate in each test . CD3+/IFN-γ+, CD8+/IFN-γ+ and CD4+/IFN-γ+ T cell populations were gated based on the scatter properties of viable 7-AAD-/CD45+/CD3+ T cells. Moreover, for selected donors, the activation state of the cells was analyzed by staining the cells with anti-CD45 Pacific Blue, anti-CD3 FITC, anti-CD8 APC, anti-CD4 AF700, anti-CD14 BV510, anti-CD19 BV510, anti-CD69 BV605, anti-CD137 PE-Cyanine 7 (PE-Cy7), and anti-CD154 APC-Cy7 mAbs (BD Biosciences and BioLegend). Dead cells were excluded from analysis by 7-AAD staining (BD Biosciences). The following gating strategy was applied: CD45+7-AAD-SSClowCD3+CD14/19- (CD3+ T cells), CD45+7-AAD-SSClowCD3+CD14/19-/CD8+ (CD8+ T cells), and CD45+7-AAD-SSClowCD3+CD14/19-/CD4+ (CD4+ T cells). The following acceptance criteria apply for donation of SARS-CoV-2-specific T cells for clinical application: CD3+/IFN-γ+ pre-enrichment ≥ 0.01% or enrichment of a clear, defined population of ≥10% CD3+/IFN-γ+.The IFN-γ cytokine secretion assay  was performed as previously described . After oMessenger RNA (mRNA) amounts of IFN-γ, perforin and granzyme B were analyzed as previously described . Brieflyn = 4 clinical-grade manufacturing processes, SARS-CoV-2-specific T cells were isolated from leukapheresis products (LP) of pretested COVID-19-recoved donors using the CliniMACS Prodigy device (Miltenyi Biotec) and the CliniMACS CCS IFN-gamma (Miltenyi Biotec) following the manufacturer’s written instructions. In brief, total nucleated cell numbers in the LP were measured  and the process was initiated using 1 × 109 total nucleated cells. The following steps were carried out autonomously by the CliniMACS Prodigy device: ex vivo restimulation  with MACS GMP PepTivator SARS-CoV-2 Select , labeling of white blood cells with the CliniMACS CCS Catchmatrix Reagent , secretion phase, labeling and immunomagnetic separation of IFN-γ-secreting cells by antibody-conjugated super-paramagnetic particles . Prior to immunomagnetic separation, a quality control sample (preDS) was taken from the quality control bag.In + leukocytes, viability, and frequencies as well as total cell numbers were determined using Trucount Absolute Counting Tubes (BD Biosciences) and the following staining reagents: anti-CD45 APC-H7 and anti-CD3 FITC mAbs and 7-AAD . After staining, erythrocytes were lysed using Lysing Solution (Beckman Coulter) and samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the Trucount beads gate. Purity, memory phenotype and cellular composition were analyzed using combinations of the following staining reagents: anti-CD45 APC-H7, anti-CD3 FITC, anti-CD4 AF700, anti-CD8 APC, anti-CD14 BV510, anti-CD19 BV510, anti-IFN-γ PE, anti-CD45RA BV605, anti-CD62L BV421, anti-CD56 AF700, anti-CD33 APC, anti-CD19 PE-Cy7 mAbs, and 7-AAD . After staining and lysis of erythrocytes, samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the CD45+ leukocyte gate.Quality control of LP, preDS, and DS was performed as previously described . Total C5 cells per well in TexMACS (Miltenyi Biotec). For feeder cells, autologous PBMCs were irradiated at 50 Gy and seeded at 1 × 107 cells per well. Cultures were supplemented with recombinant human Interleukin-2  and maintained for 7–13 days including regular media change and splitting of the cells.SARS-CoV-2-specific T cells enriched via CCS using the CliniMACS Prodigy device and MACS GMP PepTivator SARS-CoV-2 Select (Miltenyi Biotec) were seeded into 24 well plates at 1 × 10Autologous PBMCs were labeled using the CellTrace Violet Cell Proliferation Kit , seeded into 24 well plates and loaded with PepTivator SARS-CoV-2 Select overnight. Unloaded PBMCs served as control for unspecific killing. Expanded T cells were harvested, counted and seeded into 96 well plates together with unloaded or loaded PBMCs in different effector-to-target ratios . As a control for target cell viability, target cells were incubated in absence of T cells. CellEvent Caspase 3/7 Detection Reagent (Invitrogen) was added to the cultures. After 4 hours, supernatants were collected for subsequent multiplex analysis. Cells were washed and stained with 7-AAD and analyzed via flow cytometry using the BD FACSCanto 10c system.Cell culture supernatants collected from cytotoxicity assays were subjected to LEGENDPlex using the CD8/NK Cell Kit (BioLegend). Samples were prepared and acquired using the BD FACSCanto 10c system according to the manufacturer’s instruction.p-values .Data were analyzed using BD FACSDiva v8.0.1 (BD Biosciences), FlowJo v10 , LEGENDPlex Software (v8.0), and Microsoft Excel 2010 . Prism Version 8.2.0  was used to generate graphs as well as for statistical analysis using a linear regression model and the Kruskal–Wallis or Friedman test followed by multiple comparison correction. Significance levels were calculated and expressed as n = 198) were analyzed first with regard to antiviral T cell frequencies via IFN-γ ELISpot Assay using PepTivator SARS-CoV-2 Select (5 PBMCs (responders only). To further characterize the antiviral T cells, IFN-γ and TNF-α-producing T cells were detected in heparinized whole blood , values from unstimulated control were subtracted).Convalescent COVID-19 donors (2 Select . SARS-Cole blood . T cellsn = 22), SARS-CoV-2-specific T cells were detectable, with pre-enrichment frequencies of CD3+/IFN-γ+ cells ranging from 0.01 to 0.18% (mean 0.07%). Comparing the results obtained by ELISpot Assay and CSA, trends for a positive correlation were found between antiviral T cells detected by ELISpot Assay and frequencies of CD3+/IFN-γ+ cells both, pre-enrichment  and post-enrichment   .n = 22 donors tested, n = 18 fulfilled the acceptance criteria for donation of SARS-CoV2-specific T cells for clinical application  with mean pre-enrichment frequencies of CD3+/IFN-γ+ cells of 0.08% (0.01–0.18%), and a mean purity obtained after magnetic enrichment of 44.58% (12.97–76.08%). In case of the remaining n = 4 donors, the enrichment of CD3+/IFN-γ+ T cells was below 10% . These donors did not fulfill the acceptance criteria, hence are not suitable for donation of SARS-CoV-2-specific T cells.The mean purity obtained after magnetic enrichment was 37.44% , an activated phenotype and expression of cytotoxic molecules. A positive correlation between results obtained by ELISpot Assay and CSA suggests the suitability of ELISpot Assay for screening of potential donors for antiviral T cell manufacturing  were selected first based on results obtained via ELISpot Assay during donor screening and second based on results of CSA during donor pretesting. SARS-CoV-2-specific T cells responding to MACS GMP SARS-CoV-2 Select could be enriched by 174-fold  were reduced by 64-fold, while the frequencies of effector memory T cells (TEM) were markedly increased  and the final formulated T cell products (DP) were analyzed with respect to microbial sterility: No growth was observed within 7–14 days of incubation (data not shown). Endotoxin levels were assessed using Endosafe nexgen PTS and were <5 EU/ml (data not shown).Analysis of cellular impurities in the DS revealed a decrease of CD3to preDS . Moreovencreased . In persncreased . ViabiliTo estimate the reactivity of PepTivator SARS-CoV-2 Select-specific T cells towards the different VOCs, we performed ELISpot assay using peptide pools covering the Spike (S) protein from wild type (WT), and different VOCs as well as membrane (M) and nucleoprotein (N) . Samples+) was determined using flow cytometry (+) were significantly increased in co-cultures with antigen-loaded compared to unloaded target cells.SARS-CoV-2-specific T cells enriched by CCS IFN-gamma using the CliniMACS Prodigy System were expanded for up to 13 days using irradiated autologous PBMCs isolated from the LP and proved to maintain cellular function and high proliferative capacity . For detytometry . After 4ytometry . MoreoveTo investigate the cells’ capacity to produce and secrete cytotoxic mediators upon target recognition, cell culture supernatants of co-cultures with target cells loaded with PepTivator SARS-CoV-2 Select were analyzed via LEGENDPlex assay for secretion of effector molecules . Signifihttp://www.alloCELL.org) of the Institute of Transfusion Medicine and Transplant Engineering at Hannover Medical School has been expanded to SARS-CoV-2-specific T cells in donors recovered from SARS-CoV-2 infection. Memory T cell responses against SARS-CoV-2 were quantified and characterized after antigenic restimulation using ELISpot assay, intracellular cytokine staining, and quantitative realtime PCR. Furthermore, in light of clinical-grade manufacturing for adoptive T cell therapy, potential T cell donors were pretested using Cytokine Secretion Assay, largely analogous to the clinical-scale enrichment process via CCS IFN-gamma, and CliniMACS Prodigy. In contrast to ELISpot assay and intracellular cytokine staining, CSA allows for enrichment of antigen-specific T cells secreting IFN-γ upon stimulation. Moreover, frequencies of IFN-γ+ T cells obtained via CSA were comparable to those obtained during clinical-grade manufacturing, demonstrating that CSA is well-suited for selection of potential T cell donors. The four generated clinical-grade T cell products were characterized with respect to their cellular composition as well as to their proliferative and cytotoxic potential by using flow cytometry, in vitro cytotoxicity assays and multiplex analysis. In summary, these data demonstrate that the enriched clinical-grade SARS-CoV-2-specific T cells are able to replicate, specifically recognize and kill target cells, and secrete cytotoxic molecules upon target recognition.SARS-CoV-2-specific T cells developing during infection and subsequent recovery from the disease support viral clearance and control of infection. The developing antiviral T cell repertoire covers a broad range of viral antigens including both, structural and non-structural proteins. COVID-19 patients with insufficient endogenous functional antiviral T cells might benefit from adoptive transfer of SARS-CoV-2-specific T cells isolated from convalescent individuals, a concept which has been developed for viral complications with CMV, ADV, and EBV in immunosuppressed patients after transplantation . This st5, 5 × 105, and 1 × 106 memory T cells/kg body weight to hospitalized COVID-19 patients at risk for a severe course (One of the most dreaded side effects of the administration of T cell products is the development of Graft-versus-Host Disease (GvHD) triggered by alloreactive T cells in the product. The presence of naive T cells in T cell products precipitates the risk of GvHD, since naive T cells have a typically broader T cell receptor (TCR) repertoire and, therefore, a higher alloreactivity potential than memory T cell fractions . In a sie course . All thr+ T cells harbor the potential to successfully contribute to the elimination of the virus and the infected cells by inducing endogenous CD8+ T cells or vice versa. The effectiveness of CD4+ T cell responses has been demonstrated for AdV  match in HLA class I and II alleles of recipient and donor.In contrast to CD45RA-depleted memory T cell products, the cellular products generated in this study are specifically enriched for SARS-CoV-2 memory T cells. The recognition of infected cells by enriched antiviral T cells via the corresponding epitopes will lead to the generation and proliferation of endogenous virus-specific T cells in the patient and thus successfully and permanently control the virus. It has recently been shown that third party donor-derived T cells had contributed to endogenous CMV-specific T cell reconstitution in a patient . Therefo for AdV  and poly for AdV  as well  for AdV  and is dpatients . Antivir T cells . The int+ and CD8+ T cell subsets. In fact, the HLA-A, -B, and DR alleles represented in the SARS-CoV-2 Select peptide pool are present in >95%, >85% and >90% of the donors in the alloCELL registry, respectively. Of note, the majority of these peptides are identical in wild type and currently circulating VOCs, including the Delta and Omicron variants. It is assumed that future emerging VOCs will be sufficiently covered as well by PepTivator SARS-CoV-2 Select. Here, we were able to show that a large fraction of convalescent COVID-19 patients has specific T cells reactive against PepTivator SARS-CoV-2 Select and that these T cells could be magnetically enriched to high purities via IFN-γ CSA.Leung and colleagues reported successful manufacturing of SARS-CoV-2-specific T cells from six convalescent COVID-19 patients using CCS IFN-gamma and peptide pools PepTivator SARS-CoV-2 M, N, and S . They hy+ T cells/kg body weight, corresponding to the upper limit of T cells of donor lymphocyte infusion (DLI) in the haploidentical setting deemed to be safe  are patients with COVID-19, WHO clinical progression scale 4 with predefined risk factors for a severe disease course or patients with WHO clinical progression scale 5 (In large-scale GMP-compliant manufacturing runs using CliniMACS Prodigy, clinical-grade T cells products with purities comparable to those obtained in small-scale donor pretesting were achieved. To minimize the risk of GvHD, the overall T cell dose for adoptive transfer including virus-specific and contaminating T cells should be limited to total 25,000 CD3 be safe . Virus-s be safe . This is be safe  showing  be safe . Hence, observed . In cont scale 5 .The cellular immune response against SARS-CoV-2 is characterized by antiviral T cells with a broad repertoire, remaining readily detectable more than 1 year after initial infection. In contrast to antibody generation the protective effect of this T cell response is largely maintained also towards viral VOCs. Immunodeficient individuals or COVID-19 patients with insufficient endogenous T cell responses might therefore benefit from adoptive transfer of allogeneic, SARS-CoV-2-specific T cells isolated from convalescent individuals. In the present study, we have shown that PepTivator SARS-CoV-2 Select covering a broad variety of structural and non-structural SARS-CoV-2 proteins is suitable for donor selection and subsequent clinical-scale manufacturing of SARS-CoV-2-specific T cells. We performed four successful clinical-grade manufacturing processes for the generation of SARS-CoV-2-specific T cell products for adoptive T cell therapy. Enriched SARS-CoV-2-specific T cells harbored proliferative capacity and cytotoxic potential towards their target cells. By enriching for antiviral memory T cells with depletion of potentially alloreactive naïve T cells and careful consideration of partial HLA class I and II matching between donor and recipient, adoptive transfer of SARS-CoV-2-specific T cells should provide a unique and safe treatment option for COVID-19 patients at risk for a severe course of the disease.


Caenorhabditis elegans. We demonstrate that TDP-43 specifically enhances tau but not Aβ neurotoxicity, resulting in neuronal dysfunction, pathological tau accumulation and selective neurodegeneration. Furthermore, we find that synergism between tau and TDP-43 is rescued by loss-of-function of the robust tau modifier sut-2. Our results implicate enhanced tau neurotoxicity as the primary driver underlying worsened clinical and neuropathological phenotypes in AD with TDP-43 pathology, and identify cell-type specific sensitivities to co-morbid tau and TDP-43. Determining the relationship between co-morbid TDP-43 and tau is crucial to understand, and ultimately treat, mixed pathology AD.Although amyloid β (Aβ) and tau aggregates define the neuropathology of Alzheimer's disease (AD), TDP-43 has recently emerged as a co-morbid pathology in more than half of patients with AD. Individuals with concomitant Aβ, tau and TDP-43 pathology experience accelerated cognitive decline and worsened brain atrophy, but the molecular mechanisms of TDP-43 neurotoxicity in AD are unknown. Synergistic interactions among Aβ, tau and TDP-43 may be responsible for worsened disease outcomes. To study the biology underlying this process, we have developed new models of protein co-morbidity using the simple animal  Summary: TDP-43 co-pathology is a frequent feature of Alzheimer's disease. This paper shows that TDP-43 specifically enhances tau neurotoxicity, but not amyloid β, to promote pathological protein accumulation, behavioral impairment and selective neurodegeneration. Both Aβ its own . Recent ysiology .TARDBP, are causative for ALS, demonstrating that dysfunctional TDP-43 actively contributes to neurodegenerative pathways  and ∼50% of cases of frontotemporal lobar degeneration (FTLD-TDP) . TDP-43 pathways .Following its identification as a key pathologic protein in ALS and FTLD-TDP, TDP-43 pathology was also found to occur as a secondary pathology in other neurodegenerative diseases . In partin vitro or in vivo. In cell and mouse models, TDP-43 regulates mRNA splicing of tau exon 10, shifting the ratio of tau microtubule binding repeats from the normal balanced ratio of 3R/4R-tau to a higher proportion of 4R-tau . Expression of TDP-43 in this strain is maintained at very low levels by the insertion of a self-cleaving ribozyme between the TDP-43 coding sequence and the 3′ untranslated region (UTR) of the transgene, which results in the inactivation of most, but not all, transgene-derived transcripts   . In ordeC. elegans models of neurodegenerative disease (Movies 1-8). In contrast, Aβ+TDP Tg-low animals exhibited no change in motility from control strains .Visually, the tau+TDP Tg-low animals exhibit dramatically worse motility than either tau Tg or TDP Tg-low strains, manifested by increased uncoordination in their movement. In fact, impaired motility has been used successfully as a readout for neuronal dysfunction or neurodegeneration in other  disease  and repr strains C,D. To c strains . SimilarAs controls, we examined whether tau Tg or TDP Tg-low would synergize with a non-AD related neurodegenerative disease model expressing 86 repeats of polyglutamine pan-neuronally (polyQ Tg) . PolyQ TWe next examined several well-characterized behavioral outputs in the tau+TDP Tg-low model. Mechanosensation integrates signaling from dopaminergic, glutamatergic and serotonergic neurotransmitter classes of neurons to generate a stereotypical response to light touch. To evaluate this, we employed an assay evaluating responses to gentle touch with an eyelash hair ten times, alternating head and tail touches. Consistent with previous reports , tau Tg Fig. S3). Therefore, TDP-43 potentiates pathological tau protein changes, which may be the primary driver of neurotoxicity in co-expression models of tau and TDP-43.It is possible that the motility and sensory impairments observed in tau+TDP Tg-low are due to accumulation of neurotoxic pathological tau or TDP-43. To determine whether the co-expression of tau and very low levels of TDP-43 influenced their protein accumulation, we measured protein levels in tau+TDP Tg-low by immunoblotting. We found that both total and phosphorylated tau increased in tau+TDP Tg-low A-C, but,Fig. S4). Conversely, all transgenic strains surveyed had a significant decrease in glutamatergic neurons compared with non-Tg animals at day 1 of adulthood; however, by day 4 of adulthood, both tau Tg and tau+TDP Tg-low animals had lost significantly more neurons than non-Tg or TDP Tg-low animals alone . We also evaluated whether aging differentially affected these various neurotransmitter classes of neurons by assessing neurodegeneration over time at day 1 and day 4 of adulthood. This survey revealed distinct differences in neuron vulnerabilities by neurotransmitter class to co-morbid tau and TDP-43, and to aging. We found no significant difference in dopaminergic neurons at either day 1 or day 4 of adulthood between all strains  (. We found sut-2 null mutations [sut-2 (−)] robustly protected against tau+TDP Tg-low uncoordinated motility (sut-2(−) could reduce the accumulation of tau or TDP-43 protein. We found that sut-2(−) reduced both total tau protein and pathological phosphorylated tau in tau+TDP Tg-low animals (sut-2(−) did not significantly reduce levels of total or phosphorylated TDP-43 in tau+TDP Tg-low  . We founmotility A and GABmotility B,C. We t animals A-C. HoweP Tg-low A,D,E. TaC. elegans model of combined tau and TDP-43 proteotoxicity, we demonstrate that the presence of TDP-43 promotes the accumulation and hyperphosphorylation of tau, whereas tau does not have the same effect on TDP-43 protein. We also show that the combination of tau and TDP-43 differentially affects neurons of different neurotransmitter classes, indicating some degree of selective cell-type vulnerability. Further, we show that suppression of tau neurotoxicity using the genetic modifier sut-2 protects against effects of tau and TDP-43 synergism.Through a series of functional experiments in a C. elegans TDP-43 homolog, tdp-1, impacted neurotoxicity of tau Tg animals, but did not see any difference in tau-mediated motility dysfunction . The C. elegans tdp-1 gene lacks the C-terminal G-rich domain present in human TDP-43, which is the site of pathological phosphorylation, the location of most ALS-causing mutations in the TARDBP gene, and comprises the fibrillar core of aggregates in FTLD-TDP  . Notably, it has been previously shown that tau synergizes with Aβ in d Aβ Tg) . We alsoin vitro TDP-43 does not modulate the expression of APP, nor does APP affect TDP-43 expression  only modestly decreases TDP-43 protein accumulation and does not protect against TDP Tg motility impairment. The near complete amelioration of motility defects and neurodegeneration in the combined tau+TDP-low Tg model in the presence of sut-2(−) supports the concept that enhanced tau neurotoxicity drives the exacerbated phenotypes. However, the mechanism of this protection remains unknown. SUT-2 is nuclear localized, binds mRNA and regulates poly-A tail length  was maintained as previously described (bkIs1441[Paex-3::Tau WT(4R1N)+Pmyo-2::dsRED] (gnaIs2[Pmyo-2::YFP+Punc-119::Abeta1-42] (dvIs50[pCL45(snb-1::Abeta1-42::3′UTR(long))+mlt-2::GFP] (bkIs241[pF25B5.3::Q86-YFP] (oxIs12[Punc-47::GFP + lin-15(+)] +Pmyo-2::dsRED]; bkIs1943[Psnb-1::hTDP-43 WT::K4aptazyme::unc-54 3′UTR+Pmyo-3::mCherry] was generated by crossing CK1441 and CK1943. The CRISPR-generated whole-gene deletion CK3011 sut-2(bk3011) was generated as described in sut-2(bk3011) to generate NLS23 sut-2(bk3011); bkIs1441[Paex-3::Tau WT(4R1N)+Pmyo-2::dsRED]; bkIs1943[Psnb-1::hTDP-43 WT::K4aptazyme::unc-54 3′UTR+Pmyo-3::mCherry], NLS24 sut-2(bk3011); bkIs1943[Psnb-1::hTDP-43 WT::K4aptazyme::unc-54 3′UTR+Pmyo-3::mCherry] and NLS25 sut-2(bk3011); bkIs1441[Paex-3::Tau WT(4R1N)+Pmyo-2::dsRED]. Strains with GFP-marked serotonergic neurons were generated by crossing the reporter strain JPS617 [Ptph-1::GFP] with CK1441, CK1943 and NLS19. Strains with GFP marked dopaminergic neurons were generated by crossing the reporter strain WG1 [Pdat-1::GFP] with CK1441, CK1943 and NLS19. Strains with GFP marked glutamatergic neurons were generated by crossing the reporter strain OH10972 [Peat-4::GFP] with CK1441, CK1943 and NLS19. Strains with GFP-marked cholinergic neurons were generated by crossing the reporter strain LX929 [Punc-17::GFP] with CK1441, CK1943 and NLS19. Strains with GFP-marked GABA-ergic motor neurons were generated by crossing sut-2(bk3011) with CK1441, CK1943, NLS19 and the reporter strain EG1285 [Punc-47::GFP]. See Table S1 for complete list and descriptions of all strains used in this study.Wild-type escribed . Previou::dsRED] , GRU102 eta1-42] , CL2355 -2::GFP] , CK241 bQ86-YFP]  and EG12n-15(+)] . CK1943 C. elegans were transferred to 35 mm NGM assay plates seeded with 20 μl OP-50 bacteria. Animals were allowed to acclimate to conditions on assay plates at room temperature for at least 30 min before recording movements for 1 min at 7.5 frames per second. For thrashing assays, staged-matched day 1 adult C. elegans were given 30 min to acclimate to the assay room conditions. Approximately 50 animals were then transferred to a 35 mm assay plate by way of washing in 1 ml M9 buffer, given 1 min to standardize swimming behavior, followed by a 1 min recording time at 14 frames per second. Tracks were verified and repaired as needed. Figures show results from at least three independent replicates.Unstimulated activity and thrashing behaviors were assessed using the WormLab system (MBF Bioscience). For unstimulated activity assays, stage-matched day 1 adult Mechanosensation assays were adapted from EPG comparisons were performed using the NemaMetrix ScreenChip System and associated software packages NemAquire and NemAnalysis, and Microsoft Excel. Stage-matched day 1 adult animals were washed in M9 and then incubated at room temperature in 10 mM Serotonin in M9 buffer for 20 min to induce pharyngeal pumping. Animals were loaded into a ScreenChip20 cartridge under vacuum. The electrical signal  was recorded individually for 2 min. Average pump frequency and duration readouts from NemAnalysis were averaged over three independent replicates per strain.Animals were grown to day 1 or day 4 adult. Living animals were immobilized on a 2% agarose pad with 0.01% sodium azide, and intact neurons were scored under fluorescence microscopy on a DeltaVision Elite (GE) imaging system using an Olympus 60× oil objective. Scoring was conducted blinded to genotype. Figures show results from at least three independent replicates.C. elegans were harvested and snap frozen per sample. Protein was extracted by resuspending pellets in 1× sample buffer, three sessions of 10 s sonication with cooling on ice water in between sessions, and 10 min boiling. Samples were loaded and resolved on precast 4-15% gradient SDS-PAGE gels and transferred to PVDF membrane as recommended by the manufacturer (Bio-Rad). On immunoblots, human TDP-43 was detected by a monoclonal antibody, anti-TDP-43 . TDP-43 phosphorylated at pS409/S410 was detected by a monoclonal antibody, anti-phospho TDP-43 . Human tau was detected by a polyclonal antibody anti-tau . Phosphorylated tau was detected by a monoclonal antibody anti-phosphorylated tau . C. elegans β-tubulin levels were measured using monoclonal antibody E7  as a loading control. HRP-labeled goat anti-mouse IgG secondary antibody  was used at a dilution of 1:2500. HRP-labeled mouse anti-rabbit IgG secondary antibody  was used at a dilution of 1:10,000. Quantitation was completed by ImageJ software densitometry analysis of scanned film images.Approximately 10,000 stage-matched day 1 adult All statistical analyses were performed using GraphPad Prism statistical software. Statistical significance was determined using one-way ANOVA with Tukey's multiple-comparison test. Behavioral assays are graphed using violin plots showing the distribution and density of measures of worm movement in response to the particular stimulation for each assay; all other data are presented using standard bar graphs. Error bars represent standard error of the mean (s.e.m.).


Apoptosis is regulated by interactions between the BH3-only and multi-domain Bcl-2 family proteins. These interactions are integrated on the outer mitochondrial membrane (OMM) where they set the threshold for apoptosis, known as mitochondrial priming. However, how mitochondrial priming is controlled at the level of single cells remains unclear. Retrotranslocation of Bcl-XL has been proposed as one mechanism, removing pro-apoptotic Bcl-2 proteins from the OMM, thus reducing priming. Contrary to this view, we now show that Bcl-XL retrotranslocation is inhibited by binding to its BH3-only partners, resulting in accumulation of these protein complexes on mitochondria. We find that Bcl-XL retrotranslocation dynamics are tightly coupled to mitochondrial priming. Quantifying these dynamics indicates the heterogeneity in priming between cells within a population and predicts how they subsequently respond to a pro-apoptotic signal. Bcl-2 proteins regulate apoptosis through mitochondrial outer membrane permeabilisation (MOMP). The relative proximity of a cell to MOMP, termed mitochondrial priming, determines whether a cell will undergo apoptosis in response to an insult, can predict how tumours respond to chemotherapy , 2, and via binding to specific anti-apoptotic Bcl-2 proteins [Mitochondrial priming can be measured experimentally by BH3-profiling , 9. Whenproteins –13. Howeproteins –17. Deteproteins –21.Here we measure the subcellular dynamics of Bcl-2 proteins to interrogate single cell heterogeneity in mitochondrial priming. Bcl-2 family proteins constantly shuttle between mitochondria and the cytosol , 23. ForBax and Bak constitutively retrotranslocate from mitochondria, although with markedly different kinetics , 23, 27.Bax/Bak double knockout (DKO) MEFs were transiently transfected with full-length fluorescent-protein tagged Bcl-XL, Bcl-W, or Bcl-2. Fluorescent recovery after photobleaching (FRAP) allows rapid analysis of protein mobility within live cells. Mitochondrial GFP-Bcl-XL fluorescence recovered rapidly within the photobleached region of interest (ROI), indicating that the protein was dynamically exchanging between membrane bound and soluble fractions Fig. . MitochoAnti-apoptotic Bcl-2 proteins lacking their C-terminal transmembrane (TM) regions still bind BH3-domain peptides within their exposed hydrophobic pocket . HoweverBinding between BH3-only and multi-domain anti-apoptotic proteins have largely been defined in vitro , 29, 30.We asked if reduced Bcl-XL retrotranslocation was dependent on canonical BH3-domain binding. We co-expressed in DKO MEFs GFP-Bcl-XL and mCh-BH3 proteins with substitutions within the BH3-domain that reduce binding within the hydrophobic pocket. Both tBid-mCh and mCh-BimEL reduced GFP-Bcl-XL FRAP Fig. . HoweverpaGFP-Bcl-W showed behaviour distinct to paGFP-BclXL when co-expressed with BH3-only proteins Fig. . mCh-BimThese data indicate that BH3-only protein binding to anti-apoptotic proteins slows retrotranslocation rates from mitochondria, and the binding specificity of BH3-only proteins for full-length anti-apoptotic Bcl-2 proteins in live cells gives a different picture from that determined using BH3-peptides and truncated proteins in vitro.As GFP-Bcl-XL retrotranslocation was inhibited by the broadest range of BH3-only proteins, we hypothesised that we could use it to assess their activity in live cells. We generated MCF10A mammary epithelial cells stably expressing GFP-Bcl-XL using lentiviral transduction. These were then transiently transfected with mCh-tagged Bad, tBid, Puma, Noxa, or BimEL, and FRAP analysis performed Fig. . The resThe transient expression of BH3-only proteins would mimic the mitochondrial priming observed when cells are exposed to a stress that would increase their activity. We therefore hypothesised that there would be reduced GFP-Bcl-XL retrotranslocation in cells subjected to conditions such as reduced growth factor receptor (GFR) signalling, which would increase priming but not by itself induce apoptosis. MCF10A cells stably expressing GFP-Bcl-XL were cultured in either complete growth medium or in serum and growth factor (GF)-free conditions before FRAP analysis Fig. . StarvedTogether, these data indicate that Bcl-XL is rapidly exchanged between mitochondria and the cytosol in non-apoptotic cells Fig. . MembranTam. Bad links GFR signalling to priming in MECs [via 14.3.3 binding [Tam. We stably infected MCF10A cells (which are ER negative) with a lentivirus co-expressing BadERTam and GFP-Bcl-XL , which was accompanied by increased mitochondrial association of GFP-Bcl-XL , or mCh-Bak which preferentially binds Bcl-XL (MODE2) [Tam displaced tBid-mCh, increasing mCherry FRAP. In contrast, MODE2 inhibition displayed a distinct profile. mCh-Bak did not stabilise mitochondrial GFP-Bcl-XL to the extent seen with BH3-only proteins  or pore forming effectors Bax and Bak (MODE2) . We aske (MODE2) . tBid-mCins Fig. . Activats slowly , 27, andTam Fig. .These data indicate that the dynamics of Bcl-XL retrotranslocation are directly linked to apoptotic priming by BH3-only proteins.We were intrigued to observe significant cell-to-cell variation in GFP-Bcl-XL dynamics, with overlap between untreated and 4-OHT-treated conditions Fig. . QuantifTam/GFP-Bcl-XL cells. Immunoblotting ; anti-GFP, (Invitrogen); anti-oestrogen receptor alpha (Santa Cruz); anti-V5 (Serotec); anti-mtHsp70 (Affinity Bioreagents); anti-Vinculin (Sigma), anti-pBad phospho-S112 (New England Biolabs #2921S), anti-Bad  secondary antibodies (Jackson Labs). Etoposide, paclitaxel, and 4-hydroxy tamoxifen were obtained from Sigma.For immunoblotting, proteins were separated by SDS PAGE. Following transfer to nitrocellulose, membranes were incubated with primary antibodies. Proteins were detected with IrDye 800 and 680 conjugated secondary antibodies , which were detected using an Odyssey CLx imager (LiCor). The full-length blots are shown in the via site-directed mutagenesis . Bcl-XLR139D was generated via site-directed mutagenesis on GFP-BclXL. Mutagenesis was performed using either QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) or by Gibson assembly (New England Biolabs) within the full-length versions in pEGFP or pCDH vectors. mCherry-tagged tBid, BimEL and Bad, along with the BH3-domain variants, were generous gifts from David Andrews (McMaster University). mCherry-Puma and Noxa were generated by PCR amplification of the coding sequences and cloning into mCherryC1.For the ∆TM variants, Bcl-2, Bcl-W, Bcl-XL, and Mcl-1 were truncated by introducing a stop codon upstream of the hydrophobic transmembrane domain Tam was generated by cloning the coding sequence for Bad into pCDH-EF1-T2A-GFP-BclXL, and subsequently inserting the coding sequence for the oestrogen receptor hormone-binding domain [Lentivirus expression vectors were generated using pCDH-EF1-MCS-T2A (SystemBiosciences). GFP and paGFP-Bcl2 proteins were inserted downstream of the T2A sequence. For the paGFP variants, H2B-mRFP was inserted upstream of the T2A sequence . BadERTag domain  in frame2. All cells were maintained in 10 cm dishes (Corning) and passaged when appropriate.MCF10A and MDA-MB-231 cells were obtained from ATCC. Bax/Bak double knock out MEFs were originally obtained from Stanley Korsmeyer (Dana Farber). MEFs and MDA-MB-231 were grown in DMEM (Lonza) supplemented with 10% FBS (Labtech) and 1% penicillin/streptomycin (Sigma). MCF10A cells were grown in DMEM-F12 (Lonza) supplemented with 5% Horse Serum (Biosera), 25 ng/ml EGF, 0.5mg/ml hydrocortisone, 100ng/ml Cholera Toxin, 10 µg/ml insulin, 1% penicillin/streptomycin (Sigma). All lines were maintained at 37 °C with 5% COFor transient transfections, cells were seeded into 35 mm glass bottom dishes (MatTek) 24 h pre-transfection to ensure approximately 70% confluency the next day. Cells were transfected using TransIT-X2 reagent (Mirus) as per manufacturer’s instructions.For stable viral infection, cells were seeded into a six-well culture plate (Corning) 24 h prior to infection to ensure approximately 40% confluency for infection. Cells were infected in full growth medium containing 2 µg/ml Polybrene (Millipore) and the appropriate concentration of virus. The cells were then incubated overnight at 37 °C before adding fresh growth medium. Cells were passaged for two weeks and stably expressing cells selected by fluorescence-activated cell sorting (FACS).Prior to imaging, cells were washed twice in PBS and 2 ml of live-imaging medium (Ham’s-F12 supplemented with 10% FBS (Lonza), 1% penicillin/streptomycin, and 25mM HEPES (Sigma)) was added to each dish before incubating in the microscope chamber at 37 °C for a minimum of 1 h prior to imaging. 4-OHT (Sigma) was added to cells at a concentration of 10 nM for 1 h before imaging. Images were captured using a 3i Zeiss confocal system with a CSU-X1 Spinning Disc (Yokogawa), using a 63×/1.40 Plan Apochromat objective (Zeiss), an Evolve EM-CDD camera (Photometrics), and a motorised XYZ stage (Intelligent Imaging Innovations) driven by Marianas hardware. Images were captured using Slidebook 5.0 software (Intelligent Imaging Innovations).For FRAP, a region of interest (ROI) was selected within the cytosol of a cell and photobleached . Images were taken at 5 s intervals for 30 s pre-bleach and then post-bleaching. For GFP photoactivation, a ROI was selected within the cytosol and photoactivated for 10 ms using a 405 nm laser at 100% power. Images were analysed using Image J software. Briefly, background was subtracted using a rolling ball radius of 50 before measuring levels of fluorescence within the bleached ROI in each sequential image. Data were normalised to 100% fluorescence pre-bleach (FRAP) or 100% fluorescence post-photoactivation and a one-phase association or dissociation curve fitted.Cells were immunostained for active caspase 3 as previously described . For quaFor analysis of endogenous Bad, MCF10A cells were cultured either in complete growth media or serum-free growth media for 24 h. After 24 h, media was replaced for 1h, either with complete growth media (serum and recovery conditions) or serum-free growth media (serum starvation condition). After 1 h, cells were fixed in 4% PFA and stained for DAPI alongside either anti-pBad S112 or total Bad. Cells were imaged using an EVOS M7000 using a 40× Plan Fluorite 0.75NA objective  or a Zeiss Axioplan 2 using a 63× 1.4NA Plan Apochromat objective (for images). Fiji was used to determine the corrected total cell fluorescence of a minimum of 100 individual cells per condition.2. Images were acquired on an AS MDW live cell imaging system (Leica) in brightfield using a 20× HC Plan Fluotar objective, using point visiting to allow multiple positions to be captured within the same time course. Image stacks were analysed using ImageJ, single cells identified and followed manually to determine fate.Cells were cultured in 24-well tissue culture plates, and treated with 1 μM Taxol, 5 μM ABT-737, or 10 μM 4-OHT as detailed in text, and imaged at 15 min intervals for 50 h, maintained at 37 °C and 5% CODetails of statistical analysis used are provided in each figure legend. All analysis was undertaken using GraphPad Prism.Supplementary dataMarked up manuscript fileSupplementaryMovie 1SupplementaryMovie 2SupplementaryMovie 3SupplementaryMovie 4SupplementaryMovie 5SupplementaryMovie 6SupplementaryMovie 7SupplementaryMovie 8Change of authorship request formauthor checklistauthor checklist


Caenorhabditis elegans is a complex behavior with a strong mechanosensory component. C. elegans has several characterized mechanotransducer proteins, but few have been shown to contribute to mating. Here, we investigated the roles of PEZO-1, a piezo channel, and TRP-4, a mechanotransducing TRPN channel, in male mating behavior. We show that pezo-1 is expressed in several male-specific neurons with known roles in mating. We show that, among other neurons, trp-4 is expressed in the Post-Cloacal sensilla neuron type A (PCA) sensory neuron, which monitors relative sliding between the male and the hermaphrodite and inhibits neurons involved in vulva detection. Mutations in both genes compromise many steps of mating, including initial response to the hermaphrodite, scanning, turning, and vulva detection. We performed pan-neuronal imaging during mating between freely moving mutant males and hermaphrodites. Both pezo-1 and trp-4 mutants showed spurious activation of the sensory neurons involved in vulva detection. In trp-4 mutants, this spurious activation might be caused by PCA failure to inhibit vulva-detecting neurons during scanning. Indeed, we show that without functional TRP-4, PCA fails to detect the relative sliding between the male and hermaphrodite. Cell-specific TRP-4 expression restores PCA's mechanosensory function. Our results demonstrate new roles for both PEZO-1 and TRP-4 mechanotransducers in C. elegans mating behavior.Male mating in  Significance StatementCaenorhabditis elegans male copulatory behavior has emerged as a paradigm for the study of complex innate behavior in a comprehensive manner. We find that the orthologs of Piezo and TRPN account for some of the mechanosensation predicted for male mating, thereby allowing us to refine the model for this innate behavior. Most notably, we identify the key role of TRPN in the ability of the PCA sensory neuron to detect relative movement between the male and his partner. By monitoring relative movement, PCA can provide inhibitory input to the downstream circuit for vulva detection, contributing to switching between two temporally adjacent behaviors. We provide an example of how brain-wide activity imaging, genetics, and connectomics combine to understand the function of a neural circuit.Caenorhabditis elegans, including a TRPA ortholog TRPA-1, a TRPV ortholog OSM-9, a TRPN ortholog TRP-4, and a DeG/ENaC channel composed of MEC-4/MEC-10 and accessory proteins. These channels regulate diverse rapid behavioral responses  and HOok sensillum neuron type B (HOB) have ciliated sensory endings but are also embedded in a structure with a mechanosensory morphology (the hook) . The cilmec-4 gain-of-function, mec-4 reduction-of-function, and mec-10 loss-of-function mutants have turning defects, suggesting the involvement of the MEC-4/MEC-10 complex in regulating turning behavior during mating  in mating. Specifically, g mating . Anotheretection . PKD1 isetection . Here, wCaenorhabditis elegans strains were cultured at 23°C ( at 23°C . Assays Genomic information was obtained from WormBaseThis study:pezo-1(sy1113) IVPS7489 pha-1 III; him-5 V;syEx1771 [pBX-1 + pezo-1(p3)::GFP]PS8908 pha-1 III; him-5 V;syEx1792 [pBX-1 + pezo-1(p5)::GFP]PS8909 hpIs675 [Prgef-1 GCaMP6::3xNLS::mNeptune + lin-15(+)]; him-5 (e1490) VPS8040 hpIs675 [Prgef-1 GCaMP6::3xNLS::mNeptune + lin-15(+)]; pezo-1(sy1113) IV; him-5 (e1490) VPS8041 hpIs675 [Prgef-1 GCaMP6::3xNLS::mNeptune + lin-15(+)]; pezo-1(av240) IV; him-5 (e1490) VPS9157 hpIs675 [Prgef-1 GCaMP6::3xNLS::mNeptune + lin-15(+)]; trp-4 (sy695) I; him-5 (e1490) VPS9045 syEx1715[(pWP082)Peat-4(p8)::TRP-4::unc-54 3'UTR + unc-122::RFP];hpIs675[Prgef-1GCaMP6::3xNLS::mNeptune + lin-15(+)]; trp-4 (sy695) I; him-5 (e1490) VPS9669 Other strains:him-5 (e1490) VCB4088 pezo-1(av240) IVAG570 trp-4 (sy695) I; him-5 (e1490) VPS1905 hpIs675 [Prgef-1 GCaMP6::3xNLS:: mNeptune] XZM9624 aeaIs010 [Prgef-1::GCaMP6s::3xNLS + lin-15(+)]ADS1002 uuEx18 [dcr-1(wild-type) + dpy-30::mCherry]BB92 otIs377 [myo-3p::mCherry]; unc-64(e246) IIIADS1014 bxIs19 [trp-4p::GFP]OH10235 him-5 (e1490) otIs564 [unc-47(fosmid)::SL2::H2B::mChopti + pha-1(+)] VOH13105 pha-1 (e2123) III; him-5(e1490) V; otIs518 [eat-4(fosmid)::SL2::mCherry::H2B + pha-1(+)]OH13645 otIs376 [eat-4(prom4)::GFP + rol-6(su1006)]OH10972 otIs392 [eat-4(prom6)::GFP + ttx-3::DsRed]OH11152 otIs138 [ser-2(prom3)::GFP + rol-6(su1006)] X; otIs521 [eat-4(prom8)::tagRFP + ttx-3::GFP]OH16719 otIs520 [eat-4(prom11)::GFP + ttx-3::mCherry]OH14018 pezo-1, a transcriptional fusion plasmid of several predicted regulatory regions and Green Fluorescent Protein (GFP) were generated and injected into N2 hermaphrodites along with a myo-2 DsRed coinjection marker , we injected a 46,168 Crispr/Cas9 plasmid (peft-3:: Cas9-sv40_NLS:: ttb-2 UTR) along with pRB1017 plasmids (pU6:: gRNA construct) containing our guide RNA sequences  into young adult hermaphrodites  and a detection PCR with two primers external to the proposed deletion . A homozygous mutation line was determined by the absence of a band in the control PCR and the presence of a 500 bp band in the detection PCR ; him-5(e1490) mutant background, PS8040 was generated by crossing ZM9624 into him-5(e1490), and PS9045 was generated by crossing ZM9624 into trp-4(sy695); him-5(e1490). To track mating events, we used hermaphrodites that expressed red fluorescent markers in their cuticle or muscles. To encourage the richness of male behavior, we used partners with normal motility or showing only mild unc phenotypes.For pan-neuronal imaging, we used ZM9624, a strain designed to coexpress calcium indicator GCaMP6s and red fluorescent protein mNeptune in all neuronal nuclei under the promoter . PS8041 the trp-4(sy695) mutation   mutation. The resulting strain—PS9669—was screened for animals showing expression of the coinjection marker, which were selected for pan-neuronal imaging.We sought to rescue ion Fig.  by expreng eat-4 , 51. Thephrodite , 53. We x, y, and z. Imaging experiments lasted from 1.5 to 10 minutes and specifically aimed at capturing scanning, turning, and vulva detection events.To record the activity of neurons in freely moving males, we used a custom-built spinning disc confocal microscopy setup , 54. BriNeuronal activity traces were extracted from the raw data following image preprocessing and registration. Registered data were down-sampled to 5 volumes per second. Neuronal segmentation and tracking were performed using manual and semiautomated methods with the help of the Fiji plugin MaMuT 0.27 , 56. Flupezo-1(sy1113),pezo-1(av240), and trp-4(sy695) males, we extracted activities of eight neurons implicated in vulva detection and scanning  males. For trp-4 rescue males (PS9669), we quantified PCA activity. For control males, we used previously generated recordings of ZM9624 males, which were performed using the same microscopy setup . A time lag with the absolute maximum correlation across all motif instances was identified. One-sample t test was used to test if the correlation between the neuron's traces and behavior at that time lag was significantly greater than zero.Escherichia coli OP50 solution onto a 6 cm NGM agar plate and allowed to dry. Two him-5 hermaphrodites were placed onto the OP50 spot and allowed to acclimate for 5 minutes. A single male was added to the plate, and the plate was imaged with a Nikon microscope equipped with a 10x objective and a PointGrey camera. The male was tracked continuously by adjusting the stage position with a stage controller. The experiment was recorded at 5 frames/ second. The recording started when the male approached a hermaphrodite, and continued for 15 minutes or until sperm release, whichever was earlier. Mutant and control males were imaged in parallel.L4 males were picked onto a separate plate overnight and all experiments were performed on the next day. Mating plates were prepared by placing 2 μl of concentrated Collected recordings of mating were analyzed blindly. For each experiment, we tabulated timestamps for the following behavioral events: (i) ventral contact of the male tale with the hermaphrodite body, (ii) loss of ventral contact, (iii) contact with the vulva, (iv) end of vulva contact, (v) successful turns, (vi) failed turn attempts, (viii) sliding over to lateral sides of the hermaphrodite, (vii) beginnings and ends of pauses away from the vulva, and (viii) copulation and sperm release. From these timestamps, we calculated several measures of mating performance: (i) turning success rate, (ii) the number of ejaculation events per ventral contact duration, (iii) the number of ejaculation events per vulva contact duration, (iv) the ratio between spurious pausing duration and vulva contact duration, (v) the ratio between passing the vulva events and ventral contact duration, (vi) mean vulva contact, (vii) the number of “sliding over” events per ventral contact duration, and (viii) the number of ventral contact loss events per ventral contact duration.pezo-1 were previously shown to cause feeding-related phenotypes in C. elegans ] led to an expression pattern that contained all the sparser expression patterns with other transcriptional fusions.To determine ene Fig.  and B. Tene Fig.  to H. Evpezo-1 expression in several head neurons, pharynx-intestinal valve, vulval muscle, spermatheca, Canal Associated Neuron (CAN), and male tail neurons. Male-specific neurons that showed strong pezo-1 expression included PCB, PVV, and several sensory ray neurons [Fig. We observed trp-4 was previously shown to be expressed in eight pairs of A-type ray neurons  and pezo-1(av240)), a trp-4 (sy695) mutant, and control males (him-5). Virgin males were allowed to mate for 15 minutes with freely moving hermaphrodites. Freely moving hermaphrodites allow a more complete analysis of behavioral phenotypes as they present greater challenges to successful mating. For each male, we calculated the mating success index: the number of ejaculation events per courtship duration. This index is higher when the male succeeds in mating after a short period of scanning. The index is lower when the male succeeds in mating after a long period of scanning or fails to mate at all. pezo-1(av240) and trp-4(sy695) mutants, but not pezo-1(sy1113) were significantly less efficient at mating compared to control males   and 20% of trp-4(sy695) mutant males were able to mate successfully, compared to 60% and 50% of control and pezo-1(sy1113) males, respectively. We conclude that both pezo-1 and trp-4 mutations compromise mating efficiency.Given that both ly) Fig. . Only 10pezo-1 and trp-4 mutants, we analyzed video recordings of mating behavior, focusing on specific behavioral motifs: scanning, turning, stopping at the vulva, spurious spicule insertion attempts away from the vulva, sliding over the hermaphrodite's lateral side, and sperm release. We found that pezo-1(av240) and trp-4(sy695) mutants, but not pezo-1(sy1113) were significantly less efficient at multiple motifs of mating behavior. Compared to control males, pezo-1(av240) and trp-4(sy695) mutants were less likely to complete successful turns , showed more spurious spicule insertion attempts away from the vulva , and were less likely to stop at the vulva . When pezo-1(av240) and trp-4(sy695) males did reach the vulva, they were less likely to perform the full spicule insertion and sperm release   males also lost the hermaphrodite more easily (Wilcoxon test andP < 0.007). Mutations in both genes compromise multiple steps of mating, and result in partially-overlapping phenotypes.Low mating efficiency can result from defects in any one of the many steps that comprise overall mating behavior. To pinpoint the phenotypes of 26) Fig. . pezo-1(pezo-1(av240) mutant (a complete deletion of pezo-1) had much stronger mating defects compared to the pezo-1(sy1113) mutant . The C-terminal deletion in pezo-1(sy1113) removes the pore domain of PEZO-1 has a much stronger effect on the brood size compared to pezo-1(sy1199), a mutant with a Crispr/Cas-9 knock-in of a “STOP-IN” cassete (pezo-1(av240) and pezo-1(sy1113) mutants. Indeed, brood size comparisons show that the pezo-1(sy1113) mutants have a significantly larger brood size than the pezo-1(av240) mutants, ∼80 progeny per animal vs ∼20 progeny per animal over a three-day period  mutant, 32% ± 12% ,(SD) turn attempts fail and the tail loses contact with the hermaphrodite. We tested whether PVV showed different activity upon turning attempts in the wild-type males and pezo-1 (av240) mutants. PVV activity was extracted and aligned to the onset of turning attempts. Successful turns and failed turns were analyzed separately. In the wild type, PVV activity starts rising shortly before the turn and peaks 1.2 seconds (median) after the turn onset  mutants, PVV activates upon turning and its temporal dynamics is similar to the wild type  mutants, more than 50% of turning attempts were unsuccessful during brain-wide imaging experiments. In both wild-type and mutant males, PVV peaks later—at 1.8 seconds—when the turning is unsuccessful  mutants show compromised turning, and compromised turning is associated with a delay in PVV activation (both in mutants and wild type), it is uncertain if the pezo-1 mutation itself causes the delayed activation of PVV.ron Fig.  to K. Baron Fig. , 63. Theron Fig. . In the set Fig. . In the ful Fig.  to E. Alpezo-1 and trp-4 are expressed in neurons involved in vulva detection, we tested whether neurons in the previously identified vulva-detecting circuit—PCB, PCC, HOA, HOB, R2B, and PVX—exhibited different activity upon vulva contact in mutant males compared to control males. We recorded the activity of the male's entire posterior brain during mating with freely moving hermaphrodites. We extracted activities of PCB, PCC, HOA, HOB, R2B, and PVX and aligned them to the onset of vulva-detecting events. In control males, PCB, PCC, HOA, HOB, R2B, and PVX show a sharp increase in activity when the male tail contacts the vulva , pezo-1(av240), and trp-4(sy695) mutants  and trp-4(sy695) mutants frequently pause scanning away from the vulva and show spurious spicule insertion attempts , pezo-1(av240), and trp-4(sy695) mutants for such spurious activation of the vulva-detecting circuit  males, 5 of 10 (50%) pezo-1(av240) males, and 13 of 15 (86%) trp-4(sy695) males.pts Fig. . Previoupts Fig. . We screpezo-1av20 and trptrp-4 is expressed in the PCA neuron, we asked if the spurious activity in trp-4 mutants might be caused by the loss of mechanosensory function of PCA and the consequent release of the vulva-detecting circuit from glutamatergic inhibition from PCA.Previously, it was suggested that PCA mechanosensory activation with backward scanning inhibits spurious activation of the vulva-detecting circuit . When PCP < 0.001). When the male tail slides forward, PCA activity decreases . In contrast, in trp-4 males, PCA activity does not change when the direction of the relative sliding changes . Also, unlike in control males, in trp-4 mutants PCA does not become active upon ventral contact with the hermaphrodite . This suggests that mechanosensation in PCA is dependent on the TRP-4 channel. Without functioning TRP-4, PCA cannot monitor the relative sliding between the male and hermaphrodite and is thereby unable to provide glutamatergic inhibition to vulva-detecting neurons during scanning. Lack of inhibition results in the spurious activation of vulva-detecting neurons.In control males, the activity of PCA is correlated with the relative sliding between the male and the hermaphrodite. When the male tail slides backward relative to the hermaphrodite, PCA increases its activity  and A-type ray neurons (which express trp-4) have been implicated in turning ) demonstrate fewer ejaculation events, successful turns, and vulva contacts than a partial deletion, while also causing more spurious pausing, “pass vulva” events, and “loss of contact” events than the pezo-1(sy1113) mutants. Consistent with this, a larger deletion of pezo-1 has a more severe phenotype on a neuronal level: pezo-1(av240) males show more events of spurious activation of the circuit involved in vulva detection. In addition, pezo-1(sy1113) has a less severe egg-laying phenotype compared to pezo-1(av240), a result consistent with Bai et al. 2020  phenotypes might be affected by another genome feature in the pezo-1 locus, which might have been altered by the large deletion. However, given that the nonpore containing region of the human and mouse Piezo1 has been demonstrated previously to be sufficient for mechanotransduction (C. elegans PEZO-1 with the C-terminal deletion preserves some of its function (Our results suggest that al. 2020 , where aal. 2020 , 65–67. s et al.  showed tsduction , 66, it function .TRP-4 as the key mechanotransducer (Fig. trp-4 is expressed in PCA. In trp-4 mutants, PCA does not become active when the male slides backward relative to the hermaphrodite. Moreover, trp-4 mutants pause backward scanning away from the vulva, which recapitulates the phenotype caused by PCA ablation. Cell-specific TRP-4 expression in the PCA neuron restores its ability to monitor sliding between mating partners. We suggest that initiation of backward sliding of the male tail relative to the hermaphrodite activates the TRP-4 channel, which in turn activates PCA. This activation of PCA provides glutamatergic inhibition to the neurons involved in vulva detection, which is required for persistent scanning (Our results provided further evidence for a mechanosensory role of PCA in male mating behavior and suggested that its function relies on cer Fig. . Previoucer Fig. , 51 and cer Fig. . We showscanning , 58.TRP-4 remains to be studied.PCA has been implicated in multiple motifs of mating. In addition to the monitoring of backward sliding, PCA has been demonstrated to play roles in copulation , 51, andpezo-1 and trp-4 mutant phenotypes are incompletely penetrant. For example, PCB, which expresses pezo-1, shows nearly normal activity in both pezo-1 mutant alleles; A-type ray neurons R2A and R6A, which express trp-4, retain their activation by the hermaphrodite contact in trp-4 mutants. The incomplete penetrance of the phenotypes might be due to additional, yet uncharacterized components that may contribute to activation of these neurons in response to mechanical as well as chemical stimuli from the hermaphrodite. Furthermore, many sensory neurons in the posterior brain of C. elegans males are connected to each other, a property that is thought to enable recurrent excitation. It is plausible that even though mutant PEZO-1 or TRP-4 might be incapable of activating a neuron in response to a relevant mechanical stimulus from a hermaphrodite, the neuron itself might still be activated by other sensory neurons, which are tuned to correlated inputs from the mating partner.Many pgac213_Supplemental_FilesClick here for additional data file.