Site-specific attachment of radioactive cargoto antibody delivery vectors provides homogeneous, well-defined immunoconjugates.Recent studies have demonstrated the utility of oxaziridine chemistryfor site-specific labeling of methionine residues. Herein, we appliedthis approach to site-specifically radiolabel trastuzumab-derivedFab immunoconjugates with 68Ga, which can be used for invivo PET imaging of HER2-positive breast cancer tumors. Initially,a reactive azide was introduced to a single solvent-accessible methionineresidue in both the wild-type Fab and an engineered derivative containingmethionine residue M74, utilizing the principles of oxaziridine chemistry.Subsequently, these conjugates were functionalized with a modifiedDFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WTand DFO-M74 conjugates were radiolabeled with generator-produced [68Ga]Ga3+, to yield the novel PET radiotracers,[68Ga]Ga-DFO-WT and [68Ga]Ga-DFO-M74. In vitroand in vivo studies demonstrated that [68Ga]Ga-DFO-M74exhibited a higher affinity for HER2 receptors. Biodistribution studiesin mice bearing orthotopic HER2-positive breast tumors revealed ahigher uptake of [68Ga]Ga-DFO-M74 in the tumor tissue,accompanied by rapid renal clearance, enabling clear delineation oftumors using PET imaging. Conversely, [68Ga]Ga-DFO-WT exhibitedlower uptake and inferior image contrast compared to [68Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facilemethionine-oxaziridine modification approach can be simply appliedto the synthesis of stable and site-specifically modified radiolabeledantibody–chelator conjugates with favorable pharmacokineticsfor PET imaging.Bioconjugates of antibodies and their derivatives radiolabeledwith β ImmunoPET imaging has greatly increasedour understanding of tumor heterogeneity and, ultimately, has playeda vital role in guiding therapy treatment. Radiometals are frequentlyused for ImmunoPET and are most commonly attached to antibodies usingchelators.1Monoclonal antibodies (mAbs) in immunotherapyhave revolutionizedcancer treatment and prognosis in the past decade. Clinical use ofantibodies could be further personalized by integrating molecularimaging to provide whole-body information on antibody biodistributionand antigen target expression (including heterogeneity) during therapyplanning and treatment.3 Theresulting heterogeneous mixtures of immunoconjugates often demonstratesuboptimal pharmacokinetics and decreased affinities for target antigens.6 Methods have been developed enabling production of well-definedsite-specifically labeled radioimmumoconjugates: such approaches utilize(i) cysteine (Cys) engineering;8 (ii) the heavy chainglycan region of a mAb, incorporating azide-bearing sugars via chemoenzymaticmethods to facilitate “click” conjugation;13 (iii) enzymatic methods employing sortase A,16 microbial transglutaminase;19 and (iv) incorporation of an azide-containing non-natural aminoacid (AA) facilitating site-selective conjugation.20 Recently, we have demonstrated an elegant approach utilizingdibromomaleimide motifs to simultaneously attach deferoxamine (DFO)and sarcophagine (sar) at IgG hinge regions while rebridging the twocysteines.21 While each of these strategieshas their clear advantages, enzyme-based methods, for example, requireexpensive enzymes, glycan-based methods can alter the Fc-binding propertiesof the mAb and they are restricted to intact antibodies,22 and Cys engineering methods often require multistep reactions toreduce and reoxidize/rebridge disulfide bonds, potentially leadingto disulfide scrambling.23Traditionally, antibody–chelatorimmunoconjugates are createdvia stochastic conjugation between the primary amine side chain oflysine (Lys) residues and reactive esters or isothiocyanate functionalgroups, yielding amide or thiourea conjugates, respectively. However,while these methods are simple and easy to implement, the high abundanceof lysine residues results in a lack of selectivity in number andlocation of conjugation, which risks modifying the antigen-bindingregions of a mAb, hampering its immunoreactivity.Modificationof naturally occurring AAs to achieve a high levelof selectivity is intrinsically challenging. Such difficulties resultfrom a lack of site-specificity, that is, a mixture of heterogeneousproducts resulting from modification of repeated functionalities suchas in the case of Lys and Cys (from reduced disulfides) residues.Targeting low abundance amino acids provides higher chances of formingselective unique chemical handles. Moreover, in cases where modificationof an intrinsic AA is not available, genetic incorporation of canonicalAAs available for conjugation is a well-established technique.28 Most notably, the redox-based (ReACT) strategy utilizes oxaziridinereagents that convert the Met thioether side chain to a sulfimideconjugate in a single step.24 The reactionis rapid and can be performed at neutral pH and room temperature.While the ReACT method is highly selective, it was previously shownthat stability of the conjugate varies with the location of modificationin an antibody scaffold, with the hydrolytic stability inversely proportionalto the solvent accessibility of the site.29 In a parallel study by Christian et al.,30 it was demonstrated that stability of the conjugate can be vastlyincreased by incorporation of electron-donating N-substituents on the oxaziridine. Lin et al. have recently reportedthe [18F]F-labeling of Met residues in peptides and bovineserum albumin using the copper-mediated alkyne–azide coupling(CuAAC) reaction with alkyne-bearing modified Met sulfimide conjugates.31Methionine (Met) occurrences are rare in proteins, and they areoften found in buried hydrophobic pockets, making Met an excellenttarget for site-specific modification. Owing to the less reactiveside chain of Met in comparison to Lys and Cys, only a handful ofstrategies have been reported for Met-selective modification.32 visualization of targeted tissues is facilitated by rapidblood clearance, resulting in improved tumor-to-nontargeted-tissuescontrast.33 Gallium-68  is one of the leading β+-emitting radiometalsfor PET imaging due to its widespread availability from benchtop 68Ge/68Ga generators. The short half-life of 68Ga is well suited to the fast blood clearance of Fab fragments.35 We required a chelator that is capable of near quantitative radiochemicalyields with [68Ga]Ga3+ at a near neutral pH,short incubation times, and mild temperature. We have previously shownthat the siderophore, deferoxamine (DFO), is superbly suited for thesepurposes,36 and others have shown sufficientstability of the resulting 68Ga-DFO complex over shorttimeframes in a biological milieu.38A major disadvantage of IgG1 antibodies(∼150 kDa) as imagingprobes is their long circulation time, leading to the requirementfor significant time (1 day to 1 week) between administration/injectionof the tracer and imaging protocols, to optimize target–to–backgroundcontrast in resultant images. In contrast, while monovalent antigenbinding fragments (Fab) (∼50 kDa) typically have lower affinitiescompared to their full length IgG counterparts,68Ga]Ga and assess the resulting [68Ga]Ga-DFO-trastuzumabFab immunoconjugates in vitro and in vivo.Intriguedby the highly selective and facile nature of the ReACTmethod, we explored this strategy for radiometal chelator–antibodyconjugation. Herein, we describe the development of DFO-trastuzumabFab conjugation using the ReACT platform and subsequent radiolabelingwith Ga3+. The conjugates(∼150 μg in 60 μL of aqueous 0.2 M NH4OAc solution) were combined with a solution containing [68Ga]Ga3+ and an aqueous solution of NH4OAc , followedby incubation at 37 °C for 15 min and subsequent analysis bysize exclusion (SE)-HPLC , toyield [68Ga]Ga-DFO-M74 in a 36.4 ± 3.8% radiochemicalyield (n = 5) (non-decay corrected) and in >99%radiochemicalpurity as observed by SE-HPLC. In contrast, the SE-HPLC analysis ofZeba spin-purified [68Ga]Ga-DFO-WT afforded multiple signalsat 5.42, 9.08, and 10.10 min. The earlier retention time of the specieseluting at 5.42 min indicates a larger molecular-weight species andis likely due to aggregation of [68Ga]Ga-DFO-WT. We furtherpostulate that the two closely eluting signals at 9.08 and 10.10 minalso correspond to [68Ga]Ga-DFO-WT, potentially in itsnative form and also at least partially denatured. We suggest thatthis aggregation and denaturation phenomena observed for [68Ga]Ga-DFO-WT are a result of modification/derivatization of the WTFab (vide infra). [68Ga]Ga-DFO-WT was recovered in 33.8± 2.8% radiochemical yield (n = 5) (non-decaycorrected).Both DFO-WT and DFO-M74were radiolabeled with [SE)-HPLC 2A. The [68Ga]Ga-DFO-M74and [68Ga]Ga-DFO-WT were analyzed by nonreducing SDS-PAGEon bright-view imaging and autoradiography  for both [68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT. Following Zeba spin treatment, purified radiolabeledconjugates only showed 68Ga radioactive signal coincidingwith stained protein band corresponding to a molecular weight of ∼50kDa . SE-HPLCradiochromatography analysis of [68Ga]Ga-DFO-M74 indicatedthat 95% of the conjugate remained intact after 4 h while 93% of [68Ga]Ga-DFO-WT remained intact. This size exclusion chromatographymethod did not enable distinction of radiometabolite products; itis possible that they arise as a result of instability of the 68Ga-labeled complex, instability of the sulfimide linkage,or even degradation of protein components by serum proteases.Serum stabilitystudies were carried out on [68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT towardtheir HER2 target antigen, in vitro cell uptake experiments were carriedout in HER2-positive HCC1954 cells and HER2-deficient MDA-MB-231 cells.Both [68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT showedsignificant uptake in HCC1954 cells with uptake inhibited by additionof an excess (200 equiv) of unmodified trastuzumab (68Ga]Ga-DFO-M74 compared to [68Ga]Ga-DFO-WT in HCC1954 cells (p = 0.01).The observed difference is possibly related to the site of conjugationat M74 in [68Ga]Ga-DFO-M74 vs M107 in [68Ga]Ga-DFO-WT.The HC.M107 residue is located at the antigen binding region of theCDR H3 domain: modification and labeling at this site are likely todecrease the affinity of the antibody.29 It is, however, interesting to note that the binding affinity of[68Ga]Ga-DFO-WT toward HER2 is not completely ablated despiteconjugation at its antigen-binding region. It is also possible thatthe likely lower stability of [68Ga]Ga-DFO-WT  leads to lower accumulation of [68Ga]Ga-DFO-WT in HER2-expressing cells. It should also be noted thatin the case of [68Ga]Ga-DFO-WT, it is likely that unmodifiedWT Fab competed with radiolabeled and unlabeled DFO-WT-Fab for bindingto HER2 receptors. This is yet another drawback of a bioconjugationapproach that involves the formation of heterogeneous mixtures ofproducts: it is experimentally very difficult to compare uptake andaffinity of any single, defined species.To assess the relative bindingof [stuzumab 3. Minima68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT were studied in NODscid gamma (NSG) female mice bearing orthotopic HER2-positive HCC1954tumors, using PET/CT imaging and ex vivo tissue biodistribution studies.[68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT (2.1–3.6MBq) were each administered intravenously (via tail vein) to mice(n = 1 per bioconjugate), and PET/CT images wereobtained over 4 h (S14). PET/CT imaging showedthat both [68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WTaccumulated rapidly at tumors , which could be clearly delineated. However, clearance of[68Ga]Ga-DFO-WT from blood circulation and non-target organswas lower compared with that of [68Ga]Ga-DFO-M74  compared with [68Ga]Ga-DFO-M74(3.37%ID/g), leading to tumor/liver ratios of 0.16 and 0.36, respectively.The increased tumor/muscle, tumor/blood, and tumor/liver ratios ledto improved tumor contrast for [68Ga]Ga-DFO-M74 comparedto [68Ga]Ga-DFO-WT. Lastly, to assess specificity of [68Ga]Ga-DFO-M74 using PET/CT imaging, one mouse was administeredwith a dose of trastuzumab 2 days prior to administration of [68Ga]Ga-DFO-M74. As predicted, compared to the animal injectedwith [68Ga]Ga-DFO-M74, significantly reduced tumor uptakewas observed (0.56%ID/g) as well as a lower tumor/muscle ratio (2.44).The biodistribution of [-DFO-M74 4B. For e68Ga]Ga-DFO-M74and[68Ga]Ga-DFO-WT was also performed (n =3 per group) at 2 h p.i. of tracers, mice were culled, and organswere dissected, weighed, and counted for radioactivity. Consistentwith PET/CT imaging data, [68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT exhibited tumor uptakes of 1.6 ± 0.6 and 1.3 ±0.2%ID/g, respectively  compared with [68Ga]Ga-DFO-M74 .Similarly, the concentration of 68Ga radioactivity in theliver was 9.0 ± 1.6%ID/g for [68Ga]Ga-DFO-WT, comparedwith 2.1 ± 0.2%ID/g for [68Ga]Ga-DFO-M74 . This resultedin higher tumor-to-normal tissue ratios at 2 h for [68Ga]Ga-DFO-M74compared to those for [68Ga]Ga-DFO-WT, consistent withPET/CT imaging data ] complex is sufficiently stable overthe time course of the in vivo experiments. It is also noteworthythat the ovaries and uterine horn, which are healthy tissues knownto express HER2,44 showed uptake of both tracers.The ex vivo biodistribution of Ga3+, but envisagethat other pairs of radiometallic ions/chelators could similarly beincorporated into Fab derivatives. Similarly, we also envisage thatother Fab derivatives for other cancer and disease cell surface receptormarkers could also be employed.We have demonstrated that the combination of antibody engineeringto introduce a Met residue into a Fab fragment, followed by Met-functionalizationusing oxaziridine conjugate chemistry, enables homogeneous, site-specificradiolabeling using radiometals. We have used the siderophore chelator,DFO, for coordination of [68Ga]Ga-DFO-M74 exhibited higher affinity compared to [68Ga]Ga-DFO-WT. In vivo, the higher affinity and/or stability of [68Ga]Ga-DFO-M74 compared to [68Ga]Ga-DFO-WT ledto faster blood clearance, lower accumulation in liver, and slightlyhigher tumor uptake. In combination, these factors resulted in improvedcontrast for [68Ga]Ga-DFO-M74; thus, we believe that thereare clear advantages of using the M74 oxaziridine-modified Fab comparedwith the analogous WT Fab for PET imaging of HER2 expression.In vitro studies demonstratedthat, while both new radioimmunoconjugatesretained binding toward HER2-expressing HCC1954 cells, Ga-DFO-M74, similar to valuesreported with 111In- and 64Cu-DOTA-Fab at 24h p.i., the study was limited by the half-life of 68Gaand the relatively high amounts of radioactivity that remained incirculation at 2 h p.i.45 In this work,we have elected to use DFO to enable 68Ga-radiolabeling;however, other chelators such as NOTA or DOTA could enable applicationswith a wider range of radionuclides, including 44Sc, 64Cu, and 111In, which could complement the pharmacokineticsof the Fab fragments more fittingly.48 Weare currently developing new oxaziridine-functionalized chelatorsfor this purpose and comparing the bioconjugates of these with conventionallyprepared bioconjugates containing mixtures of non-site-specificallylabeled species.The trastuzumab-derived Fab fragment has generated considerableinterest for nuclear imaging due to its favorable pharmacokineticsprofile. Its derivatives radiolabeled with 68Ga to yield novel PET radiotracers[68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT. In vitroand In vivo studies revealed that [68Ga]Ga-DFO-M74 exhibitedhigher affinity toward HER2 receptors and demonstrated more favorablepharmacokinetics, resulting in improved contrast compared to [68Ga]Ga-DFO-WT. The promising results obtained with [68Ga]Ga-DFO-M74 opens up possibilities to explore the use of the ReACTplatform for other radiometals/chelators and other Fab derivatives.Antibodies modified site-specifically viathe ReACT platform giverise to immunoconjugates that are well-defined and homogeneous. Wetook advantage of this approach in combination with the highly facileSPAAC to prepare two trastuzumab Fab conjugates modified with a DFOchelator. The resulting immunoconjugates DFO-WT and DFO-M74 were successfullyradiolabeled with 31 and DBCO-PEG4-DFOwere prepared as previously described.39 TrastuzumabFab (5 mg) were incubated at 50 μM with 1  for 30 min at rt in PBS after which thereactions were quenched by addition of 400 mM l-Met (10 μL)and buffer exchanged into PBS using a 5 mL Zeba 7 kDa MWCO desaltingcolumn . Then, 10 mol equiv of DBCO-PEG4-DFO (5 mM stock in DMF) was added, and the reaction mixtureswere incubated at rt overnight. The conjugate was purified and bufferexchanged into ammonium acetate (0.1 M) using a PD-10 column and elutedin 0.5 mL fractions. Fractions containing protein were collected andfurther purified and buffer exchanged into ammonium acetate (0.1 M)by six cycles of spin filtration through Amicon Ultra-0.5 mL centrifugalfilters . M74 Fab-DFOconjugate (4.89 mg) was recovered in 100 μL at a concentrationof 967 μM while the WT Fab-DFO conjugate (4.3 mg) was recoveredin 100 μL at a concentration of 881 μM. Samples for ESI-TOFMS analysis were prepared at 10 μM in 0.1 M ammonium acetateor in H2O .Oxazridine-N68Ga]Ga3+ waseluted from a GalliAd generator where 68Ge was attachedto a tin dioxide column using 0.1 M HCl. ∼430 MBq [68Ga]Ga3+ is typically obtained in 1.1 mL and is used withoutfurther purification. Fab-DFO conjugates were diluted to 50 μMin NH4OAc (0.2 M). To 60 μL of this solution wereadded 40 μL of NH4OAc (2 M) and 75 μL of [68Ga]Ga3+ in HCl (0.1 M). The resulting solutionwas incubated at 37 °C for 15 min and purified using a Zeba spin0.5 mL 7 kDa MWCO desalting column twice . The reaction was then analyzed by SEC-HPLC, iLTC, and SDS-PAGE.iTLC-SG strips were spotted with 1 μL of radiolabeling mixtureand developed in a mobile phase of citrate buffer .For 68Ga-Fab-DFO, Rf < 0.1,and for unreacted 68Ga, Rf >0.9. SEC-RadioHPLC: [68Ga]Ga-DFO-M74 had a retention timeof 9 min while unchelated 68Ga eluted at 11.2 min; [68Ga]Ga-DFO-WT afforded species with retention times at 5.4min (aggregated species), 9.1, and 10.1 min. Radiochemical puritydetermined by SEC-HPLC: [68Ga]Ga-DFO-M74: >99% at aspecificactivity of 84.2 MBq mg–1 or molar activity of 4.25GBq μmol–1 and [68Ga]Ga-DFO-WT:>99% at a specific activity of 74.5 MBq mg–1 ormolar activity of 3.63 GBq μmol–1.Ga-DFO-M74 and[68Ga]Ga-DFO-WT were prepared as above and buffer exchangedinto HBSS buffer using Zeba spin 0.5 mL 7 kDa MWCO desalting columns;the radioimmunoconjugates were then diluted with HBSS to a final concentrationof 1.5 μM prior to uptake studies. Once harvested after trypsinization,HCC1954 and MDA-MB-231 cells were washed once with PBS and resuspendedin HBSS with 0.2% BSA to give 1 × 106 cells per tubein 500 μL. [68Ga]Ga-DFO-M74 and [68Ga]Ga-DFO-WT were added to eachtube. For blocking conditions, unmodified Trastuzumab  was added to the tubes and incubated at rt for5 min prior to addition of the radioimmunoconjugates. Tubes were thenincubated at 37 °C for 1 h. The cells were isolated by centrifugationat 400g for 5 min, aspirating the supernatant, andresuspended in PBS. The cells were washed twice by centrifuging at400g for 5 min and aspirating the supernatant. Theradioactivity associated with cell pellets and supernatant was thencounted using a gamma counter.[All animal experiments were ethicallyreviewed by an Animal Welfare & Ethical Review Board at King’sCollege London and carried out in accordance with the Animals (ScientificProcedures) Act 1986 (ASPA) UK Home Office regulations governing animalexperimentation.scid Il2rgtm1WjI/SzJ) mice (18–25 g) were obtainedfrom Charles Rivers Laboratories. Animals were housed in ventilatedcages, given food and water ad libitum, and allowed to acclimatizefor approximately 1 week before tumor cell inoculation. Approximately1.5 × 106 HCC1954 cells in a 100 μL cell suspensionof a 1:1 mixture of PBS/BD Matrigel (BD Biosciences) were injectedsubcutaneously in the mammary fat pad between the fourth and fifthnipples in the left flank. Experiments were performed approximately3 weeks after the injection of cancer cells. Mice bearing mammaryfat pad xenografts were randomly assigned to three groups . In the blocking group, 48 h before the scanning/biodistributionstudies, trastuzumab (0.5 mg in 200 μL of PBS) was administeredvia the tail vein of anaesthetized mice.Sevento nine-week-old female NOD scid gamma (NSG)  with mice under 0.8–1.5% isoflurane in oxygen anesthesiaand warmed to 37 °C for the duration of the experiment. Micewere administered radiolabeled Fab fragments  in 200 μL of PBS via intravenoustail injection. Dynamic PET scans were acquired for up to 4 h postinjection, followed by a CT scan for anatomical visualization . PET/CTdata sets were reconstructed using a Monte Carlo-based full-3D iterativealgorithm  with 4 iterations, 6 subsets, and 0.4mm isotropic voxel size. Images were coregistered and analyzed usingVivoQuant v.3.0 (Invicro). Regions of interest (ROIs) were delineatedfor the PET activity quantification in specific organs. Uptake ineach ROI was expressed as a percentage of injected dose per gram oftissue (% ID/g).n =3/group) were administered with similar amounts of conjugates thatwere used for imaging study and were maintained under anesthesia untilthe chosen time point . Mice were culled by cervical dislocation, and organswere dissected, weighed, and gamma-counted along with standards preparedfrom the corresponding sample of injected radiolabeled conjugates.Conjugate uptake was calculated as a percentage of injected dose pergram (% ID/g) of tissue.Mice (


Please see the correct version of Figure 2 at the following link: .2.)The Reagents sub-section contains three typographical errors.-In the text "The Rho C siRNA is a pool of", "Rho C" should read "RhoC".-In the text "antisense 5’-UAGUU-CUCAA- AGACAGUAGtt-3’", "A- A" should read "AA".-In the text "antisense 5’-UGUAUAAA-GUGCUGGUGUG- tt-3’", "G- tt" should read "Gtt".


Caenorhabditis elegans in response to PFT attack. We demonstrate via analyses of C. elegans mutants that inhibition of feeding by PFT requires the neuronal G protein Goα subunit goa-1, and that maintenance of this response requires neuronally expressed calcium activator for protein secretion (CAPS) homolog unc-31. Independently from their role in feeding cessation, we find that goa-1 and unc-31 are additionally required for immune protection against PFTs. We thus demonstrate that the behavioral and immune responses to bacterial PFT attack involve the cross-talk between the nervous system and the cells directly under attack.Pore-forming toxins (PFTs) are abundant bacterial virulence factors that attack host cell plasma membranes. Host defense mechanisms against PFTs described to date all function in the host tissue that is directly attacked by the PFT. Here we characterize a rapid and fully penetrant cessation of feeding of  Mycobacterium tuberculosis, Streptococcus pneumonia and Staphylococcus aureus, among others, are especially problematic because of antibiotic resistance Bacterial infectious diseases rank among the top leading causes of death worldwide. C. elegans using the PFT Cry5B and, where tested, were found to have conserved roles in mammalian cells C. elegans, suggestive of a neuronal component to PFT responses.Cells possess PFT-defense mechanisms involved in membrane resealing, as well as various molecular defenses C. eleganse.g., neuronal control of cytokine production C. elegans   . sa734 igoa-1(sa734) feeding behavior shows that after 10, 30 and 120 minutes exposure to Cry5B it has significantly higher fractions of animals feeding than wild type (goa-1(sa734) mutation also causes impaired cessation of feeding in response to VCC and Cry21A after 10, 30 and 120 minutes  shows significantly higher pumping rates than wild type on Cry5B  mutants have a N-terminal truncation of first 4 amino acids causing it to lack the consensus site for myristoylation goa-1(sa734), and a general resistance to phenotypes induced by administration of exogenous serotonin C. elegansNext, we measured pharyngeal pumping rates of several of the Goα and Gqα pathway mutants in absence or presence of Cry5B  carries a loss-of-function mutation in a regulator of G-protein signaling (RGS) protein, functioning downstream of goa-1 in the control of several behaviors eat-16(ce71) animals show a slightly defective feeding-inhibition response, albeit with less expressivity than that of goa-1(sa734) (1(sa734) .egl-30 and egl-10 are components of the Gq pathway that generally functions antagonistically to the Go pathway egl-30 or egl-10 respectively, as expected, did not affect inhibition of feeding by Cry5B (egl-10 (egl-10(xs)) or carrying egl-30 (tg26 and js126) gain of function mutations, which share many phenotypes with goa-1 loss-of-function mutants  by Cry5B . Howeverunc-31(e928), a null allele unc-31 is selectively expressed in neurons  and eat-16(ce71) mutants show significantly decreased survival compared to wild type on the three Cry5B doses tested (goa-1(n1134) mild reduction-of-function mutant also shows significant hypersensitivity at two Cry5B doses, although not to the extent of goa-1(sa734) and eat-16(ce71). This innate immunity role appears uncoupled from cessation of feeding as eat-16(ce71) mutants show only a modest defect of feeding inhibition and goa-1(n1134) mutants display a normal feeding-cessation response.For a quantitative analysis of the sensitivities of several Goα and Gqα pathway mutants, we performed dose response assays with purified Cry5B. Consistent with the qualitative assays, s tested . The goaegl-30(n686) animals, and goa-1(n1134) egl-30(n686) double mutant animals show decreased Cry5B PFT survival similar to that of goa-1(n1134) alone (goa-1(Q205L)egl-10 (egl-30 gain of function (C. elegansImmunity to Cry5B is unchanged in 4) alone . Constit)egl-10 , or egl-function . So, in V. cholerae expressing VCC and scored 24 hours later for survival. goa-1(sa734) and eat-16(ce71) null mutant animals show significantly decreased survival after 24 hours on various doses of V. cholerae expressing VCC compared to a V. cholerae strain lacking the PFT, and for eat-16(ce71) a dose-response to VCC is evident  animals are hypersensitive to infection by the bacterial pathogen Pseudomonas aeruginosagoa-1(n1134) mild reduction-of-function animals show significantly increased lethality on the highest VCC dose only  Hypersensitivity of these mutants is not likely due to general illness, as in a previous study goa-1(sa734) animals showed no hypersensitivity to the heavy metal cadmium, or high osmolarity Next, we exposed mutants lacking Goα components to  evident . V. cholild type   and egl-21 (carboxypeptidase E) mutants, which have reduced neurosecretion because of reduced dense-core vesicle (DCV) exocytosis or defects in DCV contents, are resistant to P. aeruginosa infection unc-31, egl-3 and egl-21 mutants in our screen  Hpo phenotype is not likely due to general sickness, as loss of unc-31 increases life span and confers resistance to P. aeruginosar screen . Oppositto Cry5B , Fig. 4Acontrols . The uncunc-31 is expressed in all neurons and other secretory cells, but not in the intestine unc-31(e928) animals in which unc-31 expression was selectively restored to the neurons via the rab-3 promoter C. elegans from an attack by PFT on its intestinal cells.C. elegans, and that the Goα pathway components goa-1 and downstream eat-16 are required for this feeding-cessation response. goa-1 and eat-16 are furthermore required for defense against PFTs. egl-21 and neuronal unc-31 are also required for PFT defense, and neuronal unc-31, but not egl-21, is required for maintenance of PFT-induced feeding cessation.We present data showing that bacterial PFTs rapidly and reversibly inhibit feeding in bre-3(ye28) animals showed normal feeding rates ), and exposure of C. elegans to Cry5B results in permeabilization of the intestinal cells but not the pharyngeal cells e.g., neuropeptides) between the somatic and pharyngeal nervous systems, or the pharyngeal nervous system could autonomously sense the effects of Cry5B . Consistent with this model  uncresponse , (2) goaresponse 2A, B andresponse .goa-1 results in both continued feeding on Cry5B and increased sensitivity to Cry5B, the two phenotypes could be related–i.e., increased consumption of the PFT could cause increased sensitivity because of and increased PFT dose. However, several lines of evidence suggest against such a relationship. Loss of goa-1 results in hypersensitivity to V. cholerae lacking VCC (V. cholerae lacking VCC (goa-1(n1134) reduction-of-function animals show normal inhibition of feeding (eat-16(ce71) mutant  Since loss of king VCC . Howeverking VCC . Further feeding  but are  feeding , and a l) mutant , 3B. So,P. aeruginosa resistance for unc-31 and goa-1 loss-of-function mutants C. elegans against V. cholerae expressing VCC but increases its sensitivity to V. cholerae lacking VCC The inverse correlation that was found with regard to unc-31 and goa-1 in P. aeruginosa defense unc-31(e928) stops feeding after 30 minutes on Cry5B but prematurely resumes feeding at two hours (goa-1(sa734) is feeding at all time points tested, and to a larger extent than the unc-31 mutant. In addition, the effects of goa-1 and unc-31 mutation on pumping on Cry5B appear to be cumulative, i.e., the goa-1(sa734); unc-31(e928) double mutant shows higher fractions pumping on Cry5B than either single mutant  or 30°C (V. cholerae) in LB broth, supplemented with 50 µg/mL carbenicillin where applicable.Bacterial strains used in this study were E. coli-Cry5B, E. coli-Cry21A, V. cholerae-VCC, and no-PFT control plates were prepared as described To determine fractions of animals in a population that are feeding, E. coli-Cry5B plates were prepared as above. 10-13 L4 animals were transferred to each plate, and pumping rates were measured for 30 seconds, before transfer, or after 30 minutes incubation at room temperature. Each assay was repeated independently three times. It is of note that goa-1(n1134) mutants were reported to have slightly decreased pumping rates n1134 and sa734  bacteria with empty vector control (0%) bacteria at the indicated ratios, as described For qualitative toxicity assays, including the screening , 0%, 10%Quantitative survival assays with purified Cry5B were performed as described E. coli-Cry5B dilutions. Animals that showed internal hatching of progeny (“bagging”) or the intestine protruding through the vulva (“exploding”) were censored. Pilot assays involving the use of 5-fluoro-2′-deoxy-uridine (FUdR) to prevent the development  of progeny revealed that FUdR causes V. cholerae to lose its pathogenicity. Therefore, egl-30-like strains could not be tested here, as without inhibiting the development of their progeny the terminal phenotype of most of these animals is bagging due to their low egg-laying rates. An E. coli OP50 control was included with each assay, which showed no significant lethality for any of the strains after 24 hr (data not shown). Three independent repeats were performed with 30–50 animals per treatment. After 24 hours, wild-type animals are qualitatively affected by the presence of VCC but show no lethality on any of the VCC doses . Prism 5.0 Software (GraphPad) was used to draw graphs. p values, means or medians and confidence intervals or interquartile ranges of all experiments performed for this study are provided in Figure S1Cry21A inhibits feeding, transfer does not alter feeding, and Cry5B plates retain potency over 24hr. (A) Animals transferred to E. coli expressing Cry21A rapidly stop feeding, whereas animals transferred to control plates do not. (B) Fractions of wild-type animals feeding at various time points after transfer are the same as before transfer. (C) Wild-type animals transferred to 24-hr old E. coli-Cry5B plates are not feeding 10 or 30 min after transfer. Statistics indicate difference between 24-hr old control and 24-hr old Cry5B plate. Here and in all subsequent supplemental figures graphs show mean ± standard error of 3 experiments, and statistics indicated are: ns not significant, * p<0.05, ** p<0.01, *** p<0.001. Additional statistics are provided in (TIF)Click here for additional data file.Figure S2goa-1 null mutants constitutively feed on Cry21A and Cry5B, and transferring worms does not affect their pumping rates. (A) E. coli-expressed Cry21A inhibits feeding in wild-type animals after the indicated exposure times, but does not inhibit feeding in goa-1(sa734) mutants. (B) goa-1(ep275) constitutively feeds on E. coli-expressed Cry5B. (C) 30 minutes after transfer to plates with E. coli not expressing PFT, pumping rates are the same as before transfer. Bars show mean ± standard error of 3 experiments, and dots are individual measurements of all three experiments. Additional statistics are provided in (TIF)Click here for additional data file.Figure S3Goα is required for PFT defense. After 48 hr, goa-1(ep275) mutants are qualitatively hypersensitive to E. coli-expressed Cry5B. Scale bar  = 500 µm.(TIF)Click here for additional data file.Table S1Feeding and sensitivity phenotypes of mutants on Cry5B. Includes References S1 for (DOCX)Click here for additional data file.Table S2Statistical analyses.(DOCX)Click here for additional data file.Table S3C. elegans strains used in this study.(DOCX)Click here for additional data file.


C. elegans that a moderate increase in physiological cholinergic signaling at the neuromuscular junction (NMJ) induces the calcium (Ca2+)-dependent activation of HSF-1 in post-synaptic muscle cells, resulting in suppression of protein misfolding. This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of gei-11, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR). This, in-turn, activates the voltage-gated Ca2+ channel, EGL-19, and the sarcoplasmic reticulum ryanodine receptor in response to acetylcholine signaling. The release of calcium into the cytoplasm of muscle cells activates Ca2+-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells. This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.Protein quality control requires constant surveillance to prevent misfolding, aggregation, and loss of cellular function. There is increasing evidence in metazoans that communication between cells has an important role to ensure organismal health and to prevent stressed cells and tissues from compromising lifespan. Here, we show in  C. elegans muscle cells. This involves gei-11 knockdown-dependent upregulation of acetylcholine receptors, and the release of calcium into the cytoplasm of muscle cells through cell membrane and sarcoplasmic reticulum specific channels. Subsequently, activation of the heat shock factor 1 (HSF-1) leads to the expression of cytoplasmic chaperones that suppress misfolding of metastable and aggregating proteins, restoring folding and muscle function. This reveals a new non-canonical mechanism for the cell non-autonomous regulation of the heat shock response to ensure balance between cells in a metazoan.The protein quality control machinery is responsible for preventing the accumulation of misfolded and damaged proteins and loss of cellular function. The capacity of cellular surveillance is limited however, leading to increased appearance of protein aggregates and risk for age-associated diseases. Here, we show that upregulation of acetylcholine receptors and moderate increased cholinergic activity leads to a calcium-dependent stress response that suppresses protein misfolding and restores homeostasis in  Cellular health and organismal lifespan are critically dependent upon the fidelity of the proteome and the proteostasis network C. elegans and D. melanogaster have been particularly amenable genetic models for identification of proteostasis components and modifiers of protein misfolding and toxicity Much of our understanding of the HSR and the proteostasis network has come from studies using cultured cells and model organisms. The invertebrate animals gei-11 knockdown-dependent upregulation of cholinergic receptor levels at the neuromuscular junction (NMJ) triggers activation of HSF-1. This reveals that under normal physiological conditions, the balance between cholinergic and GABAergic signaling at the NMJ regulates protein homeostasis in body-wall muscle (BWM) cells gei-11, is beneficial to muscle cells. We demonstrate that there is a critical range of cholinergic activity at the NMJ leading to the Ca2+-dependent activation of HSF-1 and expression of molecular chaperones that results in an enhanced protective state in post-synaptic muscle cells.In this study, we describe a new pathway for the heat shock response involving calcium signaling, in which C. elegans for genetic modifiers of muscle proteostasis identified gei-11 RNAi as a potent suppressor of polyglutamine (polyQ) aggregation in BWM cells  screen performed in WM cells  [30]. Kn pattern : I–IV anrn , a member of the Myb superfamily of transcription factors that is homologous to mammalian SNAPC4 (snRNA-activating protein complex subunit 4) gei-11(tm6548) is the only mutant allele available for this gene, and has a lethal phenotype . gei-11 has been proposed to have a negative regulatory effect on AChRs in C. elegans BWM cells, and is also expressed in head neurons, germ cells, somatic gonad, and intestine The gene C. elegans, two types of ACh receptors, each with distinct subunit composition and pharmacology, are expressed at the NMJ gei-11 downregulation on the expression of NMJ AChR subtypes, we monitored the expression of the L-type (levamisole-sensitive) AChR subunits unc-29, unc-38, unc-63 and lev-1, and the N-type (nicotine-sensitive) homomeric AChR, acr-16. The effect of gei-11 knockdown increased the expression of only the three essential subunits of the L-AChR  by approximately 3-fold, and had no effect on the expression of N-AChR acr-16 . The results showed that polyQ aggregation was unaffected when an essential L-AChR subunit was co-downregulated with gei-11 (unc-38(e264)), that corroborated the results obtained with RNAi , unc-63(x26), unc-29(e1072)) gei-11 RNAi treatment  animals were resistant to levamisole compared to Q35 animals upon gei-11 RNAi ) (gei-11 RNAi suppression of Q35 aggregation in a dose-dependent manner (unc-38(e264) animals ) . Additiot manner , ; the s animals .gei-11 RNAi and increased cholinergic receptor expression on muscle polyQ aggregation was a direct consequence of gei-11 knockdown in muscle cells, rather than a downstream effect from another tissue. For example, gei-11 RNAi did not have an effect on aggregation of polyQ expressed in the intestine ;myo-3p-RDE-1 (here referred to as rde-1(ne219);mRDE-1) rde-1(ne219) (gei-11 in rde-1(ne219);mRDE-1 animals increased the expression of essential L-AChR subunits ;mRDE-1 animals was suppressed by 50% upon gei-11 RNAi relative to vector control, with no effects observed in Q35;rde-1(ne219) animals  . Knockdo>3-fold, , compara compare . Likewis animals . Taken tgei-11 knockdown-mediated suppression of polyQ aggregation was selective for this class of highly aggregation-prone species or reflected a more general enhancement of proteostasis in the BWM cells, we examined the effects of gei-11 knockdown on the folding stability of four endogenous metastable proteins that function as folding sensors in muscle cells gei-11 was downregulated at the restrictive temperature, the loss-of-function phenotype of each TS protein was decreased by 50–60% , to reveal that Q35 aggregation was no longer suppressed  and small heat shock protein family  in wt animals, and show that gei-11 RNAi enhanced the expression of each chaperone gene from 2-to-10-fold (unc-29(e1072), or in HSF-1 mutant hsf-1(sy441) animals (gei-11 RNAi was absolutely dependent upon both cholinergic receptor activity and HSF-1. These results were further corroborated with the cholinergic antagonist dTBC and the L-AChR unc-63(x26) mutation ;mRDE-1 animals, To establish whether ppressed . We moni-10-fold , a level-10-fold . Moreove animals . Therefomutation  that alsgei-11 induced the HSR, we monitored the activity of the heat shock transcription factor, HSF-1, using the electrophoresis mobility shift assay (EMSA) gei-11 induced HSF-1 DNA-binding activity to the heat shock element (HSE)  : lane 6,nt (HSE) : lane 3.gei-11 knockdown by examining the expression of other components of the proteostasis network, including the expression of the UPR-regulated ER chaperones , metabolic stress FOXO/DAF-16 regulated genes (sod-3 and mtl-1), and oxidative stress regulated genes . As shown in gei-11 knockdown in wt animals. Taken together, these results demonstrate that modulation of cholinergic receptors at the NMJ reprograms post-synaptic proteostasis through the activation of HSF-1 and the selective induction of cytoplasmic chaperones.We further established the downstream regulatory effects of unc-30, the transcription factor that regulates the GABA operon, had the opposite result leading to enhanced aggregation in BWM cells gei-11, we posit that there is a critical balance between the levels and activities of AChR and GABAR )  Q35 aggregation was suppressed by >40% , whereas8(e264)) . These rR activation at the NMJ affects muscle proteostasis by combining both genetic and small molecule agonists and antagonists probes. Reducing GABAergic activity by exposure to low concentrations (25 µM) of the GABAR antagonist Lindane R expression and GABA release at the synapse by using a combination of RNAi and specific loss-of-function mutations in the GABA pathway: unc-30 (GABA synthesis), unc-47 (GABA transport) and unc-49 (GABAR). Knockdown of unc-47 and unc-49 suppressed Q35 aggregation, and this effect was less penetrant upon dilution of RNAi (1∶1) with vector RNAi (unc-30(e191) and unc-47(gk192) mutants had the opposite effect , GABA compensated for the gei-11-mediated increase in cholinergic signaling and prevented the suppression of Q35 aggregation, in a dose-dependent manner , or to the GABAR antagonist Lindane (25 µM), resulted in elevated expression of the cytoplasmic hsp70 family of chaperone genes (C12C8.1 and F44E5.4), consistent with enhanced proteostasis. Likewise, and as expected, genetic reduction of GABA signaling using unc-47 or unc-49 RNAi , unc-49(e407) or unc-30(e191) that no longer express GABA or GABAR, led to a massive upregulation of hsp-70 , EGL-19, and flux of Ca2+ into the cytoplasm egl-19(n582)), a weak hypermorphic mutant (egl-19(n582ad952)), and a stronger hypermorphic mutant (egl-19(ad695)) in the background of Q35 2+ flux into the muscle, had opposing effects: the strongest hypermorphic mutant ad695 enhanced Q35 aggregation, whereas the weak hypermorphic n582ad952 and weak hypomorphic n582 mutants both suppressed Q35 aggregation (2+ suppressed aggregation (egl-19(n582) and egl-19(n582ad952); hsp-70  were observed in animals with Ca2+-mediated enhanced aggregation (egl-19(ad695), 2+ influx on muscle cell folding environment, reflected by the effect on aggregation. Whereas a mild imbalance in Ca2+ influx, achieved with the weak hypermorph and hypomorph mutants, activated a protective HSR (corresponding to a moderate upregulation of hsp-70), the EGL-19 strong hypermorphic mutation, resulted in a much larger imbalance in signaling and accentuated stress response (corresponding to higher levels of hsp-70). These results provide further support for the importance of a critical physiological stimulation range to establish proteostasis.Cholinergic activity at the NMJ leads to activation of the muscle voltage-gated Caregation  [29]. Cogei-11 knockdown was dependent on the VGCC, we treated Q35;egl-19 mutant animals with gei-11 RNAi (gei-11 in hypomorphic egl-19(n582) mutant animals had no effect on Q35 aggregation  was additive to the beneficial effects of gei-11 RNAi on folding (egl-19(ad695) effect on Ca2+ levels was deleterious  at the sarcoplasmic reticulum (SR), releasing additional Ca2+ into the cytosol   hsp-70 levels and significantly reduced Q35 aggregation by more than 60%  , supportthan 60% , S6B. Atthan 60% . These r2+ regulates many signaling cascades, we examined the specificity of gei-11 RNAi-dependent Ca2+ release by the RYR on induction of hsp-70 and suppression of polyQ aggregation, by testing a RYR-specific antagonist and a RYR mutant. We employed dantrolene sodium (DS), a clinically relevant muscle relaxant that selectively targets RYR and blocks Ca2+ release from the SR during muscle contraction hsp-70 and suppression of Q35 aggregation by gei-11 RNAi (unc-68(kh30) ) with the RYR modulators had no significant effect on aggregation   [58]. Frregation  supportichannels . Taken tcapacity .We demonstrate that cholinergic-dependent calcium signaling across the synaptic junction induces an atypical heat shock response that promotes protein homeostasis and suppresses misfolding and aggregation in post-synaptic muscle cells. These molecular events are dependent upon HSF1, but are distinct from the classical HSR regulated by transient exposures to acute heat shock stress. The key feature of this novel neuromuscular stress signaling mechanism, centers around the balance between cholinergic and GABAergic signaling at the NMJ. The importance of balanced signaling is highlighted by observations that muscle overexcitation caused by the complete absence of GABA is deleterious to the folding environment and results in a proteotoxic cellular environment gei-11 as a new genetic modifier of protein folding reveals a new strategy by which metazoans ensure proteostasis across tissues. Our biological observations suggest that the regulation of receptor expression in muscle cells can initiate a protective mechanism against stress and degeneration, such as age-dependent sarcopenia gei-11, could suggest novel therapeutic targets for proteostasis maintenance with possible benefit for the patients suffering from somatic wasting diseases. Overall, it emphasizes the importance of dissecting neuronal signaling pathways that affect organismal stress responses and cellular function.The identification of gei-11 was identified from a genetic screen for proteostasis regulators that enhanced the cellular folding environment 2+ influx in muscle cells homeostasis also occurs at a critical range. The activation of both EGL-19 and RYR channels by cholinergic signaling , Q35 AM140 (rmIs132[Punc-54::q35::yfp]I), Q37 AM470 (rmIs225[Punc-54::q37::yfp]II) G93A AM265 (rmIs177[Punc-54::G93Asod1::yfp]) vha-6::q44::yfp + rol-6(su1006) +pBluescript II] unc-15(e1402)], CB1157 [unc-54(e1157)], HE250 [unc-52(e669su250)] and CB286 [unc-45(e286)] rde-1(ne219)] and WM118 ] unc-29(e1072)], CB904 [unc-38(e264)], ZZ26 [unc-63(x26)], RB918 [acr-16(ok789)], VC311 [unc-47(gk192)], CB845 [unc-30(e191)], CB407 [unc-49(e407)] egl-19(n582)], DA952 [egl-19(n582ad952)], DA695 [egl-19(ad695)] unc-68(kh30)], PS3551 [hsf-1(sy441)]. Where indicated, genetic crosses between mutant animals and Q35 animals were generated.Animals were maintained according to standard methods, at 20°C on nematode growth media (NGM) with OP50 C. elegans was performed as described previously, using the commercial RNAi library  n≥3). As a negative control, animals were fed bacteria carrying the L4440 empty vector (vector). Liquid RNAi treatment was performed in 96-well plates, with a total volume of 60 µl per well, consisting of 15–20 worms and RNAi bacteria. Bacteria was grown overnight (∼16 h), induced with isopropyl β-D-thiogalatoside , pelleted and resuspended in S-medium complete  so that the final OD595 nm was 0.9 in the well. RNAi assays on plates were performed as described previously RNAi gene knockdown in Animals were mounted on a 3% (w/v) agar pad on a glass slide, immobilized with 2 mM levamisole (Sigma), and subjected to FRAP analysis using the Zeiss LSM510 confocal microscope  as previously described n≥3). Results are shown relative to wt animals' speed on L4440-RNAi vector control plates (100%).The movement of 6 day old animals grown on RNAi-seeded NGM plates  were digitally recorded using a Leica M205 FA microscope with a Hamamatsu digital camera C10600-10B , and the Hamamatsu Simple PCI Imaging software. Animals were tracked using a custom ImageJ plugin wrMTrck T Method , normalized to actin (act-1) in the same sample, and relative to the non-treated or vector control sample. Measurements were performed for ≥3 biological samples.RNA from ∼50 animals was extracted with Trizol (Invitrogen), followed by DNase treatment (Applied Biosystems #AM1906). mRNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad #170-8891). cDNA real-time PCR amplification was done using the iQ-SYBR Green Supermix (Bio-Rad #170-8880) and the iCycler system (Bio-Rad) see . Expressn = 3). Compound stock solutions: 800 mM levamisole (Sigma) in water, 300 mM nicotine (Sigma) in ethanol.Five day old animals grown on RNAi-seeded NGM plates at 20°C (≤40) were transferred onto NGM plates, equilibrated at 20°C, containing 1 mM Levamisole (Sigma), 30 mM Nicotine (Sigma) or the solvent . Sensitivity to the drugs was followed by visual inspection every 2 to 5 min and defined as paralysis, or lack of movement in response to prodding on the nose and tail of the animal . Replicates of each condition were included in each assay/plate. Animals were incubated with each drug, at the concentrations indicated in the respective Figures, from L1 stage , L2 stage  or L4 stage , until day 6 of age, at 20°C (n≥3) (see Compound assays were performed in liquid culture as described previously n≥3) see . At thisunc-15(e1402) and unc-54(e1157), stiff paralysis for unc-52(e669su250), and egg-laying phenotype for unc-45(e286)  (n = 3) Temperature sensitive (TS) mutant animals were age-synchronized to L1 stage, grown on RNAi-seeded NGM plates  from day 1 at a sensitized temperature of 23°C (to maintain the RNAi suppressor effect on aggregation), or at the control restrictive (25°C) and permissive (15°C) temperatures, and scored for phenotypes on day 5, as previously described 32P]-labeled probe containing the proximal heat shock element (HSE) from the C. elegans hsp-70 (C12C8.1) gene promoter. Nuclear extracts (40 µg) were incubated with the [32P]-labeled probe (HSE or mutant) for 20 min at room temperature. For heat shock treatment (HS) the samples were pre-incubated at 35°C for 30 min. For competition experiments, a 100-fold molar excess of the same unlabeled oligonucleotide was added to the mixture. The samples were analyzed by electrophoresis on a 4% (w/v) polyacrylamide native gel that was dried and scanned using a PhosphorImager . Oligonucleotide probes: HSE-F: taaattgtagaaggttctagaagatgccaga; HSE-R: tctggcatcttctagaaccttctacaattta; HSEmut-F: taaattgtaaaaggaaataaaagatgccaga; HSEmut-R: tctggcatcttttatttccttttacaattta.Native nuclear protein extracts were prepared from 200 µl of pelleted worms (grown on NGM RNAi-seeded plates), with the commercial Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit (# 78835), as described previously Figure S1gei-11 RNAi. (A) gei-11 RNAi suppression of Q37 (I-IV) and SOD1G93A (V-VIII) aggregation in BWM cells of 5 day old animals, shown by the diffuse fluorescent pattern in III, IV, VII and VII; in contrast to a foci-like pattern in the vector control I, II, V and VI. Boxed areas refer to magnified images to the right . (B) RT-PCR amplification of q35-yfp (top panel). mRNA from wt animals is a negative control for transgene amplification (left); yfp-RNAi is a positive control for reduced q35 mRNA levels. Actin (bottom panel) is the control for total mRNA. Band intensities were used to determine mRNA levels relative to vector control (n = 3).  SDS-PAGE and western blotting analysis of Q35 protein levels, with anti-YFP (top) and anti-α-tubulin (bottom) antibodies. Q35 protein levels relative to tubulin were calculated from band intensities on (B), and are shown as a relative percentage of the vector control . (E) gei-11 knockdown did not affect polyQ aggregation in the intestine (iQ44).Suppression of protein aggregation in BWM cells by (TIF)Click here for additional data file.Figure S2gei-11 RNAi is abolished in the L-AChR unc-38(e264) mutant background.  Cholinergic sensitivity assays with 5 day old animals treated with gei-11 or vector RNAi and scored for paralysis on (B) 1 mM Levamisole plates  and (C) on 30 mM nicotine plates . N-AChR mutant acr-16(ok789) and L-AChR mutant unc-38(e264) were used as controls for receptor specificity. (D) Dose-dependent effect of AChR antagonist dTBC (in water) on Q35 aggregation (±SD). Student t-test **p<0.01 and ***p<0.001; data and statistics are relative to Q35;vector (±SD). (E) Real-time qPCR analysis of gei-11 levels in wt, rde-1(ne219) and rde-1(ne219);mRDE-1 animals, treated with gei-11 or vector RNAi. Data are relative to wt animals (±SD).Suppression of aggregation through AChRs. (A) Suppression of Q35 aggregation by (TIF)Click here for additional data file.Figure S3gei-11 knockdown on stress responses. (A) Real-time qPCR analysis of hsp expression levels in heat-shock treated wt animals . Data are relative to wt animals in control temperature (20°C) (±SD). (B) Real-time qPCR analysis of wt animals treated with gei-11 RNAi did not show induction of UPR-regulated ER chaperones, metabolic stress FOXO/DAF-16 regulated genes or oxidative stress regulated genes .Effect of (TIF)Click here for additional data file.Figure S4p<0.01, ***p<0.001.Dose-dependent effect of levamisole on muscle cells Q35 aggregation. Relative foci count upon levamisole (in water) treatment, and statistics relative to Q35;vector (±SD). Student t-test **(TIF)Click here for additional data file.Figure S52+-dependent.  Suppression of Q35 aggregation and hsp induction by gei-11 RNAi were prevented upon co-treatment with the Ca2+ chelator BAPTA . Data are relative to Q35;DMSO control (±SD). (C) RNAi of Ca2+-dependent kinases and calmodulins tested for effect on Q35 aggregation, by double RNAi with gei-11 or vector control (±SD). Student t-test ***p<0.001.Suppression of Q35 aggregation is Ca(TIF)Click here for additional data file.Figure S6gei-11 RNAi. Data are relative to Q35;DMSO in vector RNAi (±SD).  Dose-dependent effect of the RYR agonist 4-CmC and the antagonist DS on Q35 aggregation. % of foci are relative to Q35;water control for 4-CmC and Q35;DMSO for DS (±SD). Student t-test ***p<0.001 and **p<0.01.EGL-19 and RYR agonists and antagonist dose-dependent effect on muscle cells Q35 aggregation. (A) Dose-dependent effect of Nemadipine A on suppression of Q35 aggregation by (TIF)Click here for additional data file.Protocol S1Supplementary (DOC)Click here for additional data file.Table S1Control values for RNAi gene knockdown.(DOC)Click here for additional data file.


Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2–mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in  Caenorhabditis elegans, integrin PAT-2/PAT-3 functions as an engulfment receptor in muscle cells. The integrin α subunit PAT-2 mediates both the recognition and subsequent phagocytosis of apoptotic cells. PAT-2 signals through UIG-1 for CDC-42 activation, leading to the cytoskeletal reorganization as the engulfing muscle cell extends pseudopods around the apoptotic cell. Furthermore, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, both of which appear to act upstream of small GTPase CED-10 (RAC1), predominantly function in epithelial cells to mediate cell corpse removal. Therefore, epithelial cells and muscle cells employ different engulfment receptors for apoptotic cell recognition, downstream signaling, and specific GTPase activation during apoptotic cell removal.When cells undergo apoptosis, their corpses are quickly recognized and phagocytosed by engulfing cells. Although many cell types, such as muscle cells and epithelial cells, possess the ability to remove apoptotic cells, little is known about the receptors and signaling pathways used for apoptotic cell uptake by these “amateur” phagocytes. We show that, in  During the development of multicellular organisms, cells that are unnecessary, damaged, or harmful undergo programmed cell death (apoptosis) in vivo role of most of these receptors in the clearance of apoptotic cells and the tissues in which they act at the whole organism level have not been defined.In flies and mammals, engulfment of apoptotic cells is mediated by “professional” phagocytes, such as mobile macrophages and dendritic cells, or by “amateur” phagocytes, such as muscle cells, epithelial cells, and endothelial cells C. elegans adult hermaphrodite, 1090 somatic cells are generated, 131 of which undergo apoptosis During the development of a C. elegans has two integrin α subunits, INA-1 and PAT-2, and a single β subunit, PAT-3 pat-3 knockout mutant has a stronger defect in cell corpse engulfment than the ina-1 mutant pat-2 may also mediate the removal of apoptotic cells. In this study, we examined and characterized the role of pat-2 in cell corpse engulfment and showed that it functions in the muscle-mediated internalization of apoptotic cells and acts through a pathway distinct from the previously known pathways.Integrins are transmembrane αβ heterodimers that make connections to the extracellular matrix and cytoskeleton and activate several signaling pathways required for multiple cellular processes, including cell adhesion, cell migration, and cell survival pat-2(st567) mutants pat-2 RNAi are embryonic lethal and show a phenotype of paralyzed arrest at the two-fold stage (Pat), as PAT-2 plays an essential role in body wall muscle assembly and function during embryogenesis pat-2 in apoptosis by counting the number of apoptotic cells at the comma and 1.5-fold stages, the two stages at which the majority of embryonic apoptosis occurs pat-2 mutant embryos are still developing normally, and found that both pat-2(st567) and pat-2(RNAi) embryos had a Ced  phenotype with increased numbers of apoptotic cells (pat-2(st567) mutant was rescued by the transgene pat-2pat-2::gfpP, in which the pat-2::gfp translational fusion construct is expressed under the control of the endogenous pat-2 promoter pat-2P (pat-2(st567) mutant was specifically caused by pat-2 loss of function. The pat-2pat-2::gfpP transgene also rescued the Pat phenotype of the pat-2(st567) mutant  mutant .ced-3(n717) and ced-4(n1162), strong mutations in the pro-apoptotic genes ced-3 and ced-4 that block almost all programmed cell death pat-2(st567) or pat-2(RNAi) embryos  or pat-2(RNAi) mutants was not suppressed by either the ced-3 or ced-4 mutation  microscopy analysis of cell corpses in the wild-type and pat-2(st567) mutant during embryogenesis prior to the 2-fold stage. We found that, although the timing and number of cell death events were similar in the two types of embryo  mutation further increased the number of cell corpses in the ina-1(RNAi) mutant, but not in the pat-3 mutant  or pat-2(RNAi) mutation and strong loss-of-function or null alleles of the engulfment ced genes for the two pathways. Interestingly, the pat-2(st567) or pat-2(RNAi) mutation further enhanced the engulfment defect in mutants defective in either pathway  or pat-2(RNAi) mutation could increase the engulfment defect in double mutants between the two pathways, such as the ced-1(e1735); ced-5(n1812) and ced-12(tp2); ced-7(n1892) double mutants. In the first case in which pat-2 would act in both pathways, we would expect that pat-2(st567) or pat-2(RNAi) would not enhance the engulfment defect of the double mutants. However, we found that the pat-2(st567) or pat-2(RNAi) mutation significantly increased the number of cell corpses in both double mutants (pat-2 probably functions in a pathway distinct from these two pathways to promote the engulfment of cell corpses.We next determined whether  pathway . This su mutants . We therabi-1 (abl-1 interactor 1) acts in parallel to the two major engulfment pathways during cell corpse removal pat-2(RNAi) or pat-3(st564) enhanced the engulfment defect in the abi-1(null) or abi-1(RNAi) embryo  embryo , showingpat-2pat-2::gfpP or pat-2pat-2::mcherryP transgene, which rescued the Ced and Pat phenotypes of the pat-2 mutant  and M-lines of muscle cells  embryo  embryo . Punc-54ic cells . These rpat-2P and unc-54P promoters, respectively, to monitor the PAT-2-mediated and muscle-mediated engulfment processes simultaneously in the 1.5-fold embryos. Approximately 22.7% and 18.1% of the cell corpses were enclosed by the PAT-2::mCherry and PAT-2::GFP circles, respectively, and most of the mCherry and GFP circles were co-localized, suggesting that most, if not all, PAT-2-mediated engulfment involves muscle cells  mutant embryos, the MYRI::mRFP circle formation took approximately 21 minutes to complete, more than three times longer than that in the wild-type, indicating that the internalization process was compromised.The persistence of cell corpses in the  process . The MYRpat-2 mutant using the unc-54myri::mrfpP transgene. As shown in pat-2 mutants, no apoptotic MSpppaaa cells had the MYRI::mRFP circle at the time when grinder formation had just finished, and nearly half of MSpppaaa cell corpses still persisted and showed no MYRI::mRFP circle at the time of pharyngeal pumping ::mcherryP, in which the coding sequence for the PAT-2 extracellular domain with a signal sequence [PAT-2(ex)] was fused to that of mCherry under the control of the heat-shock promoter hspP. The transgene was then introduced into the wild-type and ced-1(e1735); ced-5(n1812) double mutant embryos. The ced-1(e1735); ced-5(n1812) double mutant embryos contain many persistent apoptotic cells, resulting in a greater chance of seeing PAT-2(ex)::mCherry binding, especially during late embryogenesis when very few cell corpses are present in the wild-type embryos. We found that secreted PAT-2(ex)::mCherry clustered on the surface of apoptotic cells (ced-1(e1735); ced-5(n1812) double mutant embryos at the 4-fold stage. A similar percentage (16.6%) was observed in the wild-type embryos at the 1.5-fold stage ::mCherry and observed PAT-2(ex)::mCherry clustering around apoptotic cells including the apoptotic MSpppaaa cell (hspP promoter. This result shows that PAT-2(ex)::mCherry expressed in and secreted from muscle cells recognizes, and binds to, the surface of apoptotic cells. Embryos with PAT-2(ex)::mCherry overexpression by either hspP or unc-54P resulted in increased numbers of apoptotic cells at the comma and 1.5-fold stages (We further examined whether PAT-2(ex)::mCherry bound to specific apoptotic cells. Approximately 20% of the MSpppaaa cell corpses had a PAT-2(ex)::mCherry circle , but nonaaa cell , despited stages  and delad stages , indicathsppat-2(ex)::mcherryP and hsp ina-1(N)::gfpP. We found that PAT-2(ex)::mCherry was co-localized with INA-1(N)::GFP on some apoptotic cells  as in reference ic cells . Thus, tpat-2(st567) mutants. We generated pat-2pat-2Δcyto::gfppat-2(st567); P transgenic animals that expressed the PAT-2Δcyto::GFP fusion protein under the control of the pat-2 promoter. To our surprise, PAT-2Δcyto::GFP fully rescued the Pat phenotype of the pat-2(st567) mutant, but failed to rescue the Ced phenotype (pat-2pat-2Δcyto::gfppat-2(st567); P transgenic embryos contained increased numbers of cell corpses (pat-2(st567) embryos because of developmental arrest. The pat-2(st567) embryos carrying the pat-2 pat-2Δcyto::gfpP transgene also had increased numbers of cell corpses at the 2-, 3- and 4-fold stages ; pat-2 pat-2Δcyto::gfpP transgenic embryos than those in wild-type embryos. Approximately 61% of the MSpppaaa cell corpses still persisted at the stage of pharyngeal pumping, while none remained in the wild-type embryos at this stage  embryos at this stage (pat-2pat-2Δcyto::gfppat-2(st567); P embryos. This result and the aforementioned data together indicate that the PAT-2 cytoplasmic domain is required for the muscle-mediated engulfment of apoptotic cells, but is dispensable for its subcellular localization in muscle cells and function during muscle development.PAT-2Δcyto::GFP was localized to the surfaces, dense bodies and M-lines of muscle cells in the wild-type and  mutants , similaris stage . Thus, tpat-2 mutants, but failed to induce the Pat phenotype (Expression of the PAT-2Δcyto::GFP fusion protein in the wild-type embryos resulted in increased numbers of cell corpses at the comma and 1.5-fold stages comparable to those seen in henotype . In addihenotype . For exahenotype . This reced-10 mutation enhanced the engulfment defect in the pat-2(RNAi) mutant (ced-10 overexpression failed to rescue the engulfment defect of the pat-2(RNAi) mutant  mutant  and that) mutant  suggest Drosophila and C. elegans to specifically mark the filamentous form of actin unc-54moesin::gfpP in which MOESIN::GFP was expressed in muscle cells using unc-54P showed a MOESIN::GFP circle around apoptotic cells  allele has a deletion that eliminates part of the 5′ regulatory sequence, the entire first exon, and part of the first intron of the cdc-42 gene (C. elegans Gene Knockout Consortium), while the uig-1(ok884) mutation deletes the region coding for the DH/PH domain, which has the GEF activity uig-1 or cdc-42 embryos contained increased numbers of cell corpses at the comma and 1.5-fold stages  embryos revealed a cell-corpse engulfment defect. In the wild-type, 97% of cell corpses disappeared within 40 minutes and none lasted longer than 60 minutes (cdc-42(gk388) mutants, only 40% of the cell corpses disappeared within 40 minutes and almost 40% persisted longer than 60 minutes  specific for CDC-42, function downstream of PAT-2/PAT-3 signaling for muscle assembly d stages , similar minutes . However minutes .cdc-42 acted in the same pathway as pat-2 to promote apoptotic cell engulfment. We found that neither cdc-42 nor uig-1 increased the number of cell corpses in pat-2 mutants at the comma and 1.5-fold stages  mutants, whereas ajm-1gfp::cdc-42P, which expressed GFP::CDC-42 in hypodermal cells, did not  embryos  or ced-10(tm597) mutant (pat-2(RNAi) embryos  mutants  or ina-1(gm144) mutants, respectively  mutant , Table 6pat-2 by the ajm-1P promoter in hypodermal cells partially rescued the defective engulfment of the ina-1 mutants and, reciprocally, overexpression of ina-1 by the unc-54P promoter in muscle cells also partially rescued the engulfment defect of the pat-2 mutants  mutants  fully rescued the Pat phenotype of the t defect  argues a M-lines , which a M-lines  nor its  M-lines , muscle pat-2(st567) mutants show a weak engulfment phenotype compared with that of the ced-1 mutants during embryogenesis ::mCherry to apoptotic cells.The PAT-2 extracellular region bound to apoptotic cells when PAT-2(ex) was fused to mCherry and overexpressed using the heat shock promoter . BecauseLike PAT-2, CED-1 and INA-1 are expressed in muscle and hypodermal cells et al. and Zou et al.We showed that the muscle-mediated internalization of cell corpses took apporximately 6 mintues . Similarv in the mouse immune system revealed that this protein is essential for the engulfment of apoptotic cells by gut-associated macrophages and dendritic cells v, are defective in the removal of apoptotic B cells by tingible body macrophages in the spleen germinal centers C. elegans provides a good system for studying the amateur phagocyte-mediated engulfment of apoptotic cells, as it does not have professional phagocytes. Our data showed that PAT-2 acts in muscle cells and transduces the engulfment signal through a novel signaling pathway for apoptotic cell removal. Recently, a mouse lacking ELMO1 showed a defect in Sertoli cell-mediated engulfment of apoptotic germ cells, but no engulfment defect was detected in macrophages or fibroblasts Recently, conditional deletion of integrin αEscherichia coli OP50 as the food source at 20°C unless otherwise noted ced-1(e1735), ced-12 ; LGII, cdc-42(gk388), mIn1[mIs14 dpy-10(e128)]; LGIII, abi-1(ok640), ced-4(n1162), ced-6(n1813), ced-7, unc-79(e1068), pat-2(st567), dpy-17(e164), pat-3(st564), qC1 dpy-19(e1259) glp-1(q339); LGIV, ced-2(n1994), ced-3(n717), ced-5(n1812), ced-10; LGIV, uig-1(ok884). dpy-17(e164) was used to balance unc-79(e1068) pat-2(st567). The homozygous unc-79(e1068) pat-2(st567) mutants were also maintained using the extrachromosomal pat-2pat-2::gfpP or pat-2pat-2::mcherryP transgene. In either of the transgenic lines, the unc-79(e1068) pat-2(st567) mutant embryos that lost the transgene were both Pat and Ced. The numbers of cell corpses in the unc-79(e1068) mutant at the comma and 1.5-fold stages were similar to those of the wild-type at the same stage, indicating that unc-79(e1068) does not affect apoptosis. qC1 dpy-19(e1259) glp-1(q339) was used to balance pat-3(st564). mIn1 was used to balance cdc-42(gk388). The pat-2(st567) allele has a G441D alternation in the extracellular domain. The information of the transgenic strains used in this work is listed in The N2 Bristol strain was used as the reference wild-type strain. All strains were maintained on nematode growth medium (NGM) plates with gfp vector pPD95.75, the mcherry vector pYW806, and the mrfp vector pYW897. pYW806 and pYW897 were respectively generated by replacing gfp in pPD95.75 with mcherry or mrfp via the KpnI/EcoRI sites. To generate pat-2pat-2::gfpP (pYW903) or pat-2pat-2::mCherryP (pYW950), the 10 kb fragment containing the 4 kb upstream sequence and the full-length pat-2 coding region without the stop codon was PCR-amplified from the cosmid F54F2  using the primers TCCCCCCGGGTTTATGACTCACAGAC and GGGTACCGATGCATTTGTC CGTGACGT, and cloned into pPD95.75 or pYW806, respectively, via the KpnI site. The full-length pat-2 cDNA construct (pYW901) was generated by inserting into yk616b4 (Dr. Yuji Kohara) via the PstI site a 0.6 kb pat-2 cDNA which was amplified by RT-PCR using the primers AACTGCAGATGCGAGAGGGTAGTTTTCC and GATTCTTCTTTCCTGGAACTGCAGC. To generate pat-2nls::gfpP (pYW949) or pat-2gfpP (pYW903), the 4 kb upstream sequence of pat-2 was first amplified by PCR from the cosmid F54F2 using the primers TCCCCCCGGGTTTATGACTCACAGAC and TCCCCCCGGGATCTACTGG AAATTTG and inserted into pPD95.67 or pPD95.75, respectively. The unc-54gfpP (pYW899) or unc-54mcherryP (pYW900) construct was generated by inserting the 1 kb HindIII/KpnI fragment of pPD30.38 containing unc-54P into pPD95.75 or pYW806, respectively. The ajm-1gfpP (pYW902) was generated by inserting into pPD95.75 via the SalI/BamHI sites a 5.5 kb ajm-1P fragment which was amplified by PCR from pBR980 CGTCGACCGATTTGACCGTTCGATAAG and CGGATCCTCGTCGGTA GTACTCGTCC. ced-1gfpP (pYW898) was generated by inserting into pPD95.75 via the SphI site a 5 kb ced-1P fragment which was PCR-amplified from genomic DNA using the primers GGCATGCATACCTCCTGATATG GGGTGA and GCATGCTTGC GGCTGCAAAAAAACAGGG. ced-1ced-1::gfpP (pYW904) was generated by three-piece ligation. The 6 kb PCR-amplified fragment from ced-1ced-1::gfpPAGGTACCATGCGTCTCATTCTCCTTGTGC and GGTCGA CGTGATTGTTCAGATGA and the 2.4 kb fragment PCR-amplified from genomic DNA using the primers CGTCGACCTCTATTAGAAGAGCATGACG and TGGTACCGAGGTGTACAAATTGTCCTGAGC were inserted into pYW898 via the SalI and KpnI sites. ced-1ced-1::gfpP fully rescued the engulfment defect of the ced-1(e1735) mutant (data not shown). To generate pYW901 containing pat-2 cDNA without the stop codon, pat-2 cDNA was PCR-amplified from the full-length pat-2 cDNA clone using the primers CGGTACCATGCGAGAGGGTAGTTTTCC and GGGTACCGATAGCATTTGTC CGTGACGT and inserted into the pGEM-T Easy vector (Promega) via the KpnI site. To generate unc-54pat-2::gfpP (pYW913) or unc-54pat-2::mCherryP (pYW916), the 3.7 kb KpnI pat-2 fragment from pYW901 was inserted into pYW899 or pYW900, respectively, via the KpnI site. To generate unc-54ced-1::gfpP (pYW905), the 8.5 kb KpnI ced-1 fragment from pYW904 was inserted into pYW899 via the KpnI site. To generate unc-54 ced-1::mrfpP (pYW941), a 8.6 kb SpeI/BamHI fragment, containing unc-54P and the first 4.9 Kb of ced-1, and a 3.6 kb BamHI/KpnI fragment, corresponding to the rest ced-1 sequence, of pYW905 were fused to a 1.5 kb KpnI/SpeI fragment, containing the mrfp sequence, of pYW897 by three-piece-ligation. To generate unc-54moesin::gfpP (pYW940), the 0.6 kb SmaI/NcoI fragment containing the moesin actin-binding sequence was inserted into pYW899 via the NheI/NcoI sites. The moesin plasmid was a gift from Dr. Fabio Piano unc-54 gfp::cdc-42P (pYW906), two plasmids gfp_Ntag_TA and cdc-42_Ntag_TA were generated first. To generate the gfp_Ntag_TA plasmid, PCR-amplified gfp, using oligonucleotides CGGTACCATGAGTAAAGGAG AAGAACT and GTCTAGATTTGTATAGTTCATCCATGCC as primers and pPD95.75 as template was inserted to the vector pGEM T-Easy. To generate the cdc-42_Ntag_TA plasmid, PCR-amplified cdc-42 from k1101h01(Y. Kohara) using primers GTCTAGAATGCAGACGATCAAGTGCG and CGTTAACCTAGAGAATATTGCACTTCTTC was inserted into the pGEM T-Easy vector. The 1 kb HindIII/KpnI fragment of pPD30.38 containing unc-54P, the KpnI /XbaI gfp fragment from the gfp_Ntag_TA plasmid, and the XbaI/EcoR1 cdc-42 fragment from the cdc-42_Ntag_TA plasmid were inserted to the pPD95.75 vector previously digested with HindIII and EcoRI in a four-piece-ligation reaction to generate pYW906. To generate ajm-1pat-2::gfpP (pYW948) or ajm-1ced-1::gfpP (pYW914), the 3.7 kb KpnI pat-2 sequence from pYW901 or the 8.5 kb KpnI ced-1 sequence from pYW904 was inserted, respectively, into pYW902. To generate ced-1pat-2::gfpP (pYW942), the 3.7 kb KpnI pat-2 sequence from pYW901 was inserted into pYW898 via the KpnI site. To generate pat-2pat-2Δcyto::gfpP (pYW964), pat-2Δcyto was PCR-amplified from pYW901 using the primers CGGTACCATGCGA GAGGGTAGTTTTCC and TTGGT ACCGTC CTATAGAATAATGCAA and cloned into pYW903 via the KpnI site. To generate Pemphtypepat-2(ex)::mcherry (pYW917), the DNA fragment corresponding to pat-2(ex) was PCR-amplified from pYW901 using the primers CGGTACCATGCGAGAGG GTAGTTTTCC and CGGTACCAGATCTCTTCC TTCTTCAGA and cloned into pYW900 via the KpnI site. To generate hsppat-2(ex)::mcherryP (pYW966), the 4.2 kb NheI/PvuI fragment of the unc-54 pat-2(ex)::mCherryP plasmid containing pat-2(ex)::mCherry was inserted into pPD49.78 via the NheI/PvuI sites. To generate hspgfp::cdc-42P (pYW959), the 1.5 kb KpnI/HpaI fragment of pYW906 corresponding to gfp::cdc-42 was inserted into pPD49.78 and pPD49.83 (different tissue specificity) via the KpnI/EcoRV sites. To generate the unc-54ina-1::gfpP plasmid, unc-54P was PCR-amplified using pYW913 as template and oligonucleotides GCATCCGCCAAGCTTGTCTTCTTC and GGATCCGGTACCGT CGACGCTAC as primers and used to replace the ced-1P region of ced-1ina-1::gfpP via the SphI/BamHI sites. To generate the pat-3pat-3::gfpP (pYW1091) plasmid, the 10.2 kb pat-3 genomic DNA was amplified by PCR and subsequently cloned into the pPD95.75 vector via the XmaI/KpnI sites. To generate the unc-54myri::mrfpP (pYW1092) construct,the myri::mrfp cDNA of unc-86myri::gfpP (from C. Bargmann) was inserted into the unc-54mrfpP vector via the KpnI site. To generate the ajm-1gfp::cdc-42P (pYW1093) plasmid, the gfp region of the pPD95.75 plasmid was replaced by gfp::cdc-42 from the hspgfp::cdc-42P construct via the KpnI site, and the resulting construct was then digested with BamHI and SalI and subsequently inserted with the BamHI and SalI fragment containing the Pajm-1 promoter from pYW902.Three vectors were used to generate constructs expressing fluorescent proteins, the pat-2 RNAi clone, the 1.3 kb PstI/HindIII fragment of pat-2 cDNA was inserted into the pPD129.36 vector. The pat-3 RNAi plasmid was obtained from the J. Ahringer RNAi library. All RNAi experiments were carried out using a bacterial feeding protocol To generate the rol-6 (su1006)], pTG96[sur-5::gfp] or sur-5rfpP plasmids (50 ng/µL) as coinjection markers hspced-10V12P was injected with the coinjection marker pTG96[sur-5::gfp] to unc-79(e1068) pat-2(st567)/dpy-17(e164), and no rescue of the Pat phenotype was observed. To score cell corpses, transgenic embryos carrying the transgene hspced-10V12P and pTG96[sur-5::gfp] were scored and only the data for those which later exhibited the Pat phenotype were used.Transgenic animals were generated by microinjection of the indicated plasmid(s) (3–50 ng/µL), using the pRF4 mothers, GFP-negative embryos at the indicated developmental stages were analyzed. To analyze cell death events during embryogenesis, wild-type or mutant embryos were filmed during the period 200–460 min after the first cleavage and the time point at which each cell corpse appeared was noted and was reported relative to the first cell death for comparison between the wild-type and mutants. About 40–50 serial Z-sections were recorded at 0.4 µm intervals every 1 min. To measure the duration of cell corpses in the wild-type and mutants, cell corpses appearing between 360 to 410 min after the first cleavage during embryogenesis were followed, and about 50–60 serial Z-sections were recorded at 0.3 or 0.4 µm intervals every 1 min. To monitor the muscle-mediated internalization of apoptotic cells, the fluorescence images of wild-type or mutant embryos carrying the unc-54myri::mrfpP transgene were recorded using the DeltaVision microscope  equipped with a digital camera (Photometrics Cascade II 512 EMCCD) at 1 (for wild-type) or 3 (for mutant)-minute intervals for about 120 minutes.Embryos were mounted on a 4% agar pad in M9 buffer in the presence (four-fold stage embryos) or absence (comma and 1.5-fold stage embryos) of 30 mM sodium azide at 20°C. Cell corpses were analyzed using DIC microscopy, as previously described hsppat-2(ex)P::mcherry or embryos carrying the transgenes hsppat-2(ex)P::mcherry and hspina-1(N)P::gfp were subjected to heat shock at 33°C for 60 min and transferred to 20°C to recover for 4–5 hours, then embryos were examined using fluorescence microscopy (for mCherry and/or INA-1(N)::GFP signal) or DIC microscopy (for cell corpses). To overexpress GFP::CDC-42, CED-10V12 or Annexin V::mRFP by heat-shock, the embryos carrying the respective transgene were subjected to heat shock at 33°C for 30 min and transferred to 20°C to recover for 2 hours, then embryos were examined using fluorescence microscopy  or DIC microscopy (cell corpses).For the heat shock rescue experiments, transgenic embryos were subjected to heat shock at 33°C for 30 min and transferred to 20°C to recover for 2 hours, then cell corpses in embryos at the indicated stages were counted. To test the binding of PAT-2(ex)::mCherry to apoptotic cells and its effect on cell-corpse engulfment when overexpressed, embryos carrying the transgene Figure S1pat-2 expression is not detectable in embryonic apoptotic cells. (A) GFP, Annexin V::mRFP, merged GFP and mRFP, and DIC images of a wild-type early embryo co-expressing pat-2nls::gfpP and hspannexin V::mrfpP transgenes after heat shock treatment. Annexin V::mRFP, which is secreted and clusters around the surface of apoptotic cells, was used to label apoptotic cells. Apoptotic cells are indicated by arrows. The scale bar represents 5 µm. (B) GFP, Annexin V::mRFP, merged GFP and mRFP, and DIC images of a wild-type late embryo co-expressing pat-2nls::gfpP and hspannexin V::mrfpP transgenes after heat shock treatment. Apoptotic MSpppaaa cells are indicated by arrows. The scale bar represents 5 µm.(TIF)Click here for additional data file.Figure S2pat-2pat-2::mcherryP and pat-3pat-3::gfpP. (A) PAT-2::mCherry and PAT-3::GFP were co-localized to the dense bodies (arrows) and M-lines (arrowheads) in muscle cells. The scale bar represents 10 µm. (B) PAT-2::mCherry and PAT-3::GFP were co-localized around an apoptotic cell (arrows). The scale bar represents 5 µm.PAT-2 and PAT-3 are co-localized in muscle cells and apoptotic cells. A wild-type larva (A) and an embryo (B) co-expressing the transgenes (TIF)Click here for additional data file.Figure S3ced-1 or ced-5 mutation. PAT-2::GFP (left) and DIC (right) images of a ced-1(e1735); ced-5(n1812) embryo carrying the transgene pat-2pat-2::gfpP. Apoptotic cells are indicated by arrows and shown enlarged in insets. The scale bar represents 5 µm.PAT-2::GFP localization around cell corpses is not disrupted by either (PDF)Click here for additional data file.Figure S4unc-54nls::gfpP or unc-54pat-2(ex)::mcherryP transgene in embryos. (A) DIC and GFP images of a ced-1(e1735) mutant embryo expressing unc-54nls::gfpP. Arrows indicate apoptotic cells. (B) DIC and PAT-2(ex)::mCherry images of a wild-type embryo expressing unc-54pat-2(ex)::mcherryP. Arrows indicate MSpppaaa cells. Both scale bars represent 5 µm.Expression of (TIF)Click here for additional data file.Figure S5ajm-1pat-2::gfpP and unc-54pat-2::mcherryP. MSpppaaa cell corpses are indicated by arrows and shown enlarged in insets. The scale bar represents 5 µm.The MSpppaaa cell corpse is engulfed by a muscle cell. PAT-2::GFP, PAT-2::mCherry, and DIC images of an embryo co-expressing (TIF)Click here for additional data file.Figure S6hsppat-2(ex)::mcherryP at the time of pharyngeal pumping. Apoptotic MSpppaaa cells are indicated by arrows. (B) Localization of CED-1::GFP around the apoptotic MSpppaaa cell. DIC and CED-1::GFP images of a wild-type embryo expressing ced-1ced-1::gfpP.  The PAT-2(ex)::mCherry signal is not observed around C1, C2 and C3 cell corpses. DIC and PAT-2 (ex)::mCherry images of ced-1(e1735); ced-5(n1812) double mutant embryos expressing the transgene hsppat-2(ex)::mcherryP. C1, C2 and C3 cell corpses are indicated by arrows. (E) Co-localization of PAT-2 (ex)::mCherry and INA-1(N)::GFP on some apoptotic cells. DIC, PAT-2 (ex)::mCherry, INA-1(N)::GFP and merged images of a ced-1(e1735); ced-5(n1812) double mutant embryo co-expressing the transgenes hsppat-2(ex)::mcherryP and hspina-1(N)::gfpP. Apoptotic cells are indicated by arrows. All scale bars represent 5 µm.Localization of PAT-2 (ex)::mCherry, CED-1::GFP and INA-1(N)::GFP around apoptotic cells. (A) Localization of PAT-2 (ex)::mCherry around the apoptotic MSpppaaa cell. DIC and PAT-2 (ex)::mCherry images of a wild-type embryo expressing (TIF)Click here for additional data file.Figure S7pat-2(st567) adult worms expressing the transgene pat-2pat-2Δcyto::gfpP. PAT-2Δcyto::GFP is localized to muscle cell boundaries , dense bodies (indicated by arrows) and M-lines (indicated by close arrowheads). The scale bar represents 10 µm. (B–C) PAT-2::GFP and PAT-2Δcyto::GFP are localized to the surface of muscle precursor cells. The DIC, PAT-2Δcyto::GFP (B) and PAT-2::GFP (C) images of wild-type and pat-2(st567) embryos carrying the unc-54pat-2Δcyto::gfpP transgene (B) or the wild-type carrying the pat-2pat-2::gfpP transgene (C). All scale bars represent 5 µm.Deletion of the cytoplasmic domain of PAT-2 does not affect its localization to muscle cell surfaces, dense bodies and M-lines. (A) The DIC and PAT-2Δcyto::GFP images of wild-type and (TIF)Click here for additional data file.Figure S8pat-2pat-2Δcyto::gfp]pat-2(st567); Ex[P embryos. The DIC and PAT-2Δcyto::GFP images of a wild-type and a pat-2(st567) embryos expressing pat-2pat-2Δcyto::gfpP at the stage when grinder formation had finished. A PAT-2Δcyto::GFP circle was detected around the MSpppaaa cell corpse (indicated by an arrow) in the wild-type (A) but not in the pat-2(st567) (B) embryo. The engulfing muscle cell for the MSpppaaa cell corpse is indicated by an arrowhead. The scale bar represents 5 µm.The internalization of MSpppaaa cell corpses is defective in the (PDF)Click here for additional data file.Table S1Localization of PAT-2 or PAT-2(ex) around apoptotic cells.(DOC)Click here for additional data file.Table S2pat-2 and cdc-42 mutant embryos.The engulfment of C1, C2 and C3 cell corpses appears normal in (DOC)Click here for additional data file.Table S3pat-2Δcyto::gfp on the Ced phenotype during mid and late embryogenesis.Effects of (DOC)Click here for additional data file.Table S4List of transgenic strains and transgenes used in this work.(DOC)Click here for additional data file.Text S1pat-2 does not appear to be expressed in embryonic apoptotic cells.(RTF)Click here for additional data file.