Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis. Actin polymerization-driven migration and invasion is coordinated by the proto-oncogenes c-Src and c-Abl kinases and cytoskeletal regulatory proteins including Rac GTPase, the Scar/WAVE complex and Ena/VASP proteins.7Breast cancer metastasis is one of the leading causes of cancer-associated mortality in women worldwide.14 Mena is upregulated in breast cancer and promotes invasion.16 We identified Lamellipodin (Lpd) as a binding partner of Ena/VASP proteins.18 Lpd localizes to lamellipodia, thin membrane protrusions at the leading edge of migrating cells.17 The Lpd-Ena/VASP interaction is positively regulated by Abl kinase-mediated Lpd phosphorylation, which drives Ena/VASP recruitment to lamellipodia by Lpd.19Ena/VASP proteins  enhance processive filament elongation.17 and binds directly to the Scar/WAVE complex.20 Scar/WAVE activates the Arp2/3 complex to nucleate branched actin networks during lamellipodia formation.7 Surprisingly, Lpd-driven random cell migration in 2D requires Lpd binding to Scar/WAVE, but not to Ena/VASP.20Lpd is required for lamellipodium formationThe mechanisms by which actin regulators coordinate the interplay between actin-elongation and actin-branching factors to promote cancer cell invasion remain incompletely understood. Here, we report that Lamellipodin mediates invasive 3D migration of cancer cells via selective, regulated interactions with Ena/VASP and Scar/WAVE. Our findings point to key roles for increased Lpd levels in breast cancer invasion and metastasis.21 to examine whether Lpd mRNA levels correlated with occurrence of distant metastases in breast cancer patients. Lpd was overexpressed in several types of breast tumors compared with matched healthy tissue 25 generated from 312 patients with invasive breast cancer with anti-Lpd antibodies. Moderately, but not highly, increased abundance of Lpd in the cytoplasm  ): 1.765 (1.026–3.036); 20 we observed a significant association between highly, but not moderately, increased Lpd staining intensity at the plasma membrane and reduced disease-free (Histoscore 3: HR (95% CI): 1.652 (1.24–2.428)) and metastasis-free survival of breast cancer patients (Histoscore 3: HR (95% CI): 1.515 (1.054–2.178); We observed higher Lpd levels in invasive and metastatic basal cell lines compared with noninvasive, luminal tumor cell lines . Therefoy tissue . High ley tissue .22, 23, 26 on their ability to metastasize from an orthotopic mammary tumor to the lungs. We generated stable LM2 cell lines with two independent Lpd-targeting shRNAs or a non-targeting control shRNA, all three retroviruses also conferring the cis-linked marker ZsGreen , a highly metastatic derivative of MDA-MB-231 breast cancer cells, ZsGreen . Lpd kno ZsGreen . Importa ZsGreen . In addi ZsGreen .in vivo.We then explored whether Lpd promotes cancer cell invasion during metastasis. In fixed samples, tumors generated from control shRNA LM2 cells prominently invaded the surrounding stroma. However, tumors from Lpd knockdown cells were markedly less invasive . We inve28 Overexpression of GFP-Lpd in MDA-MB-231 cells enhanced the frequency of seeding of these breast cancer cells compared with GFP-expressing control cells .dia size , similaron speed . We choson speed . In agreon speed  but did on speed , compareon speed . Lamellion speed . Taken t6 To determine how Lpd depletion influences actin polymerization, we used a G-actin incorporation assay30 to measure the abundance and distribution of polymerization-competent, free (uncapped) F-actin barbed-ends in lamellipodia of living cells. Silencing of Lpd significantly reduced free barbed-end formation 1 min after EGF stimulation, relative to Ctrl-shRNA-expressing cells , could respond to EGF gradients in the micropipette assay and discovered that, while GFP-Lpd effectively rescued the chemosensing defects in Lpd-depleted cells, GFP-LpdEVmut conferred no significant phenotypic rescue  digestion of ECM, we tested whether Lpd increases invasion via MMP-dependent or migration-dependent mechanisms. MMP inhibitor treatment of GFP-expressing control cells reduced invasion, as expected. Similarly, MMP inhibitor treatment reduced invasion of Lpd overexpressing MDA-MB-231 cells. However, Lpd overexpressing cells treated with MMP inhibitors invaded significantly further compared with GFP control cells treated with the inhibitor, suggesting that Lpd functions to increase invasion by increasing migration and potentially MMP-dependent ECM degradation .39 We observed that Lpd co-localized with the invadopodial marker cortactin at invadopodia, at sites of matrix degradation , all Scar/WAVE-binding sites (GFP-LpdS/Wmut), or all Ena/VASP and all Scar/WAVE-binding sites (GFP-LpdEV+S/Wmut) had been mutated. All of these mutants localized to the leading edge of MDA-MB-231 cells  to efficiently pull down the Scar/WAVE complex and associated proteins. GST-Abi pulldowns from cells co-expressing GFP-Src contained higher levels of endogenous Scar/WAVE2 co-precipitating with endogenous Lpd compared with GFP controls  and the eight c-Abl phosphorylation sites (GFP-LpdY8F) to phenylalanine, rendering them non-phosphorylatable. Overexpression of GFP-Lpd induced low levels of Lpd tyrosine phosphorylation, which was markedly enhanced by stimulation with 100 ng/ml EGF for 5 min. In contrast, neither GFP-LpdY6F nor GFP-LpdY8F tyrosine phosphorylation was enhanced when cells expressing these constructs were stimulated with EGF On the basis of our findings, we propose that Lpd functions as an essential component of a pro-metastatic signaling pathway composed of Src and Abl kinases, Lpd, Ena/VASP and Scar/WAVE that promotes metastatic progression.34 Myc-Pir121, -Nap1, -Abi1d, -WAVE2 in pRK5-Myc-DEST20 and Lpd17 in pENTR (Invitrogen) mutated using Quikchange (Agilent), transferred into pCAG-DEST-EGFP (Gateway)  and pCB6-Src-WT-EGFP, pCB6-Src-KI (kinase-inactive)-EGFP49 (M Way).GFP-VASP, pMSCV-mRFP1-FP4/AP4-mito,S/Wmut: 20(AAS82582.1) Site 1: (aa 968–978) GKKP(P>A) (P>A) T(P>A)Q(R>A)N; Site 2: (aa 1119–1129) APP(P>A)TR(P>A)K(R>A)ND; Site 3: (aa 1230–1244) RRGP(P>A)A(P>A)(P>A)(K>A)(R>A)DQNT.GFP-LpdEVmut: 20(AAS82582.1) FP4-1: aa 869 F>A; FP4-2: aa 916 F>A; FP4-3: aa927 F>A; FP4-4: aa 1064 F>A; FP4-5: aa1073 F>A; FP4-6: aa1082 L>A; FP4-7: aa1202 L>A.GFP-LpdY4F: 19(AAS82582.1) Y>F: aa 426, 456, 513, 1226; GFP-LpdY6F: (AAS82582.1) Y>F: aa 366, 426, 456, 466, 481, 510; GFP-LpdY8F: (AAS82582.1) Y>F: aa 366, 426, 456, 466, 481, 510, 513, 1226.GFP-LpdpLJM1-H2BK-mCherry: histone H2BK amplified from A431 cell by RT–PCR cloned into pLJM1-mCherry .50 or MSCV-ZsG-2A-Puro-miR30 retroviral vector.51 Cells were FACS sorted or puromycin (Invitrogen) selected and knockdown tested by western blotting. shRNA (5′–3′) used:shRNAs were cloned into pLL3.7-Puro, miR30-MLS EGFP/mCherryHuman Lpd-shRNA1: TTTCCCCAAAAGATAATTCTG, humanLpd-shRNA2: TTCCCATACTTTGCAATGCGG, ratLpd-shRNA2: TAGAGCTCACAGTACTTTGGG, ratLpd-shRNA3: AAGAGGTCCAATCATAAGCTG and Control-shRNA (targeting luciferase): TTAATTAAAGACTTCAAGCGG . Human L53 HEK cells expressing GST-Abi2 and GFP-Src were treated for 1 h with 100 μm KB SRC 4 (Tocris) before lysis. Abl1/Abl2 double knockout MEFs , MDA-MB-231 cells (gift of A Ridley) and MDA-MB-231 LM2 cells26  were cultured in high-glucose DMEM, penicillin, streptomycin, 10% FBS. MTLn3 and MDA-MB-231 transfection was done using Lipofectamine 2000 (Invitrogen). Abl1/2DKO MEFs were serum starved (18 h) and treated with 10 μm Bosutinib (Cambridge Biosciences) (2 h) in DMSO or DMSO (control), then stimulated with 20 ng/ml PDGF-BB (2 min).HEK293, BT549, MCF7 and T47D cells obtained from ATCC and maintained according to ATCC's protocol. SUM-159 and MTLn3 cells cultured as described.17 Mena mab A351F7D9,54 Wave2 , p34Arc , Tubulin (DM1A), Hsc70 mab (Santa Cruz), GFP mab (Roche), Myc mab , pTyr mab  and Vimentin . Alexa-conjugated secondary antibodies, phalloidin  diluted 1:50–1000.Lpd pab 3917,20Immunoprecipitation, GST-pulldowns and western blotting were performed as described.19Peptide array assay was done using Src kinase (NEB) as described.19 Imaged using Deltavision microscope  . Olympus IX-81 microscope  was used. Leading edge localization (n=32–38 cells for each mutant).Cells were plated on glass coverslips, fixed and stained as described.lization  was quan5 MDA-MB-231 cells stably expressing mCherry-H2B were seeded onto underside of 8 μm pore-size transwell inserts (Greiner Bio-One Ltd) containing matrigel ). Inserts were inverted after cells adhered (4 h), placed in serum-free medium, and normal growth medium containing EGF (25 ng/ml) placed on top. Inhibitors used were GM6001 μm; STI571 10 μm and Dasatinib 10 nm. Seventy-two hours later, cells that did not cross the transwell filter were removed; invading cells visualized by confocal microscopy; 2.5 μm sections. The number of nuclei of invading cells above 40 or 80 μm was automatically quantified using the Volocity software.In all, 5 × 1016 Kymographic analysis was performed to analyze the protrusion parameters including: protrusion persistence, distance, velocity and protrusion initiation after EGF stimulation.Membrane protrusion assay for EGF-treated cells was performed as described.55For membrane protrusion assay in 3D matrigel, MDA-MB-231 cells were stained with CellTracker Green dye  embedded in matrigel (BD Biosciences) in μ-slide chambers . Four hours after plating, 5 min movies, one frame every 15 s; × 40; Olympus IX-81 were generated and protrusive activity around the entire circumference between frames automatically quantified from thresholded movies using ImageJ plugin ADAPT.56The micropipette assay was performed as described.m ATP, 5 mm MgCl2, 50 mm KCl, 50 mm Tris/HCl, pH 8.0), labeled with Rhodamine-X succinimidyl ester , depolymerized in G-actin buffer  to G-actin, and passed through PD-10 columns  to eliminate free rhodamine. The barbed-end assay in MTLn3 cells was performed as described.16 Images taken with deconvolution microscope; the ratio of the barbed-end intensity to phalloidin intensity along the edge (the zone between 0 and 0.66 μm inside the cell) quantified as described above.G-actin was extracted from rabbit muscle acetone powder and standard techniques used to gel-filter over a Superdex-200 gel filtration column. The G-actin was polymerized to F-actin in F-actin buffer (1 m57MDA-MB-231 cells used in this study were cultured on FN/gelatin matrix (for 8 h) or thin gelatin matrix (for 4 h), and treated as described.28The zebrafish tumor cell dissemination assay was done as described.5 cells in Hank's Balanced Salt Solution (Gibco); killed 6±0.5 weeks post injection; tumors dissected, weighed, flash frozen, stored (−80 °C) or fixed: 3.8% formaldehyde, imaged with a fluorescence microscope, and embedded in paraffin and sectioned. Lungs were collected, fixed: 3.8% formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). ZsGreen-positive foci were counted in left pulmonary lobe using ImageJ.Tumor growth and spontaneous metastasis formation assayed by injecting tumor cells orthotopically into inguinal mammary fat pads  . Mice anesthetized with isoflurane, injected with 1.5 × 1058 Five mice per group were used. Collagen I fibers were imaged by second harmonic generated polarized light. Cell motility observed by time-lapse imaging: 30 min in 2-min cycles. Three-dimensional time-lapse videos were analyzed using Image J. Tumor cell motility was quantified manually. A cell was scored as motile if the translocation of the cell body was visible over the course of a 30-min video within a visual field that is defined in three dimensions as 50 μm by 512 × 512 pixels. Protrusion was defined as tumors cells showing a dynamic lamellipodia-like morphology. A protrusion was defined to be at least 5 μm long, but less than half the length of the cell. Directionality was calculated as described.58Female NOD/SCID/IL2Rγ-null mice (6–8 weeks old) injected in the mammary fat pad with Ctrl-shRNA or LpdhsRNA2-expressing LM2 cells. Experiments were performed as described previously.post hoc tests (see figure legends), or Student's t-test using Prism 5 (GraphPAD Software). P-values <0.05 considered as significant.Statistical analysis performed by ANOVA with appropriate www.oncomine.org) used for Lpd mRNA expression from microarray data.21 Statistical survival analysis (Kaplan–Meier plots) was done with ROCK (http://www.rock.icr.ac.uk). Expression value of Lpd was upper quartile (25% against rest) for the NKI29524 and Loi22 data sets or upper tertile (33% against rest) for the Miller23 data set. The log-rank P-value was used for statistical significance.Oncomine  with clinicopathological data . Automated immunohistochemistry (VENTANA Discovery UTLRA) of 3 μm TMA sections was performed, deparaffinized  , antigen retrieval in automated slide stainer  . Affinity purified polyclonal rabbit Lpd antibody in PBS (1:250)  was used. Slides counterstained with hematoxylin II and bluing reagent  (4 min each), dehydrated: 1 × 70% IMS, 1 min, 2 × 100% IMS, 1.5 min, 3 × xylene, 1 min, mounted (Eukitt) and imaged .TMAs were performed with 0.6 mmIntensity of Lpd in cytoplasm and membrane assessed on TMAs using weighted histoscore (H-Score) method: intensity in majority of cells assessed as negative and weak (1), or moderate and strong (2), then multiplied by the percentage of cells within this category. The weighted histoscore: 0–200 divided into thirtiles: Cytoplasma: histoscore 1 (0–88.75), histoscore 2 (88.75–170) and histoscore 3 (170–200); Membrane: histoscore 1 (0–25), histoscore 2 (25–95) and histoscore 3 (95–200). TMAs were assessed by two independent observers (UP and CG). Intensity scores that varied by more than a factor of one or a proportion by more than 20% were jointly reassessed and consensus reached. For all other cases, the mean score was used. Disease-specific survival curves generated using the Kaplan–Meier method. The log-rank test was used to compare statistically significant differences between subgroups. Univariate and multivariate analyses Cox proportional hazards regression models used to evaluate overall and breast cancer-specific death by histoscore of Lpd intensity in cytoplasm and at membrane. All analyses were performed using Statistical Analysis Systems (SAS) 9.4 .The study approval was obtained for TMA from NHS Research Ethics Committee, King's Health Partners Cancer Biobank, London, UK. Mice: all animal experiments and husbandry were approved by MIT's Department of Comparative Medicine and Committee on Animal Care. The study approval was obtained for Zebrafish from Northern Stockholm Experimental Animal Ethical Committee.


In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic  Picornaviridae. EV-A71 is a non-enveloped virus with a single stranded, positive-sense RNA genome of approximately 7.4kb. EV-A71 causes mild hand, foot and mouth disease (HFMD), characterized by fever, vesicular lesions on the palms and soles, and oral ulcers. Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with potentially fatal neurological complications such as encephalitis, acute flaccid paralysis and aseptic meningitis, especially in children below 5 years old [Human enterovirus A71 (EV-A71) belongs to the Enterovirus genus within the family of ears old . Since tears old , Singapoears old ,7, Bruneears old  and Chinears old –11, raisears old .The EV-A71 genome comprises a single open reading frame encoding four structural proteins (VP1-VP4) and seven non-structural proteins (2A-2C and 3A-3D), flanked by 5’ and 3’ untranslated regions (UTRs). These structural proteins form an icosahedral structure, with VP1 to VP3 capsid proteins exposed to the surface and responsible for host receptor binding and antigenicity, whereas VP4 is internal .Children between 6 months and 3 years are at highest risk of acquiring EV-71 infection due to lack of protective immunity . NeutralIn the present study, we aimed to characterize human antibody responses against the structural and non-structural proteins of EV-A71 using serum samples from EV-A71-infected children. A total of 63 synthetic peptides  covering the structural and non-structural proteins of EV-A71 were also screened to identify EV-A71-specific IgM and IgG immunogenic epitopes.A total of 43 human serum samples collected from children clinically diagnosed with HFMD during HFMD outbreaks in 2000 and 2012–2013 were used in the present study. All serum samples were confirmed as positive for EV-A71 or non-EV-A71 enteroviruses by virus culture or RT-PCR from throat swabs, vesicle swabs and/or rectal swabs. EV-A71 specific-IgM and -IgG antibodies in sera samples were detected using an EV-A71 IgM-capture enzyme-linked immunosorbent assay  , and wesThe non-HFMD samples used as negative controls consisted of 10 residual serum samples previously tested positive for dengue virus IgM. All non-HFMD samples were confirmed negative for anti-EV-A71 IgM and/or IgG . Sera from 5 healthy adult volunteer donors with EV-A71 neutralization titers ≥1:32 were also used. All sera samples were obtained from the diagnostic virology laboratory, University Malaya Medical Centre in Kuala Lumpur. This study was approved by the Medical Ethics Committee of the University Malaya Medical Centre, Kuala Lumpur, Malaysia (reference number: 932.17) and conducted as per the guidelines of the International Committee on Harmonization of Good Clinical Practice (ICH-GCP) and Declaration of Helsinki. Our institution does not require written informed consent for retrospective studies of HFMD and non-HFMD patients’ serum samples.Human rhabdomyosarcoma  and human embryonic kidney  cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The EV-A71 virus isolate UH1/PM/1997 (GenBank accession number: AM396587) was propagated in RD cells using maintenance medium supplemented with 2% FBS. Virus stock was prepared by harvesting the infected cells, lysing the cells with one round of freeze-thawing and purification by sucrose cushion ultracentrifugation before storage at -80°C. The virus titers were determined by plaque assay.Codon-optimized cDNA clones encoding the entire EV-A71 proteome  based on consensus sequence alignments of different EV-A71 sequences were synthesized . An additional cassette with domain linker, StrepTag II, FLAG Tag and 8X His-Tag functions was designed and synthesized. The synthesized DNAs were subcloned into pEGFP-N1 vector to form pEGFP-N1-cassette-VP1, -VP2, -VP3, -VP4, -3C and -3D expression plasmids. The pEGFP-N1-2B, -2C and -3AB expression plasmids had been previously synthesized by our laboratory. The synthesized plasmid DNA was also subcloned into pET-52b(+) to form a pET-52b(+)-2A expression plasmid. All positive clones were screened by restriction enzyme digestion analysis and confirmed by DNA sequencing.6 cells in Opti-MEM  for 24 hours at 37°C. This was replaced with fresh maintenance medium supplemented with 10% FBS and cells were incubated for a further 24 hours at 37°C.Enhanced green fluorescent protein (EGFP)-expressing recombinant EV-A71 proteins were expressed in HEK293 cells using Lipofectamine LTX reagent . Cells were transiently transfected with the pEGFP-cassette-VP1, -VP2, -VP3, -VP4, -3C and -3D and pEGFP-N1-2B, -2C and -3AB plasmids with a ratio of plasmid DNA to Lipofectamine LTX reagent of 1:3. Cells were transfected with 16 μg of plasmid DNA per 4 × 10Recombinant EV-A71-EGFP proteins were harvested at 48 hours post-infection. Cells were washed with PBS twice and lysed with ice cold lysis buffer (20 mM HEPES (pH 7.5), 280 mM KCl, 1 mM EDTA, 1% Triton X-100) containing protease inhibitors . The recombinant EV-A71-EGFP protein lysate was purified using the μMACS GFP isolation kit  according to the manufacturer’s instructions. Briefly, the protein lysates were incubated with μMACS anti-GFP microbeads on ice for 30 minutes, then washed, and the denatured protein was eluted with 70 μl of elution buffer. The purified proteins were stored at -20°C for western blot analysis.Escherichia coli BL21 (DE3)  competent cells transformed with the pET-52b(+)-2A expression plasmid. Protein expression was induced with isopropyl-beta-D-thiogalactopyranoside  and harvested at 4 hours 30 minutes post-induction. Cells were lysed with denatured lysis buffer , followed by sonication, and cell debris was removed by centrifugation. The protein lysates were incubated with Profinity IMAC Ni-charged resins  at 4°C for 30 minutes, washed twice with denatured washing buffer  and the protein was eluted with 4 ml elution buffer . Purified protein was then concentrated using Amicon Ultra centrifugal filters  and stored at -20°C for western blot analysis.Recombinant EV-A71 proteins were expressed in Proteins from purified EV-A71 virions and protein lysates were mixed with Laemmli buffer and separated by 10% SDS-PAGE. The proteins were transferred onto nitrocellulose membranes, which were blocked with 5% skimmed milk in 0.05% Tween-20 phosphate buffered saline (0.05% PBST) for 1 hour at room temperature. The pooled human serum samples were pre-treated additionally with RIDA RF-Absorbens  or DTT  for IgM- and IgG-specific antibody detection, respectively. The membrane was then incubated with 1:5000 diluted anti-GFP-HRP , 1:300 diluted pooled human serum, 1:100 diluted Light Diagnostics EV-A71 monoclonal antibody 3323  or 1:1000 diluted EV-A71-specific mAb 979  for 1 hour at room temperature, followed by secondary antibody incubation with the 1:5000 diluted HRP-conjugated polyclonal rabbit anti-human IgM , 1:3000 diluted Amersham ECL human IgG, HRP-linked whole Ab from sheep  or 1:5000 diluted HRP-conjugated goat anti-mouse  antibody. The immunoblot was developed with Clarity Western ECL Substrate  and detected by chemiluminescence. The sizes of the protein bands were determined using the Precision Plus Protein WesternC Standard .Polystyrene 96-well Maxisorp Nunc-immuno plates  were coated with 10 μg/ml of purified EV-A71 virions. Wells were blocked with 3% bovine serum albumin (BSA) diluted in 0.05% PBST, and incubated at 37°C for 1 hour. Pooled human sera were pre-treated with RIDA RF-Absorbens to obtain IgM specific antibody. The pooled human sera were then diluted at 1:100 to 1:8000 in 1% BSA-0.05% PBST and incubated for 1 hour at 37°C, followed by 1 hour incubation with the 1:5000 diluted HRP-conjugated polyclonal rabbit anti-human IgM , 1:5000 diluted HRP-conjugated polyclonal rabbit anti-human IgG  or 1:500 diluted HRP-conjugated isotype IgG1 , IgG2 , IgG3 , and IgG4 antibodies . TMB microwell peroxidase substrate  was added and the reactions terminated with 1M phosphoric acid. Absorbance was measured at 450 nm with a reference filter of 630 nm. The optical densities (OD) were plotted as means ± standard deviation (SD).A total of 63 biotinylated peptides consisting of 15-mer peptides  were generated from consensus alignment of different EV-A71 amino acid sequences. The synthetic biotinylated peptides were either potential EV-A71 antibody epitopes predicted by the Emini surface accessibility scale  or publiThe crystal structure of EV-A71 was retrieved from Protein Data Bank (PDB) (identifier 3VBS) and visualized using the UCSF Chimera software version 1.10.1 . For seqp value of <0.05 was considered as statistically significant.All graphs were plotted and statistical analyses were performed using GraphPad Prism version 5.0 . One-way ANOVA with the Kruskal-Wallis test was used to compare optical density readings of serum samples tested for EV-A71-specific IgM and IgG antibodies by ELISA. A To define the antigenic recognition profiles of the antibodies from EV-A71-infected patient samples, individual expression plasmids encoding each structural (VP1 to VP4) and non-structural (2A to 3D) gene of the EV-A71 proteins tagged with EGFP were generated. After transient transfection in HEK293 cells, all recombinant EV-A71 EGFP proteins were successfully expressed, as shown by immunoblot analysis using antibodies against GFP  . EV-A71 Using cell lysates from different EV-A71 proteins, we found that EV-A71-specific IgM antibody from both groups of serum  were selected for further analysis. Results were expressed as the percent contribution of each epitope towards total antibody recognition to the positive peptides used in this study. A total of 22 EV-A71-reactive IgM dominant epitopes were identified and the average percentage of peptide recognition by the sera was very similar (range 3.5% to 6.2%) . Eight EOf the 22 EV-A71-reactive IgM dominant linear epitopes, 13 peptides were IgM-specific epitopes. These peptide sequences were subsequently aligned to the corresponding sequences from 12 different enteroviruses . Epitopes with conserved sequences were removed, and the remaining five epitopes , for which EV-A71 sequences varied from other enteroviruses , were seTo further validate the specificity and versatility of the selected IgM epitopes as suitable early detection targets, serum samples from patients infected with EV-A71 (n = 22) and non-HFMD children (n = 10) were screened. All IgM linear epitopes showed good responses for EV-A71-infected patient sera, particularly for PEP27, which had the highest mean OD values of 0.85±0.47 . The OD For the EV-A71-specific IgG epitopes, PEP23 was selected for individual serum testing, as it is the dominant epitope that was commonly recognized by the three sets of sera. All individual serum samples selected were positive for EV-A71-specific IgG. No statistical differences were observed, as shown in Epitope-containing sequences were next mapped onto the available three-dimensional crystal structures of the VP1, VP2 and VP3 proteins (PDB identifier: 3VBS). For the EV-A71-specific IgM epitopes, PEP12 is located in the solvent-exposed region of VP2, whereas PEP19 is partially embedded in the VP3 protein . SimilarUnderstanding the immune responses against EV-A71 is important for the development of diagnostic tools and potential subunit vaccine candidates. In the present study, the first comprehensive analysis of antibody responses against structural and non-structural proteins of EV-A71 was performed. Using EV-A71-infected patient serum samples from HFMD outbreaks in Malaysia , all virin vitro [The different roles of human IgG subclasses in neutralization and antibody-dependent enhancement are well documented in dengue and West Nile virus studies ,42. In Ein vitro . Interes+ T-cell epitopes SP2 and VP1-20 [IgM detection is important for early diagnosis of infectious diseases. A total of 63 biotinylated peptides were synthesized to determine EV-A71-reactive IgM linear epitopes using peptide-based ELISAs. Linear epitopes instead of discontinuous epitopes were the focus since they are more easily identifiable in a medium-throughput approach . Interesd VP1-20 ,47, suggd VP1-20 , suggestDifferent human IgG epitopes were recognized by patient serum samples from acute infection with high neutralization and convalescence. Using EV-A71-infected patient sera from acute infection with high neutralization, we observed that PEP47 and PEP62, located at non-structural proteins 3C and 3D, respectively, were identified as dominant anti-EV-A71 IgG linear epitopes. Convalescent sera showed similar EV-A71-reactive IgG epitope profiles as adult sera, as both sera strongly recognized PEP23 as the dominant IgG linear epitope, and adult sera also recognized PEP30 as an additional dominant linear epitope. Surprisingly, all the previously reported anti-EV-A71 IgG epitopes identified in rabbit and mouse sera (in our panel of 63 peptides) were not recognized by EV-A71-infected patient sera, except PEP20, which mapped to a similar location as the reported VP1-01 epitope . The synInterestingly, PEP23 mapped to VP1 41–55 was recognized by all anti-EV-A71 IgG antibodies from patient and adult sera. This PEP23 was the previously reported human IgG epitope VP1-15 . AlthougIn summary, PEP27 mapped at VP1 142–156 was identified as an EV-A71 IgM-specific immunodominant epitope, and is a candidate for use in a diagnostic IgM assay that differentiates EV-A71 from other causes of HFMD. This would be a valuable tool for early public health responses. PEP23 mapped to VP1 41–55 was identified as an IgG cross-reactive immunodominant epitope. Overall, the structural protein VP1 is the main immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies. In addition, epitopes against non-structural proteins were also detected. This study has provided useful insights into human immune responses to different EV-A71 epitopes at different stages of infection.S1 FigThe distribution of the peptides is shown in the (A) EV-A71 whole genome, and (B) in each EV-A71 gene.(TIF)Click here for additional data file.S2 Fig(A) EV-A71-specific IgM antibody titers and (B) EV-A71-specific IgG antibody titers were determined by virion-based ELISAs. EV-A71-infected patient pooled sera were assayed by serial dilution and subjected to virion-based ELISA. (C) EV-A71-specific IgG isotype titers in pooled sera were determined at dilutions of 1:100 using specific secondary antibodies. Non-HFMD children sera were used as negative controls. Data are presented as mean ± SD of 3 replicates.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Alcohol abuse is among the top causes of preventable death, generating considerable financial, health, and societal burdens. Paradoxically, alcohol... HSF-1 played in determining a stimulatory phenotype of Caenorhabditis elegans in response to physiologically relevant concentrations of ethanol . Using genetic techniques, we demonstrate that either RNA interference of hsf-1 or use of an hsf-1(sy441) mutant lacked the enhancement of locomotion in response to acute ethanol exposure evident in wild-type animals. We identify that the requirement for HSF-1 in this phenotype was IL2 neuron-specific and required the downstream expression of the α-crystallin ortholog HSP-16.48. Using a combination of pharmacology, optogenetics, and phenotypic analyses we determine that ethanol activates a Gαs-cAMP-protein kinase A signaling pathway in IL2 neurons to stimulate nematode locomotion. We further implicate the phosphorylation of a specific serine residue (Ser322) on the synaptic protein UNC-18 as an end point for the Gαs-dependent signaling pathway. These findings establish and characterize a distinct neurosensory cell signaling pathway that determines the stimulatory action of ethanol and identifies HSP-16.48 and HSF-1 as novel regulators of this pathway.Alcohol is a potent pharmacological agent when consumed acutely at sufficient quantities and repeated overuse can lead to addiction and deleterious effects on health. Alcohol is thought to modulate neuronal function through low-affinity interactions with proteins, in particular with membrane channels and receptors. Paradoxically, alcohol acts as both a stimulant and a sedative. The exact molecular mechanisms for the acute effects of ethanol on neurons, as either a stimulant or a sedative, however remain unclear. We investigated the role that the heat shock transcription factor  Alcohol is one of the most prevalent addictive substances and its overuse produces a severe burden on society . Acute eDrosophila, studies of acute ethanol intoxication have implicated a large number of proteins and signaling pathways  whose absolute values would be physiologically consistent with blood alcohol limits for impaired driving  or loss-of-function mutations of hsf-1 increase stress sensitivity, but also accelerate ageing  signaling pathway within the IL2 sensory neurons and identifies the exocytotic protein UNC-18 as a downstream effector for PKA. Although individual components of the Gαs pathway have been linked previously to the neuronal effects of ethanol, this study uniquely characterizes the entire signaling pathway in ethanol-dependent stimulation.Heat shock activates a transcriptional response to toxic insults whereby protective cellular chaperone (heat shock protein) expression is increased under the control of the heat shock transcription factor (HSF1) . The HSFC. elegans were cultured under standard conditions at 20° on Nematode Growth Media (NGM) agar plates with OP50Escherichia coli as a food source as previously described (N2 (wild-type), PS3551hsf-1(sy441), KG524gsa-1(ce94), KG421gsa-1(ce81), MT363goa-1(n363), NM1380egl-30(js126), KG1180lite-1(ce314), and NL2099rrf-3(pk1426). To investigate effects of single-copy transgenic rescue of hsf-1(sy441) mutants, we analyzed the OG532 (hsf-1(sy441);drSi13[hsf-1p::hsf-1::GFP::Cbr-unc-119(+)]unc-54 3′UTR + ) and OG580 (hsf-1(sy441);drSi28[hsf-1p::hsf-1(R145A)::GFP::Cbr-unc-119(+)]unc-54 3′UTR + ) strains (hsf-1(sy441) mutant. Basal locomotion rates for each strain can be found in Supplemental Material, Table S1 in File S1. Transgenic animals were derived by germline injection as previously described ;ulvEx[Phsf-1::hsf-1], hsf-1(sy441);ulvEx[Prab-3::hsf-1], hsf-1(sy441);ulvEx[Pmyo-3::hsf-1], hsf-1(sy441);ulvEx[Pglr-1::hsf-1], hsf-1(sy441);ulvEx[Punc-17::hsf-1], hsf-1(sy441);ulvEx[Posm-6::hsf-1], hsf-1(sy441);ulvEx[Pgcy-8::hsf-1], hsf-1(sy441);ulvEx[Pklp-6::hsf-1], hsf-1(sy441);ulvEx[Prab-3::hsp-16.48], hsf-1(sy441);ulvEx[Prab-3::hsp-16.48 Δ38-44], and hsf-1(sy441);ulvEx[Pklp-6::hsp-16.48]. Transgenic constructs in Bristol N2 were coexpressed with either a sur-5P::GFP or a rab-3P::GFP marker. To indicate appropriate cellular expression for additional cell–tissue-specific promoters, each of these transgenics used a GFP coexpression marker under the control of the same promoter. IL2 neurons were visualized with the promoter::GFP reporter hsf-1(sy441);ulvEx. To examine cell-specific RNAi, Bristol N2 worms were injected with the plasmid klp-6P::kin-1::klp-6P, which drove cell-specific expression of kin-1  in both the forward and reverse direction  strain. RNAi was induced by feeding  or cDNA (hsf-1), cloned into pDONR201 and recombined into DEST vectors to create tissue-specific expression vectors, as previously described  as per the manufacturer’s instructions. The unc-18 S322A expression vector was created using the Gene Tailor mutagenesis kit  from a vector carrying the unc-18 cDNA under the control of the unc-18 genomic flanking regions  . For glutathione-S-transferase (GST) fusion protein production, unc-18 was subcloned into pGEX-6p-1 as previously described  in place of Pmec-7. These vectors were converted into Gateway DEST vectors using a conversion cassette (Life Technologies). rab-3P , myo-3P , and hsf-1P are previously described . The klp-6P::kin-1 PCR fragment was then amplified from this construct and again subcloned into the same Pklp-6 construct as before, but in reverse orientation. Correct construction was confirmed by PCR and sequencing. Primers used were as follows:The kin-1 RNAi forward: 5ʹ-CTATCGATTCGCGGCCATCACAAGTTCGAATCGGA-3ʹ.kin-1 RNAi reverse (step 1): 5ʹ-CTTGTGGGCTTTTGTATAGTTCGTCCGGATAAC-3ʹ.kin-1 RNAi reverse (step 2): 5ʹ-CTATCGATTCGCGGCCATCACAAGTTGGATAACTAC-3ʹ.in vitro biochemistry, recombinant proteins  were produced as described previously  in a 50 μl final reaction volume of 2-(N-morpholino)ethanesulfonic acid (MES) buffer . Reactions were incubated at 30° for 3 hr before termination. To determine phosphorylation, 20 μl of the kinase reaction was separated by SDS-PAGE, stained with Coomassie Blue dye, destained overnight in a destainer , air-dried in Hoeffer Easy Breeze plastic frames (Thermo Fisher Scientific), exposed to a phosphor screen for 2–4 hr, and scanned by a PhosphorImager 425 (Molecular Dynamics). For in vitro phosphorylation site determination, phosphorylated samples were separated on NuPAGE 4–12% Bis-Tris precast gels (Invitrogen) and stained with Coomassie Blue dye before excision of protein bands. Gel plugs were destained in 50% acetonitrile (v/v)/50 mM ammonium bicarbonate and dried before incubation in trypsin (5 ng/μl in 50 mM ammonium bicarbonate) for 16 hr at 37°. Peptides were then extracted by sonication of gel plugs in 60% (v/v) acetonitrile/1% (v/v) trifluoroacetic acid. Extracts were thoroughly dried and ZipTipped  before electrospray ionization mass spectrometry (MS). Residual peptides were resuspended in 50% (v/v) acetonitrile/0.05% trifluoroacetic acid, and 5 μl of suspension was delivered into a QStar Pulsar I hybrid quadrupole time-of-flight MS (AB Sciex) by automated in-line liquid chromatography . A gradient from 5 to 48% acetonitrile/0.05% trifluoroacetic acid (v/v) in 60 min was applied at a 300 nl/min flow rate and survey scans of 1 sec were acquired for m/z 400–2000. The most intense ions were selected for tandem MS with 2 sec accumulation times and a dynamic exclusion of 30 sec. Identification and analysis was performed using MASCOT software (Matrix Science).For klp-6P in pPD117.01 (detailed above) via Gibson Cloning techniques (New England Biolabs). The optogenetic vectors were then transformed by germline injection into C. elegans as described above. The vectors were expressed in the lite-1(ce314) background to prevent any potential photophobic responses . Thrashing assays were performed under the dim red light in Dent’s solution , in the presence or absence of 17 mM ethanol, using a Leica MZ10F-stereomicroscope  equipped with a pE-300 light-emitting diode (LED) fluorescence light source . To activate opsin, worms were illuminated with a single flash of a 100% power green LED through the ET GFP filter set for 5 sec. For hRh1, opsin activation occurred as soon as they were placed in the Dent’s solution and thrashing was quantified after 10 min acclimation (as below). For JellyOp, worms were illuminated following the 10 min acclimation in Dent’s solution just prior to thrashing quantification, as the JellyOp activation is more transient  and a negative control (distilled water) were pipetted on opposite sides of the plate. The number of worms on either side of the plate was calculated following 90 min. The chemotaxis index (C.I.) was calculated as C.I. = (# worms at attractant − # worms at control)/total number of worms. For worm avoidance assays, a 0.9-cm ring of a substance was made by a stamping procedure on unseeded NGM plates. Immediately after stamping, 30 worms were placed within the center of the ring, completed within 2 min. Following 10 min exposure, the number of worms that had crossed the ring was counted. In preliminary experiments, 0.9 cm was found to be sufficient to permit most worms to cross a neutral substance (distilled water) within 10 min. All data are expressed as mean ± SE. As indicated, significance was tested by Student’s t-test, Mann–Whitney U-test, or ANOVA with Tukey post hoc comparisons where appropriate.Phenotypic analysis was performed in a temperature-controlled room on young adult hermaphrodites from sparsely-populated plates grown at 20°. Unless otherwise indicated, experiments were conducted at an ambient temperature of 20°. Here, locomotion rate was measured as thrashing  allele is a viable loss-of-function point mutation in the hsf-1 gene that acts as an inhibitor of HSF-1 transcriptional activity  mutation would also hypersensitize worms to the effects of low concentrations of ethanol. Surprisingly, we found that the stimulatory effect of 17 mM external ethanol was completely absent in worms containing the hsf-1(sy441) mutation (hsf-1(sy441) mutant is well established to have a temperature-sensitive phenotype and we verified that the low-dose ethanol phenotype was not affected by the ambient temperature  in the hypersensitivity to 400 mM external ethanol is dependent upon pan-neuronal hsf-1 expression (hsf-1(sy441) mutant was also pan-neuronal in nature by tissue-specific transgenic rescue. To allow for a direct comparison of the stimulatory effects of ethanol independent of basal locomotor rates, the data are presented as thrashing rate normalized to untreated worms of the same strain as done in previous studies  under its own promoter (hsf-1P::hsf-1) or specifically under the control of a pan-neuronal promoter (rab-3P::hsf-1) completely rescued the stimulatory ethanol phenotype (hsf-1(sy441) using a body wall muscle promoter (myo-3P::hsf-1) was insufficient to rescue. As a complementary approach to investigate hsf-1 function in the ethanol phenotype, we cloned and overexpressed hsb-1 in Bristol N2. hsb-1 is a negative regulator for hsf-1 transcriptional function and its overexpression in C. elegans results in similar phenotypes to loss-of-function of hsf-1  also resulted in a loss of ethanol stimulation of locomotion similar to that seen in the hsf-1(sy441) mutant (hsf-1 function.henotype . However) mutant . From thrab-3 promoter drives expression throughout the nervous system, we next determined whether the stimulatory ethanol phenotype could be localized more precisely in the nervous system. Intriguingly, the 17-mM ethanol stimulatory phenotype of hsf-1(sy441) worms could be reestablished by transgenic expression of wild-type hsf-1 specifically in cholinergic neurons (unc-17P::hsf-1) or in ciliated sensory neurons (osm-6P::hsf-1), but not in interneurons (glr-1P::hsf-1) or the AFD thermosensory neurons (gcy-8P::hsf-1)  could be reversed by transgenic expression of wild-type hsf-1 specifically in the IL2 neurons. For this, we cloned the promoter region of the klp-6 gene, which in hermaphrodites is expressed specifically in the IL2 neurons  mutant (hsf-1 in the IL2 neurons (klp-6P::hsf-1) alone could restore the stimulatory ethanol phenotype of the hsf-1(sy441) mutant to a level indistinguishable from wild-type worms (IL2-specific overexpression of hsb-1 (klp-6P::hsb-1), the negative regulator for hsf-1, was able to block the ethanol-induced stimulation in Bristol N2 (As the ::hsf-1) . The loc-1)  mutant , althougpe worms . The comistol N2 .hsf-1(sy441) worms have a slightly lower basal locomotor rate (File S1). For example, IL2 neuron overexpression of hsb-1 in Bristol N2 does not reduce basal locomotion rate at all, but does block the low-ethanol phenotype. Alternatively, overexpression of wild-type hsf-1 in IL2 neurons in the hsf-1(sy441) mutant background does not rescue the locomotor defect of the mutant, but did restore the low-ethanol phenotype. Notwithstanding this lack of correlation, we were interested in determining whether the reduced locomotor rate in the hsf-1(sy441) mutant was a consequence of the point mutation. The hsf-1(sy441) strain was originally isolated in a genetic screen of suppressors of heat shock-induced expression, where it was both backcrossed and outcrossed (File S1) and elsewhere  . The incomplete rescue of the locomotion defect was reminiscent of a reported incomplete rescue of the hsf-1(sy441) temperature-sensitivity  to a level similar to that seen with transgenic overexpression. In contrast, the OG580 strain, a single-copy rescue of hsf-1(sy441) with a DNA-binding defective mutant hsf-1 (File S1) and did not rescue the ethanol phenotype . These data indicate a potentially complex role for hsf-1 in basal locomotor rate, which is perhaps unsurprising given the broad cellular functions of the HSF-1 transcription factor and its downstream transcriptional targets. Critically, however, our results show that the basal locomotor rate is unrelated to the ethanol stimulatory effect and is instead a consequence of the hsf-1(sy441) mutation.tor rate ; however1(sy441) . The OG5sitivity . Reassurnt hsf-1 , impaireHSF-1 and its effect on sensitivity to 400 mM external ethanol indicated that the main effector for HSF-1 was the small heat shock protein HSP-16.48  mutants. Finally, overexpression of hsp-16.48 greatly reduced wild-type worm sensitivity to high external ethanol in a manner dependent upon an intact seven amino acid region of the N-terminus (amino acids 38–44)  or in IL2 neurons specifically (klp-6P::hsp-16.48) was sufficient to restore completely the stimulatory ethanol phenotype in the hsf-1(sy441) mutant (hsp-16.48 in Bristol N2 wild-types (rab-3P::hsp-16.48) increased the effect of 17 mM external ethanol, whereas the N-terminal truncation mutant (rab-3P::hsp-16.48 Δ38-44) appeared to act as neomorphic or as dominant-negative (hsf-1(sy441) background did not result in an enhancement of the ethanol-dependent reduction in locomotion in comparison to hsf-1(sy441) alone  mutant . Finallynegative . However1) alone . TherefoHSF-1/HSP-16.48 was acting in a chemosensory neuron to increase locomotion, we were interested to determine whether this increase in motility was reflective of either positive or negative chemotaxis. Indeed, the performed thrashing assays immersed the animals directly into ethanol where it would be impossible to determine any directionality to movement. We tested for this possibility by a performing a chemotaxis assay on wild-type animals toward or away from ethanol. Ethanol has been previously reported to have mild to negative effects on chemotaxis only at very high concentrations  as has been previously described . In contrast, worms were averse to octanol at concentrations as low as 1%.As IL2 chemosensory neurons to stimulate nematode motility through a G-protein-coupled receptor (GPCR)-dependent signaling mechanism. Physiological effects of ethanol are known to be modulated by various GPCRs, for example the GABAB or G-protein-coupled corticotropin-releasing factor (CRH) 1 receptors  and Gαq (egl-30) mutants, but was absent specifically for mutants of Gαs, gsa-1(ce81) and gsa-1(ce94)  .αs is a G-protein that, upon activation, stimulates adenylyl cyclase to synthesize the cAMP signaling molecule from ATP  mutants, but was completely restored in both the full (hsf-1P::hsf-1) or IL2 neuron-specific transgenic rescues (klp-6P::hsf-1)  or in IL2 neurons specifically (klp-6P::hsb-1) in Bristol N2 worms. As seen with ethanol, the effect of forskolin was restored in the OG532 single-copy rescue of hsf-1(sy441), but not in the OG580 DNA-binding defective rescue . The forskolin effect was also blocked in the Gαs gain-of-function mutants gsa-1(ce81) and gsa-1(ce94) (rab-3P::hsp-16.48) or IL2-specific (klp-6P::hsp-16.48) overexpression of hsp-16.48 alone could completely rescue the forskolin phenotype of the hsf-1(sy441) mutation (hsp-16.48 in Bristol N2 controls (rab-3P::hsp-16.48) enhanced the effect of forskolin, whereas the N-terminal truncation mutant (rab-3P::hsp-16.48 Δ38-44) appeared to act negatively (hsf-1(sy441) background  mutant.Next, as previously shown for the stimulatory effects of ethanol, we verified that stimulation by forskolin was also dependent upon ::hsf-1) . Complem-1(ce94) . Furthermutation . Also idgatively . The truckground . Taken tαs in IL2 neurons by 17 mM external ethanol; however, our evidence was obtained by pharmacological activation of the entire nematode. We next wanted to activate Gαs in the IL2 neurons directly. To accomplish this, we genetically expressed a photoactivatable Gαs-linked jellyfish opsin, JellyOp  . The created transgenic worms appeared qualitatively normal and had only a small quantitative reduction in basal locomotion rate (Table S1 in File S1). While we cannot quantify definitive cell specificity of the RNAi, if the PKA knockdown had spread greatly to other cells it would be expected to be lethal . These data support the interpretation that the effects of ethanol to stimulate C. elegans locomotion are regulated in IL2 neurons by a Gαs signaling pathway.The data thus far pointed to an activation of Gexposure . In cont pathway . Next, wn (hRh1) , which a ethanol . As an aαs-cAMP-PKA signaling pathway. PKA is a serine/threonine kinase that would be expected to phosphorylate many downstream targets in response to activation. Sec1-Munc18 (SM) proteins are essential components of the exocytotic machinery thought to function primarily through interactions with soluble N-ethylmaleimide-sensitive factor receptor proteins (C. elegans SM protein UNC-18 (D214N) was also able to block the low-ethanol phenotype , which act in a dominant fashion to endogenous unc-18  or in IL2 neurons specifically (klp-6P::unc-18 S322A) was able to block completely the stimulatory effects on motility of either ethanol or forskolin  whose absolute values are physiologically consistent with blood alcohol limits for impaired driving. Through a combination of genetics, pharmacology, and optogenetic manipulation, we have uniquely delineated the entire cell signaling pathway in response to ethanol specifically in IL2 chemosensory neurons  and not sensory neurons such as the IL2 neurons. Our data use combinations of optogenetic, genetic, and pharmacological manipulation to trace the involvement of the entire Gαs -cAMP-PKA signaling pathway in the IL2 neurons in ethanol-dependent stimulation. Additionally, we extend this pathway to the downstream phosphorylation of the exocytotic protein UNC-18 and implicate HSP-16.48 as a novel regulatory protein.Our data support the interpretation that a Gin vitro. Phosphorylation of Munc18 has been demonstrated in vivo, primarily by Protein Kinase C  mutant phenotype (hsp-16.48 can completely restore the stimulatory phenotype for the hsf-1(sy441) mutant. Although there is little constitutive HSP-16.48 expressed, even in response to 400 mM ethanol, our RNAi experiments show that these small quantities are sufficient to affect whole-animal behavior. Small HSPs can be directly phosphorylated by PKA (HSP-16.48 regulates the Gαs -cAMP-PKA signaling pathway. It may be that HSP-16.48 is acting simply as a chaperone to preserve either structural conformation of individual proteins, protein–protein interactions, or possibly to assist in the phosphorylation event itself. Alternatively, HSP-16.48 may directly interact with specific components of the signaling pathway independent of its chaperone ability. Nonetheless, the correct functioning of HSP-16.48 in ethanol-dependent phenotypes explicitly required a seven amino acid region of the protein’s N-terminus, a region not found in other small HSPs linked to temperature stress tolerance (Our data demonstrate that the α-crystallin ortholog C. elegans on agar (Our experiments have used thrashing as a measure of locomotion (IL2 neurons, with dendritic projections directly to the exterior of the body (C. elegans in response to ethanol at levels that would be seen in intoxicated humans. The stimulation of locomotion has been reproducibly demonstrated in C. elegans (Drosophila (C. elegans increase their movement in response to 17 mM ethanol? The ecology of the species is not fully understood; however, some evidence points to C. elegans feeding on bacteria associated with decomposing material. However, unlike longer chain alcohols, evidence for ethanol acting as a chemotactic cue signaling a potential nutrient source is not well supported. Although we were unable to reproduce the results, some data point to ethanol acting as a low-level chemorepellent (The internal concentration of ethanol in nematodes is reported to be ∼10% of external experimental levels due to poor penetration through the cuticle (HSP-16.48 and both the sedative (HSP-16.48 expression biphasically both enhances ethanol-induced stimulation and decreases ethanol-induced sedation. Importantly, the expression of the α-crystallin chaperone is upregulated in mice strains with a high alcohol intake preference (UNC-18, therefore presents novel targets for future pharmacological intervention that could be exploited to control the devastating physiological effects of alcohol.Alcohol acts paradoxically as both a depressant and as a stimulant. These stimulatory effects in humans are thought to be more rewarding than sedative effects and thus may play a more prominent role in determining addiction. The differentiator model for risk to alcoholism is associated with a trade-off between stimulant and sedative effects (www.genetics.org/lookup/suppl/doi:10.1534/genetics.117.300119/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


The aim of this study is to investigate the role of Galectin-3 in human thyroid cancer migration.The expression of Galectin-3 in surgical specimens was investigated using immunohistochemistry and western blot. A papillary thyroid cancer cell line (B-cpap) and an anaplastic thyroid cancer cell line (8305c) were transfected with short-hairpin RNA against Galectin-3 . Low-molecular citrus pectin (LCP) was also used to antagonize Galectin-3. The migration and invasion of the cell lines were examined. The related signaling pathways were investigated to explore the Galectin-3 mechanism of action.Galectin-3 was highly expressed in metastasized thyroid cancers. Knocking down and antagonizing Galectin-3 significantly suppressed the migration of thyroid cancer cells. Knocking down Galectin-3 inhibited the activity of Wnt, MAPK, Src and Rho signaling pathways. Galectin-3 was up-regulated via HIF-1α in a hypoxic environment. Galectin-3 knockdown could reduce cell motility in hypoxic environments.This study suggests that Galectin-3 could act as a modulator of thyroid cancer migration, especially in hypoxic microenvironments. This regulation function of Galectin-3 may work through multiple signaling pathways. Thyroid cancer is one of the most common carcinomas in human beings, with its incidence growing rapidly over the last 20 years. Among the emerging cases, most are papillary cancers . PapillaGalectins are a family of carbohydrate-binding proteins which have high affinity and specificity for β-galactoside . Among GFor thyroid cancers, detection of Galectin-3 expression has been widely used in Fine Needle Aspiration Cytology (FNAC) to help distinguish benign and malignant thyroid nodules , 14. OveIn this study, we used surgical specimens and two different thyroid cancer cell lines to investigate how Galectin-3 was associated with thyroid cancer cell migration. The potential signaling pathways through which Galectin-3 regulating migration were also explored. Our results mainly revealed that Galectin-3 regulated thyroid cancer cell migration in normoxic and hypoxic microenvironments. The signaling pathways involved included Wnt, MAPK, Src and Rho.To determine the expression characteristics of Galectin-3 in different thyroid tissues, the protein levels of Galectin-3 in surgical specimens from 14 papillary thyroid cancer patients were analyzed by western blot. 5 of the 14 patients were diagnosed with metastasized lymph nodes. Galectin-3 was highly expressed in papillary tumor tissues compared to its adjacent noncancerous thyroid tissues. Furthermore, all metastases in lymph nodes also showed high Galectin-3 expression Figure .p < 0.05). Through investigating these slides carefully, different distributions of Galectin-3 could be defined. On Figure To further confirm the expression patterns of Galectin-3 in thyroid cancer, we conducted immunohistochemistry in 20 surgical specimens, including 19 pathology-confirmed papillary thyroid cancer samples and 1 anaplastic cancer sample. The related information is listed in Table Based on our findings in immunohistochemistry and western blots of surgical samples, we first supposed that Galectin-3 played a role in the migration and invasion of thyroid cancers. Papillary thyroid cancer cell line B-cpap and anaplastic thyroid cancer cell line 8305c were chosen to knock down Galectin-3 Figure . Wound hp < 0.05; average diameter (μm) per sphere: 32.6 vs 25.7, p < 0.05)  Figure . Accordi) Figure . AltogetTo explore the mechanisms of Galectin-3 regulating the migration and invasion of thyroid cancer cells, we further investigated the effect of Galectin-3 knockdown on several signaling pathways related to cell migration. Since MAPK/ERK signaling is the most commonly studied signaling pathway in thyroid cancers , we examIt has been proven that Src kinase-dependent caveolin-1 (Cav1) phosphorylation increased tumor cell migration in a Galectin-3-dependent manner . The eff2 (100μM) to the culture medium. After 36 hours, Galectin-3 expression was markedly increased  were suppressed after Galectin-3 knockdown. Caveolin-1 (Cav-1) is a multifunctional protein with multiple binding partners that is associated with cell surface caveolae and the regulation of lipid raft domains . The dilution ratios of the primary antibodies were as follows: Galecin-3(mouse), 1: 1000; p44/42 MAPK, 1:1000; p-p44/42 MAPK, 1:2000; Src, 1:1000; p-Src, 1:1000; FAK, 1:1000; p-FAK-TyrCell proliferation was detected using Cell Counting Kit8 (Dojindo) according to the manufacturer's instructions. Briefly, 8305c and B-cpap cells were plated in 96-well plates at a density of 3,000 cells per replicate. Reagent was added at the same time for the following days. After incubation for 2 hours, the absorbance of each well was determined at 450 nm using a Gen5 system (BioTek).For the wound healing assays, cells were cultured in 6-well plates. When 100% confluence was reached, the cell monolayers were wounded with a pipette tip. Then the cells were washed by PBS and photographed. Five fields of view were chosen in each plate, and all assays were performed in duplicate.4/ml. 200 μl of cell suspension was added into the upper chamber, and 600 μl of culture medium with 10% FBS was added into the lower chamber. After 20 h, the cells on the upper side of the membrane were removed with cotton swabs, and the cells that migrated to the lower surface were fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min. The cells were counted in five fields of view in duplicate chambers.Matrigel was diluted in serum-free culture medium at a ratio of 1:8 and tiled on the upper chambers of a 24-well transwell plate . Once the Matrigel solidified, the cells were trypsinized and suspended in serum-free medium to a final concentration of 5×10The sphere formation assays were carried out in 96-well plates. Briefly, an average of 300 cells were plated in each well. The culture medium was Dulbecco's modified Eagle's and F12 media supplemented with B27 (Invitrogen), 2 μg/ml heparin (Sigma), 20 ng/ml EGF (Chemicon) and 20 ng/ml FGF-2 (Chemicon). The total number of formed spheres and their average diameters were counted after 14 days.397 (CST8556), rabbit anti-p44/42 MAPK (CST4695), rabbit anti-p-p44/42 MAPK Thr202/Tyr204 (CST4370), rabbit anti-β-catenin (CST8480), rabbit anti-GSK-3β (CST9315), rabbit anti-p-GSK-3β Ser9 (CST9336), rabbit anti- Src (CST2108) and rabbit anti-p-SrcFamily Tyr416 antibodies (CST2101) were obtained from Cell Signaling Technology. The rabbit anti-Oct3/4 (Sc9801), mouse anti-Cav-1 (sc53564) antibodies were obtained from Santa Cruz Biotechnology. The rabbit anti-GLUT1 (PA5-16793) antibody was obtained from Thermo Fisher. Rho activation assay kit (17-294) and PP2 were obtained from Merck Millipore.The mouse anti-Galectin-3 (BD556904) antibody was obtained from BD Biosciences. The rabbit anti-Galectin-3 (Ab31707) antibody was obtained from abcam. The rabbit anti-RhoA (CST2117), rabbit anti-RhoC (CST3430), rabbit anti-FAK (CST3285), rabbit anti-pFAK-Tyrp values < 0.05 were considered statistically significant.Statistical analysis was performed using SPSS 21. Comparisons of categorical data were carried out by unpaired two-tailed Student's t test. Data are presented as the mean ± S.D.


Caenorhabditis elegans embryonic cells by ectopically expressing HLH-1 in all cells of the embryo and monitoring chaperone expression. We found that HLH-1-dependent myogenic conversion specifically induced the expression of putative HLH-1-regulated chaperones in differentiating muscle cells. Moreover, disrupting the putative HLH-1-binding sites on ubiquitously expressed daf-21(Hsp90) and muscle-enriched hsp-12.2(sHsp) promoters abolished their myogenic-dependent expression. Disrupting HLH-1 function in muscle cells reduced the expression of putative HLH-1-regulated chaperones and compromised muscle proteostasis during and after embryogenesis. In turn, we found that modulating the expression of muscle chaperones disrupted the folding and assembly of muscle proteins and thus, myogenesis. Moreover, muscle-specific over-expression of the DNAJB6 homolog DNJ-24, a limb-girdle muscular dystrophy-associated chaperone, disrupted the muscle chaperone network and exposed synthetic motility defects. We propose that cellular differentiation could establish a proteostasis network dedicated to the folding and maintenance of the muscle proteome. Such cell-specific proteostasis networks can explain the selective vulnerability that many diseases of protein misfolding exhibit even when the misfolded protein is ubiquitously expressed.Safeguarding the proteome is central to the health of the cell. In multi-cellular organisms, the composition of the proteome, and by extension, protein-folding requirements, varies between cells. In agreement, chaperone network composition differs between tissues. Here, we ask how chaperone expression is regulated in a cell type-specific manner and whether cellular differentiation affects chaperone expression. Our bioinformatics analyses show that the myogenic transcription factor HLH-1 (MyoD) can bind to the promoters of chaperone genes expressed or required for the folding of muscle proteins. To test this experimentally, we employed HLH-1 myogenic potential to genetically modulate cellular differentiation of  Caenorhabditis elegans. We demonstrated that HLH-1 regulates the expression of muscle chaperones during muscle differentiation. Moreover, we showed that HLH-1-dependent expression of chaperones is required for embryonic development and muscle function. We propose that cellular differentiation results in cell-specific differences in the chaperone network that may be detrimental in terms of the susceptibility of neurons and muscle cells to protein misfolding diseases.Molecular chaperones protect proteins from misfolding and aggregation. In multi-cellular organisms, the composition and expression levels of chaperones vary between tissues. However, little is known of how such differential expression is regulated. We hypothesized that the cellular differentiation that regulates the cell-type specific expression program may be involved in establishing a cell-type specific chaperone network. To test this possibility, we addressed the myogenic commitment transcription factor HLH-1 (CeMyoD) that converts embryonic cells to muscle cells in  In. Inhlh-1os >60%, . This ph control . Thus, eQ35;hlh-1(cc561) young adults for muscle function and myosin organization. Although we excluded deformed or paralyzed animals, motility of Q35;hlh-1(cc561) young adults was reduced 2.5-3-fold, as compared to Q0;hlh-1, Q0 or Q35 young adults  young adults exhibited severe UNC-54 disorganization, while Q0;hlh-1(cc561) myofilaments maintained their striated structures and were only mildly disorganized  embryos was not mitigated in adult animals.To examine whether reduced HLH-1 levels also impacted muscle proteostasis capacity later in life, i.e., after muscle development has completed, we monitored g adults . In agrerganized . Myofilag adults  11]. Th. ThQ35;hQ35-expressing animals begins at the transition to reproductive adulthood [Q35 animals at the first larval stage (L1) (n = 430). Following the onset of reproduction, (day 5) Q35 animals had an average of ~4 foci per animal. In contrast, foci were observed in ~10% of the Q35;hlh-1(cc561) animals (n = 425) even by the L1 stage, while by day 5, Q35;hlh-1(cc561) animals had an average of ~50 foci per animal  animals were ~50% lower than in Q35 animals  later in life could be due to HLH-1 function after embryogenesis but could also stem from defects acquired during myogenesis. Indeed, Q35;hlh-1(cc561) L1 animals were already affected at 15°C  animals with smg-2(RNAi) at the L1 stage to rescue hlh-1 expression levels after embryogenesis was completed. We found that motility and aggregation of Q35;hlh-1(cc561) young adults treated with smg-2(RNAi) from L1 were partially rescued as compared to those treated with the empty vector control (hlh-1(cc561) to 25°C at the L1 stage to reduced hlh-1 expression levels past embryogenesis, did not significantly affect its motility as compared to wild type  and UNC-45 were shown to compete for myosin binding in vitro [unc-54(e1301ts) (unc-54(ts)), shows temperature-dependent misfolding [unc-54(ts) with animals that specifically over-express DAF-21(Hsp90) in body-wall muscle cells (strain AM780). These animals express daf-21(Hsp90) tagged with GFP (daf-21::GFP) under the muscle specific unc-54 promoter (HSP90M). We then monitored embryonic arrest and UNC-54 localization in wild type, HSP90M, unc-54(ts) and HSP90M;unc-54(ts) embryos laid at 20 or 25°C. HSP90M did not induce arrest at the two-fold stage when the animals were grown at 20 or 25°C . unc-54(ts) embryos showed a mild arrest at 20 and 25°C . In contrast, HSP90M;unc-54(ts) embryos were severely delayed  embryos showed defective myofilament and muscle elongation. Immuno-staining with anti-UNC-54 antibodies of HSP90M;unc-54(ts) embryos grown at 20°C exhibited strongly reduced UNC-54 staining  embryos grown at 25°C but only about half of these embryos hatched  embryos grown at 20°C , thereby facilitating embryo elongation (3-fold stage) . In contin vitro , we postsfolding  but onlysfolding . We cros delayed , with th delayed . HSP90M;staining . Althougstaining . Similar hatched . In cont at 20°C . Our datdnj-24(Hsp40), encoding the C. elegans homolog of DNAJB6. DNAJB6 is a ubiquitously expressed chaperone linked to limb-girdle muscular dystropy type 1D (LGMD1D) [dnj-24(Hsp40) (DNJ-24M) on synthetic motility defects induced by chaperone knock-down. We reasoned that if DNJ-24M perturbed chaperone interactions in muscle cells, then this might exacerbate the effects of knocking-down the levels of other muscle chaperones [dnj-24(Hsp40) in body-wall muscle of C. elegans did not result in notable motility defects  induced a strong larval arrest in wild type, HSP90M and DNJ-24M animals, yet only in the DNJ-24M animals did such treatment induce 100% paralysis (rme-8(Hsp40) and dnj-8(Hsp40) resulted in no motility phenotype in wild type or HSP90M animals, while knocking-down the expression of these genes in a DNJ-24M background resulted in motility defects , rme-8(Hsp40) and dnj-8(Hsp40) knock-down. Cell type-specific regulation of chaperone expression could, therefore, impact tissue-specific chaperone networks.The expression of aggregation-prone proteins was suggested to disrupt proteostasis by engaging chaperones and competing for their substrates , 11. Dif(LGMD1D) . LGMD1D (LGMD1D) . To addraperones . If so, aperones , over-ex defects . However animals . RNAi knaralysis . Of noteC. elegans myogenesis. We found that muscle chaperones that have HLH-1 occupancy sites in their promoter are induced in myogenic-converted embryos. This muscle-specific induction was fully dependent on HLH-1, as no induction was observed for most chaperones without HLH-1 occupancy sites or when HLH-1 expression was down-regulated. Moreover, we showed that disrupting the putative HLH-1 binding sites in two different chaperone promoters inhibited their myogenic-induced expression and muscle expression later in life. Thus, HLH-1 is required for the expression of muscle chaperones with HLH-1 occupancy sites in cells undergoing differentiation into body-wall muscle cells. While a HLH-1 differentiation-independent function in embryonic muscle cells is possible, we instead propose that muscle chaperone genes are regulated by HLH-1 together with other muscle genes during myogenesis. Linking the regulation of chaperone expression to the differentiation program could result in a distinct chaperone network, ensuring that chaperones are expressed at the required levels and with proper timing. Indeed, we found that down-regulation of HLH-1 strongly restricted proteostasis capacity, leading to misfolding of muscle protein and myogenesis arrest.In the present study, we asked whether the cellular chaperone network is regulated in a cell type-specific manner. Specifically, we asked whether muscle chaperones are regulated by the myogenic transcription factor HLH-1 during in vitro, their relative levels are critical for myosin folding and can abrogate myogenesis. We, therefore, suggest that physiological tissue-specific chaperone networks can enable cells to respond to the folding requirement of their unique proteomes, leading to distinct responses to folding challenges, such as acute stress or chronic expression of misfolded proteins.Tissue-specific differences in the expression levels of chaperones can explain why down-regulation of ubiquitously expressed chaperones led to a tissue-selective activation of the heat shock response  and why We found that muscle proteostasis can also be critical for muscle differentiation and can, as in the case of UNC-45 and Hsp90, lead to embryonic arrest and lethality. In support of this claim, a recent study showed that activation of Janus kinase 2 (JAK2) signaling associated with myeloproliferative neoplasms (MPNs) resulted in reduced expression of proteostasis component AIRAPL (arsenite-inducible RNA-associated protein-like) and led to increased insulin/insulin-like growth factor 1 (IGF1R) stability and disrupted hematopoietic differentiation . Thus, cpha-4 occupancy site in the daf-21(hsp90) promoter, as identified by modEncode, was shown to be functional in cell non-autonomous expression of daf-21(Hsp90) in adult C. elegans muscle, intestinal and neuronal cells [hlh-1(cc561) background that could be alleviated by blocking the nonsense mRNA decay pathway and thus, hlh-1 mRNA clearance. Indeed, hlh-1(cc561) exacerbated the effect of a dystrophin mutation associated with Duchenne’s muscular dystrophy, leading to muscle degeneration in adulthood [Our data demonstrate that the myogenic factor HLH-1 regulates the expression of chaperones in muscle cells. By extension, chaperones can be differentially regulated in different cell types to meet the needs of a specific proteome. The large available ChIP-seq dataset (ModEncode)  shows thal cells . For HLHdulthood , 67. We,dulthood , the regMyogenic-dependent regulation of chaperones suggests that the proteostatic requirements of the muscle proteome might dictate the expression of other quality control machineries to fit functional and folding characteristics of that proteome. Careful analysis of HLH-1 targets revealed that most were not muscle-enriched . One intAlthough the expression of many disease-associated proteins is not tissue-specific, many protein misfolding diseases exhibit tissue-specific vulnerability , 72. Foret al. [-6) peak call data, were provided in the manuscript as supporting information [mex-3 or mex-3;skn-1;elt-1 RNAi) and an overlap set identified in both [The chaperone list was complied based on the work of Brehme et al. , focusinet al. . Three cormation  and two  in both . The lat in both , kindly  in both , 40. TheHLH-1 binds to E-Box motif (CANNTG) , 80. Forhttp://www.cmbi.ru.nl/cdd/biovenn/ (BioVenn) [C. elegans embryonic development gene expression was provided by Dr. Itai Yanai [P values in Figs Venn diagrams were plotted using the BioVenn diagram generator BioVenn) . Microarai Yanai . Data weai Yanai . The proEscherichia coli OP50-1 strain at 15°C, unless indicated otherwise. Cross-strains were generated using standard C. elegans procedures.The list of strains used in this work is provided in daf-21(Hsp90) and hsp-12.2(sHsp), a 2492 bp fragment for daf-21(Hsp90) and a 921 bp fragment for the Hsp-12.2(sHsp) promoter were amplified from N2 genomic DNA and assembled into plasmid pNU106 to create plasmids pNU314 and pNU374, respectively, using Gibson ligation. Mutated promoter reporters for daf-21(Hsp90) and hsp-12.2(sHsp), pNU315::Pdaf-21(mut)::gfp and pNU375::hsp-12.2p(mut)::gfp, respectively, were generated by site-directed mutagenesis of the putative HLH-1-binding motifs at -432 bp and at -921 bp and -266 bp to ACGCGT. Plasmids were validated by DNA sequencing and injected into animals expressing unc-119(ed3);hsp-16.41::hlh-1. Promoter reporter constructs were generated and injected by Knudra Transgenics. Stable transgenic lines are listed in To generate the promoter reporter constructs for Synchronized animals were grown at 15°C for five days or transferred to 20 or 25°C at the L2 stage for 24–48 h to reach adulthood. These synchronized gravid adults (first day of egg laying) were allowed to lay eggs for 45 min and then removed. Synchronized embryos were heat shock-treated, allowed to grow for 6 h to pass the comma stage or allowed to grow for 24–48 h and complete embryogenesis.Embryos laid at 15°C were moved to new plates and were untreated or subjected to heat shock at 34°C for 30 min. To determine RNA levels, embryos were frozen immediately following heat shock. To examine expression patterns, embryos were collected after a 6 h recovery.hlh-1(cc561) embryos laid at 15°C or 25°C for 45 min were allowed to grow for 6 h. The embryonic developmental stage was examined to determine whether embryos grown at 15°C had passed the comma stage. To determine the effect of hlh-1(cc561) on gene expression, we compared the relative mRNA levels (25°C/15°C) of the genes examined. In Wild type and E. coli strain HT115(DE3) transformed with specified RNAi or empty (pL4440) vectors and allowed to lay eggs, as above. Otherwise, synchronized L1 larvae stage worms were washed from regular NGM plates, transferred to RNAi plates and grown at 15°C for five days until day 1 of adulthood.RNAi knockdown treatments were performed as previously described . RNAi co-ΔΔCT method) to T07A9.15 or tbc-10, determined to be stably expressed during embryogenesis [RNA extraction from synchronized embryos, cDNA synthesis and quantitative real-time PCR were performed as previously described . Samplesogenesis . The lisTo determine embryo arrest, synchronized embryos were grown at 15–25°C. Embryos that did not hatch until their siblings reached L2 (24–48 hours) or hatched arrested at the two-fold state were counted as arrested embryos.Age-synchronized young adults were moved to a new plate and their movement was monitored after 10 min. Animals that did not move one body length were scored as paralyzed. Otherwise, age-synchronized young adults were placed in wells containing M9 and allowed to acclimate for 5–10 min. Each animal was monitored for 15 sec and thrashes (changes in direction of bending at mid-body) were counted.Embryos were fixed in methanol and 2% paraformaldehyde and permeabilized by freeze-thawing and then immuo-stained as described . Adult aThe number of bright foci of age-synchronized animals expressing Q35::YFP was counted using an LEICA M165FC stereomicroscope.Age-synchronized animals were collected and lyzed in SDS sample buffer (95°C for 10 min). Samples were separated by SDS-PAGE and analyzed by western blot (LF PVDF membrane), using primary anti-tubulin (Sigma) and anti-GFP (Enco Scientific) and secondary DyLight 488 and DyLight 549, anti-mouse and anti-rabbit antibodies, respectively (Jackson Immuno Research). Membranes were imaged using the ChemiDoc MP Imaging System (BioRad).S1 Figth-7th AB cell divisions, ventral enclosure (VE), comma stage (cs), first movement, and L1) [Hierarchical clustering of the relative expression of 35 muscle-specific genes across 10 developmental stages  . MI mark(TIF)Click here for additional data file.S2 Fig(A) Representative images (>90%) of the expression pattern of chaperones in wild type embryos untreated or subjected to heat shock after a 6 h recovery. Scale bar is 25 μm. (B-E) Relative chaperone mRNA levels in heat shock-treated wild type (gray) or HLH-1(ec) (red) embryos. Data are relative to values obtained with untreated embryos  and are presented as means ± SEM of at least 5 independent experiments.(TIF)Click here for additional data file.S3 Figdaf-21(Hsp90) promoter, without myogenic induction. Arrows indicate body-wall muscle cells.Representative images of HLH-1(ec) animals expressing GFP under the regulation of the wild type or a mutant (TIF)Click here for additional data file.S4 Fig(A) Representative images (>90%) of the expression pattern of the indicated chaperones in wild type embryos grown at 25°C. (B-C) Relative mRNA levels (25/15°C) of wild type (gray) or hlh-1(cc561) (green) embryos . Data are presented as means ± SEM of 5 independent experiments.(TIF)Click here for additional data file.S5 Fig(A) Embryonic arrest scored for Q35;hlh-1(cc561) or Q35 embryos treated with smg-2, smg-7 or empty vector control RNAi. Data are presented as means ± SEM of at least 3 independent experiments. (B) Representative confocal images of Q35;hlh-1(cc561) muscles. Scale bar is 10 μm. (C-D) Extracts of age-synchronized (day 4) Q35 or Q35;hlh-1(cc561) animals were separated on a SDS-PAGE gel and probed with anti-GFP (top) and anti-tubulin (bottom) antibodies. Relative levels were determined by quantification of Q35::YFP protein bands. Data are presented as means ± SEM of at least 3 independent experiments. (E) The number of body movements per minute scored on the first day of adulthood in age-synchronized Q35;hlh-1(cc561) animals treated with smg-2 or empty vector control RNAi from L1. (F) The average number of visible foci scored in age-synchronized Q35;hlh-1(cc561) young adults treated with smg-2 or empty vector control RNAi from L1. (G) The number of body movements per minute scored in wild type or hlh-1(cc561) young adults shifted to 25°C at L1.(TIF)Click here for additional data file.S6 Fig(A) Images of a population of wild type, unc-54(ts), HSP90M or HSP90M;unc-54(ts) embryos laid at 20°C. (B) Representative confocal images (>90%) of unc-54(ts), and HSP90M;unc-54(ts) embryos laid at 25°C and stained with anti-UNC-54 antibodies. The scale bar is 25 μm.(TIF)Click here for additional data file.S7 Fighsp-1(Hsc70), rme-8(Hsp40) or dnj-8(Hsp40) RNAi and stained with anti-MYO-3 antibodies. Scale bar is 25 μm.Representative confocal images of age-synchronized DNJ-24M animals treated with control, (TIF)Click here for additional data file.S1 Table(A) Flowchart outlining the filtering analyses of the chaperone list. (B) Summarized data from bioinformatics analyses.(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye.Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing  Drosophila eye is derived from a field of equivalent progenitors that develop into photoreceptor neurons as well as non-neuronal cone and pigment cells. The zinc finger transcription factor Glass was thought to solely determine the photoreceptor identity. Here we show that Glass acts not only in photoreceptors, but also in the non-neuronal support cells of the eye, promoting the normal differentiation of each cell type. Loss of Glass in non-neuronal cells results in developmental defects, and these defects can be rescued by restoring Glass specifically to those cell types. We used misexpression assays to show that Glass is sufficient to induce markers of photoreceptors, cone cells and pigment cells, and we identified certain transcription factors that may cooperate with Glass to specify these non-neuronal cells. These results suggest that the role of Glass is to give each cell type its identity as a component of the eye. The transcriptional code that defines cell identities must thus include factors such as Glass that mark the cell as belonging to a specific organ.Distinct cell types within an organ are specified by the activity of cell type-specific transcription factors, which activate distinct gene networks that confer the appropriate cellular identities. The  Cell fate specification is achieved by integrating extrinsic signals with intrinsic transcriptional and epigenetic networks –3. CertaDrosophila eye consists of photoreceptor neurons and non-neuronal support cells that develop from a single field of uncommitted progenitor cells in the larval eye imaginal disc [The nal disc , 13, whinal disc . Differenal disc , 15, 16.nal disc , 18. Thenal disc , 20. Signal disc . Each cenal disc –24.gl mutants, presumptive photoreceptors acquire neuronal characteristics, but fail to differentiate and do not express Rhodopsins or the phototransduction machinery [gl mutants, their numbers and morphology are abnormal, giving rise to the “glassy” phenotype that was the basis for the original identification of gl, and pigmentation is reduced [The zinc finger transcription factor Glass (Gl) has been assigned a central role in specifying photoreceptor identity downstream of the retinal determination gene network –27. In gachinery , 27, 28.achinery , 29. Whi reduced , 30. Thechaoptin (chp) both in neuroblasts in the brain and in non-neuronal epithelial cells of other imaginal discs, independently of the retinal determination gene network. Transcriptomic analysis revealed distinct but overlapping sets of genes induced in these two cellular contexts, including genes characteristic of non-neuronal cone and pigment cells. Gain-of-function, rescue and loss-of-function experiments all supported autonomous roles for Gl in the normal differentiation of these non-neuronal cells of the eye as well as of photoreceptors. Furthermore, we identified several elements of a transcription factor network that cooperates with Gl to induce genes characteristic of cone and pigment cells. These results indicate that Gl is not a photoreceptor-specific factor, but a determinant of organ identity that is reiteratively used to promote the terminal differentiation of multiple cell types in the eye.Misexpression of Gl in the larval central nervous system has been shown to induce the expression of a subset of photoreceptor-specific genes . As a fichaoptin (chp)   in cells Flybase . gl-RA e binding , 31, and in vivo , suggestnctional . We usedasense (ase)-GAL4, deadpan (dpn)-GAL4 or inscuteable (insc)-GAL4 drivers, together with a temperature-sensitive form of GAL80 to bypass early lethality, led to induction of Chp in some cells in the central nervous system (CNS) of the developing embryo -GAL4 or in sensory organ precursors with atonal (ato)-GAL4 did not result in ectopic Chp expression  specifically in neurons [Ultrabithorax (Ubx)-FLP and the MARCM system to express Gl in clones of epithelial cells in the wing imaginal disc  driver, we observed ectopic Chp expression in Elav-negative cells , a retinal determination factor that forms a compound transcription factor with Eyes absent (Eya) [gl can also be induced by the combination of Eya and Dachshund [eya mutant clones  , 34; gl achshund . Consistachshund , we fount clones . This regl mutant third instar larval brains in which Gl expression was driven in neuroblasts by insc-GAL4 in combination with tubulin (tub)-GAL80ts  and prospero (pros) [Surprisingly, Gl induced many more genes in the wing disc than in the brain . Some ofnditions , 40. Altnditions , which vo (pros) , 28, 30.The ability of Gl to induce genes that contain Gl binding motifs is also likely to depend on their responsiveness to other transcriptional activators and repressors present in the cell. A search for motifs that were enriched in the regulatory regions of genes induced by Gl, relative to predicted targets that were not induced, identified a motif matching the binding site for Grainyhead (Grh) , a transPph13, Eye-enriched kainate receptor (Ekar) and the phototransduction component inactivation no afterpotential C (inaC) [sallimus (sls), obstructor-B (obst-B), peste (pes) and the glial marker wrapper [gl mutant clones  and rosy (ry) [eyeless (ey)-FLP or Ubx-FLP led to the appearance of ectopic red pteridine pigment, normally only produced by pigment cells in the eye [Our observation that Gl could activate gene expression in non-neuronal cells raised the possibility that its expression in the non-neuronal cell types in the eye , 29 mighC (inaC) –43, Gl a wrapper , 44. We  wrapper  Fig 5A)Pph13, Eyt clones . Expresst clones . In addiosy (ry) , 48. Con the eye , on the gl mutants exhibit numerous defects in cone and pigment cell differentiation. The number of cone cells is reduced (gl transgene to restore Gl to specific cell types in a gl mutant background. Expressing gl in cone cells and primary pigment cells with sparkling (spa)-GAL4 [54-GAL4 [gl mutant eyes with no rescue construct -GAL4  or in in[54-GAL4  each proonstruct . These rentation , indicatgl function from specific cell types. As the gl transgenic RNAi lines available did not fully reproduce the gl phenotype even when expressed ubiquitously throughout eye development, we used the CRISPR-Cas9 method to generate somatic gl mutations in a cell-type specific manner [gl  in the wild-type pupal retina, it is present during cone cell specification and in pupal primary pigment cells [spa-driven CRISPR mutagenesis   , which epression  but not pros and homothorax (hth) expression in the wing disc  into pUASTattB using EcoRI and XhoI. A 1.5 kb NdeI/XhoI fragment lacking the intron was generated by overlap PCR and used to replace the corresponding region of UAS-gl-RB to generate UAS-gl-RA. Both constructs were integrated into the VK37 attP site at position 22A3. The gl sgRNA sequences identified on www.flyrnai.org/crispr2/ [gl gRNA 1) and TACCCACCGCTGCTGAGTCC (gl gRNA 2) were cloned into pCFD4 [attP40 site at 25C6. Injections and screening of transgenic flies were carried out by Genetivision.UAS-crispr2/ , GCAGGATto pCFD4  by PCR agl-RA; FRT82, gl60j (2) UAS-gl-RB; FRT82, gl60j (3) UAS-CD8-GFP, ey-FLP; tub-GAL4, FRT82, tub-GAL80/TM6B (4) UAS-CD8-GFP, Ubx-FLP; tub-GAL4, FRT82, tub-GAL80/TM6B (5) UAS-gl-RB; FRT82, P(w+) (6) FRT82, gl60j (7) UAS-glRB; FRT82, DlRevF10 (8) FRT42; UAS-Pax2 (9) FRT42, UAS-glRB; UAS-Pax2 (10) UAS-CD8-GFP, Ubx-FLP; FRT42, tub-GAL80; tub-GAL4/TM6B (11) FRT42, UAS-glRB (12) FRT42, UAS-glRB; UAS-eya (13) FRT42; UAS-eya (14) UAS-glRB; FRT82, UAS-lz (15) FRT82, UAS-lz (16) FRT42, UAS-glRB; UAS-esg (17) UAS-esg; FRT82 (18) UAS-glRB; FRT82, hthB2 (19) esg66B, FRT40, UAS-glRB (20) eyaE18B, FRT40, UAS-glRB (21) UAS-CD8-GFP, Ubx-FLP; FRT40, tub-GAL80; tub-GAL4/TM6B. Stocks used for misexpression were (1) dpn-GAL4 (2) elav-GAL80; dpn-GAL4 (3) elav-GAL4 (4) repo-GAL4 (5) ase-GAL4 (6) ato-GAL4 (7) insc-GAL4 (8) UAS-CD8-GFP; UAS-gl-RB; tub-GAL80ts (9) Dll-GAL4 (10) insc-GAL4; tub-GAL80ts, gl60j. Stocks used for rescue were (1) spa-GAL4; gl60j (2) 54-GAL4, UAS-lacZ; gl60j (3) UAS-gl-RB, UAS-lacZ; gl2. Stocks used for CRISPR were (1) ey3.5-FLP, Act>CD2>GAL4; UAS-Cas9P2, gl60j/TM6B (2) elav-GAL4; UAS-Cas9P2, gl60j/SM6-TM6B (3) spa-GAL4; UAS-Cas9P2, gl60j/TM6B (4) 54-GAL4, UAS-lacZ; UAS-Cas9P2, gl60j/SM6-TM6B (5) gl sgRNAs (attP40). Transgenic lines other than UAS-gl and gl sgRNAs are described in Flybase.Stocks used to generate clones were (1) UAS-4/1mM EGTA for most antibodies but 45 min in 2% formaldehyde in 75mM lysine/370mM sodium phosphate pH 7.2/10mM NaIO4 for anti-Gl. Pupal retinas were fixed and stained as described [Embryos and larval eye discs, wing discs and brains were stained as described , 94, fixescribed . Antibodescribed , rat antescribed  and mous480 values were obtained using the homogenizing solution as a blank.Pigment quantification was performed as described  with thehttp://www.bioinformatics.babraham.ac.uk/projects/fastqc) and sequencing adapters were removed with Trimmomatic [Drosophila reference genome (dm3) with Tophat2 v2.1.1 1. The Picard CollectRnaSeqMetrics program (https://broadinstitute.github.io/picard/picard-metric-definitions.html#RnaSeqMetrics) was used to generate QC metrics including ribosomal RNA content, median per-gene coverage, bases aligned to intergenic regions, 5’/3’ biases, and the distribution of the bases within exons, UTRs, and introns. Per sample gene expression profiles were computed using Cufflinks v2.2.1 1 and the RefSeq genome annotation for the Drosophila reference genome dm3 [gl gene were visualized with Integrative Genomics Viewer . Genes were considered significantly changed and included in the heat map if the log2 fold change for either wing or brain misexpression was >1, p<0.01, average cpm for that misexpression condition was >1, and standard deviation/mean of the three replicates was <0.5. Intron sizes were obtained from Flybase and compared for targets predicted with high confidence by [Larval tissue was isolated from 30 animals of each genotype in triplicate and RNA was extracted in Trizol (Invitrogen). Library preparation and sequencing was carried out by the NYU Genome Technology Center. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50. For each RNA-seq sample, sequence quality was assessed with FastQC  and UAS-gl are not expressed (A) or expressed with dpn-GAL4 (B), elav-GAL4 (C), repo-GAL4 (D) or ato-GAL4 (E). Expressing Gl in either neuroblasts or neurons induces ectopic Chp in the CNS, but expressing Gl in glial cells or in the peripheral nervous system has no effect. Scale bars: 50μm.All panels show late stage embryos stained with anti-Chp . (A) is stained with anti-Elav (green). (TIF)Click here for additional data file.S2 Figinsc-GAL4 in (B-D), with tub-GAL80ts to bypass early lethality. Animals were reared at 18°C for three days and then shifted to 29°C. Cells that express Gl or Chp but not GFP are indicated by arrowheads in . These cells express Elav and are therefore likely to be differentiated neuronal progeny of the Gl-expressing neuroblasts. Scale bars: 100μm in ; 50μm in .All panels show third instar larval brains stained with anti-Gl , anti-Chp  and anti-Elav (blue). mCD8GFP (green) and Gl are driven by (TIF)Click here for additional data file.S3 FigHisCl1, Ekar, inaC, chp, nrm) and 6 in cone cells .(A) Gl target genes predicted by  based on the presence of Gl binding motifs in a region consisting of 5 kb upstream and the first intron are binned according to the size of their first intron and plotted as induced by Gl in one or more tissues (black) or induced in neither (grey). (B) A plot of the log fold change in Gl-expressing wing discs (non-patterned bars) or brains (patterned bars) for the 13 predicted targets that were induced by Gl, divided according to the size of their first introns (intron size 0–500 (red), 501–1000 (green), 1001–5000 (yellow), >5000 (blue)). Interestingly, 5 of these genes are highly enriched in mature photoreceptors ((TIF)Click here for additional data file.S4 Figgl gene targeted by the two sgRNAs. Noncoding regions are shown in gray and the zinc fingers in white. (B) wild-type control and  ey3.5-FLP, Act>CD2>GAL4; gl sgRNAs; UAS-Cas9P2/gl60j.  show larval eye discs stained with anti-Gl  and anti-Elav (green), and (D) shows an adult eye. Expressing Cas9 throughout the eye disc in a gl heterozygote results in mosaic loss of Gl by the third instar and a moderate gl mutant phenotype in the adult. (E) wild type;  elav-GAL4/gl sgRNAs; UAS-Cas9P2/gl60j.  show 42h APF pupal retinas stained with anti-Gl , anti-Elav and anti-Ecad (both in green) and (G) shows an adult eye. Insets are enlargements of single boxed ommatidia. Gl staining is reduced in photoreceptors but still present in pigment cells. Expressing Cas9 in photoreceptors results in a weaker, mosaic gl mutant phenotype (G). (H-J) show third instar larval eye discs stained with anti-Pax2  to mark cone cells and anti-Gl . (H) wild type; (I) spa-GAL4; gl sgRNAs; UAS-Cas9P2/gl60j; (J) gl60j. Gl is lost from some Pax2+ cone cells (circled by yellow dashed line in I). (K) 54-GAL4, UAS-lacZ; (L) 54-GAL4, UAS-lacZ /gl sgRNAs; UAS-Cas9P2/gl60j 42h APF pupal retinas stained with anti-Gl  and anti-β-galactosidase (green). Gl is lost from some pigment cells. Insets show enlargements of single boxed ommatidia. Scale bars: 50μm in ; 10μm in ; 20μm in ; 30μm in (J).(A) Schematic showing the positions in the (TIF)Click here for additional data file.S5 Figspa-GAL4; gl sgRNAs; UAS-Cas9P2/gl60j (B-E) and 54-GAL4, UAS-lacZ /gl sgRNAs; UAS-Cas9P2/gl60j (G-J). Loss of Gl in cone cells or pigment cells results in ommatidial patterning defects. Scale bars: 5μm. (K) Quantification of Elav+ cells per ommatidium observed in cell-specific CRISPR experiments compared to gl sgRNAs; UAS-Cas9P2, gl60j/+ control. Loss of Gl in non-neuronal cells does not affect photoreceptor numbers.(A-J) show individual ommatidia from 42h APF pupal retinas, stained with anti-Ecad. Wild-type , (TIF)Click here for additional data file.S1 Tablegl was misexpressed with insc-GAL4 in gl mutant larval brains compared to gl mutant brains, genes induced when gl was misexpressed in clones made with Ubx-FLP in wing discs compared to wild type wing discs, genes more highly expressed in wild type third instar eye discs than wing discs, and genes more highly expressed in wild type third instar eye discs than brains, all using the same cutoffs . The final sheet shows predicted direct targets of Gl according to [2 fold changes and p values for the indicated comparisons.Separate sheets show the genes induced when rding to . Columns(XLSX)Click here for additional data file.S2 Tablegl mutants and controls.Numbers of the indicated defects in photoreceptor number, cone cell, pigment cell or bristle cell number or arrangement observed in cell type-specific (XLSX)Click here for additional data file.