An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution. Fatty-acylated peripheral proteins such as members of the small G-protein Ras family, heterotrimeric G-proteins, the neuronal proteins PSD-95 and growth-associated protein-43 (GAP-43) Among all posttranslational lipid modification of proteins, including isoprenylation and myristoylation, the addition of fatty acid to the sulfhydryl group of a cysteine to form a thioester bond  is the only readily reversible linkage having a much shorter half-life than that of the protein PATs have been identified both in yeast and mammalian In this work, we demonstrated for the first time that lysophospholipase II or APT-2 is a cytosolic protein thioesterase involved in GAP-43 deacylation. GAP-43 was early identified as a functional growth cone marker participating in the mechanisms of axonal outgrowth and regeneration Although several groups have described the molecular determinants involved in the polarized sorting of GAP-43 in neural cells Using in vivo confocal fluorescence microscopy, biochemical analyses and bioinformatic approaches, we characterized the deacylation kinetics of GAP-43 and identified the thioesterase involved in this process. It was found that GAP-43 is deacylated in vivo when expressed in Chinese hamster ovary (CHO)-K1 and human HeLa cells through a mechanism mediated by APT-2, but not by APT-1. Inhibition of deacylation caused GAP-43 to accumulate in perinuclear structures while APT-2 overexpression increased the cytoplasmic pool of GAP-43. Additionally, it was also demonstrated that APT-2 overexpression affected the steady-state subcellular localization of acylated H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.full) and the acylation motif  of GAP-43 fused to spectral variants of green fluorescent protein (GFP) [cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP)] Previously, we characterized the acylation, membrane association and intracellular trafficking of full-length , N13GAP-43(C4S), N13GAP-43] and GAP-43full  which were then characterized by in vivo confocal microscopy and biochemical analyses. As previously observed, GAP-43full and N13GAP-43 localized, at steady-state, at the RE, plasma membrane and TGN  fraction after ultracentrifugation. 20% and 70% of P fraction-associated GAP-43full and N13GAP-43, respectively, were found enriched in the TX-114 detergent (D) phase, reflecting a higher hydrophobic character for N13GAP-43. In contrast, a drastic reduction in membrane association (<5% in P fraction) of newly synthesized GAP-43 was observed when cells were incubated with the PAT inhibitor 2-BP (see nonacylated N13GAP-43 and nonacylated GAP-43full in full(C3S) and 35% of N13GAP-43(C3S) were found to be associated with the P fraction. Additionally, 20% and 43% of P fraction-associated GAP-43full(C3S) and N13GAP-43(C3S), respectively, were found enriched in the D phase, reflecting a higher hydrophobic character for N13GAP-43(C3S) (N13GAP-43(C3S) is the single acylation mutant having the highest proportion of acylation and, consequently, membrane association.The membrane association and the extent of posttranslational modification of these constructs was further characterized by ultracentrifugation and Triton X-114 (TX-114) partition assays  [47]. Mo-43(C3S) . AltogetN13GAP-43(C3S) in order to investigate single deacylation events occurring on GAP-43. CHO-K1 cells transiently coexpressing N13GAP-43(C3S) and N27GalNAc-T (TGN marker) were treated with 50 µM 2-BP (2-BP) or dimethylsulfoxide  and GAP-43 subcellular distribution monitored at different times by live cell confocal fluorescent microscopy  since all experiments were performed at 20°C, a condition which drastically decreases this process Next, we evaluated the deacylation kinetics of croscopy . CHX andcroscopy  clearly croscopy . The obsN13GAP-43(C3S) were determined at the same experimental conditions by subcellular fractionation and TX-114 partition assay. It was found that 2-BP, when compared to control conditions, caused a significant decrease of membrane bounded-N13GAP-43(C3S) (full(C3S) (N13GAP-43(C3S) is deacylated with a similar kinetics both in HeLa and CHO-K1 cells  . A similull(C3S) . OverallK1 cells , precludN13GAP-43(C3S) is deacylated in vivo and that GAP-43 deacylation occurs at the TGN, besides the plasma membrane, revealing the importance of an acylation/deacylation cycle operating in GAP-43 subcellular distribution These results demonstrate that N13GAP-43(C3S) is deacylated with similar kinetic when it is expressed in CHO-K1 and HeLa cells. APT-1 is the unique described cytosolic protein thioesterase able to catalyze the deacylation of several substrates including H-Ras and G alpha subunit proteins Up to now, results indicate that To investigate whether APT-1 deacylates GAP-43, we first analyzed mRNA expression of APT-1 both in CHO-K1 and HeLa cells by reverse transcriptase-polimerase reaction chain (RT-PCR). The results showed that a PCR product of the expected size was obtained using mRNAs from HeLa cells as template . The samN13GAP-43(C3S) in CHO-K1 cells overexpressing or not human APT-1 fused to mCherry fluorescent protein -TGN-membrane association decayment after 2-BP treatment. Results clearly indicated that APT-1 overexpression in CHO-K1 cells did not significantly modify the decay rate of N13GAP-43(C3S) at the TGN compared to cells only expressing N13GAP-43(C3S). Additionally, we also found that APT-1 overexpression did not significantly modify membrane association and deacylation kinetic of diacylated N13GAP-43 and GAP-43full (N13GAP-43(C3S), we performed an in vitro deacylation assay (N13GAP-43(C3S) or a fusion protein containing the C-terminal hypervariable region of K-Ras fused to YFP (K-RasC14), and particulate fractions from nontransfected cells N13GAP-43(C3S) in the fractions evaluated by Western blot. Results indicated that the incubation with recombinant APT-1 did not significantly increase the amount of N13GAP-43(C3S) recovered in the soluble fraction. As expected, the amount of nonpalmitoylated K-Ras isoform present in the soluble fraction was independent of the presence of APT-1 (result not shown). Thus, these results strongly support the concept that APT-1 does not catalyze both in vitro and in vivo N13GAP-43(C3S) deacylation.Thus, results demonstrated that APT-1 is not expressed in CHO-K1 cells. To discard the possibility that very low endogenous expression level of APT-1 could be deacylating GAP-43, we evaluated the deacylation kinetics of P-43full . To furton assay . For theAs additional control of APT-1 activity, we evaluated the effect of ectopically expressed APT-1 on H-RasG12V deacylation and its consequences on subcellular distribution. APT-1 has been identified as one of the thioesterases in the acylation cycle of H-Ras both in vivo http://pfam.sanger.ac.uk/) [#PF02230 (APT-1) and #PF02089 (PPT-1)]. After careful classification and removal of redundant sequences, the in silico analysis allowed us to identify 21 potential acyl thioesterase transcripts, which are codified for 5 different genes  and epithelial (CHO-K1) cells, respectively. Finally, transcripts coded by gene ID 10434 were found in all analyzed cell lines, except CHO-K1 cells. Thus, the transcription profile of genes ID 127018, 339168 and 10434 did not explain for the presence of acyl-protein thioesterase activity both in CHO-K1 and HeLa cells. Consequently, results strongly suggest that gene ID 11313 potentially code for an acyl-protein thioesterase for GAP-43.To identify transcripts derived from gene ID 11313 potentially coding for GAP-43 acyl-protein thioesterases, we evaluated their expression both in CHO-K1 and HeLa cells by RT-PCR using specific primers for each transcript . ResultsN13GAP-43(C3S) deacylation in CHO-K1 cells by live cell confocal microscopy as described in N13GAP-43(C3S) deacylation in cells which did not overexpress APT-2 (N13GAP-43(C3S) was 6 min (see N13GAP-43(C3S) deacylation was reduced to 3 min (N13GAP-43(C3S).Bioinformatic search and mRNA expression experiments suggested that APT-2 is a potential acyl-protein thioesterase for GAP-43. To test this hypothesis, we evaluated the effect of APT-2, fused to mCherry fluorescent protein (hereafter APT-2), overexpression on ss APT-2 . As prev min see . In a clto 3 min , demonstfull and N13GAP-43 deacylation. For this, we analyzed the effect of 2-BP treatment on GAP-43 subcellular distribution in cells expressing GAP-43 alone or coexpresing GAP-43 and APT-2 (N13GAP-43 and GAP-43full) deacylation was partially evident after 3 h of 2-BP treatment (compare Control with 2-BP condition), maintaining a significant amount of plasma membrane-associated GAP-43. In contrast, after 6 h of 2-BP treatment, we observed nuclear accumulation of soluble GAP-43 and a clear reduction of GAP-43 at the plasma membrane, which indicate that it is highly deacylated under this condition. On the other hand, the overexpression of APT-2 caused a significant reduction in the plasma membrane content of GAP-43full at 3 h of 2-BP treatment  indicates that APT-2 is likely a soluble protein. By fusing the cDNA encoding this protein to fluorescent proteins, we found that APT-2 is mostly cytosolic and there is not any apparent membrane association. This fact strongly suggests that APT-2 can deacylate GAP-43 by transient binding to the membrane-cytosol interface probably in a widely distributed process. In particular, we observed that deacylation of monoacylated N13GAP-43 occurs as early in the acylation cycle as at the Golgi complex. In agreement with this result, it was recently demonstrated that palmitate removal of H-Ras mutants  takes place at the Golgi complex Mouse APT-2, which shares 64% identity with rat and mouse APT-1, was found to be ubiquitously expressed in mouse tissues and able to hydrolyze several lipid substrates with varied efficiency iα1) or at the C-terminus (i.e. H-Ras) full and the acylation motif of GAP-43 fused to YFP.Although some patterns of S-acylation exist, there is no specific consensus sequence for S-acylation. Similarly, it has not been defined so far a consensus for the sequences surrounding the thioacyl group which could be recognized by APT-1. In vitro, APT-1 can depalmitoylate structurally different proteins , having different subcellular distributions and S-acylated either at the N- . Expression plasmids for N13GAP-43-(YFP), GAP-43full-(YFP) and its acylation mutants, (YFP)-K-RasC14, YFP-H-Rasfull, N27GalNAc-T-CFP and N52Gal-T2-CFP have been previously described G12V was performed by directed mutagenesis of YFP-H-Rasfull using PCR. Both Cherry-APT-1 and Cherry-APT-2 constructs were obtained by RT-PCR using purified mRNA from HeLa and CHO-K1 cells, respectively, and the amplified products were cloned into pmCherry-C1 using EcoRI and SalI restriction sites.The expression vectors pECFP-C1 , pEYFP-N1 , pECFP-C1, pEYFP-C1, pEGFP-F, pmCherry-C1 and the plasmid encoding GFP-H-Ras2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics . To induce SH-SY5Y differentiation, cells were incubated with 30 µM retinoic acid for 6 days. Cells grown on Petri dishes were used for both live cell imaging and Western blot experiments. Cells were transfected with 0.6–1.2 µg/35 mm dish of the indicated plasmid using cationic liposomes  or polyethyleimine . At 24 h after cell transfection, cells were processed for Western blot experiments or plated onto coverslips, incubated for 24 h, and used in live cell imaging.CHO-K1, HeLa, SK-Mel 28 and SHSY-5Y cells  were maintained at 37°C, 5% COThe stock solution of 2-BP  was prepared in DMSO. CHO-K1 and HeLa cells expressing recombinant proteins were incubated with 50 µM 2-BP or vehicle (DMSO) for control cells for the indicated times. To analyze the effect of 2-BP on the steady state subcellular distribution of fluorescent proteins, CHO-K1 and HeLa cells were treated with 60 or 150 µg/ml CHX (Sigma-Aldrich), respectively, and inhibitors of protein degradation (60 µM cloroquine and 7.5 µM MG132). These inhibitors were added to the culture medium 24 h after cell transfection and 1 h before 2-BP treatment (Western blot experiments) or 36 h after platting transfected cells onto coverslips (live cell imaging). In live cell imaging experiments, coverslips were mounted on the microscope stage and 2-BP was added during time series acquisition.To analyze the effect of acylation inhibition on membrane binding properties of GAP-43, 50 µg/ml CHX was incorporated to the culture medium during the transfection. Then, 5 h after transfection, 10% FBS was added to the medium and 2h later 50 µM 2-BP was also incorporated. After 1 h of 2-BP treatment, CHX was removed by three washes with DMEM containing FBS, antibiotics and 2-BP. Finally, after 4 h of CHX withdrawal, cells were scrapped and used for biochemical experiments.N13GAP-43 Kp value determined as follow:Electrophoresis, transfer onto nitrocellulose membranes and protein immunodetection were performed as described by Kp were normalized to those observed in control conditions.Where S and P refer to the amount of GAP-43 present in S [Aqueous (A) plus Detergent (D) fractions] and P (A plus D fraction), respectively, after background subtraction. Values of 2HPO4, 1.6 mM NaH2PO4, pH 7.5); and harvested by scrapping in PBS containing a protease inhibitor mixture (PIM) containing 5 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.7 µg/mL pepstatin (PBS-PIM). Extracts were centrifuged at 4°C for 5 min at 13000 g and resuspended in 400 µl of 5 mM Tris-HCl (pH 7.0) (buffer T) in the presence of protease inhibitors (T-PIM). Pellets were dispersed by repeated pipetting and vortexing. After 30 min of incubation in T-PIM, pellets were passed 60 times through a 25-gauge needle. Nuclear fractions and unbroken cells were removed by twice centrifugation at 4°C for 5 min at 600 g. Supernatants were then ultracentrifuged at 4°C for 1 h at 400000 g using a TLA 100.3 rotor . The supernatant (S1 fraction) was removed, and the pellet (P1 fraction) was resuspended in 400 µl of T-PIM. Both fractions were further ultracentrifuged at 400000 g and the supernatant of S1 fraction was removed (S fraction) and the pellet of P1 fraction was resuspended in 400 µl of T-PIM (P fraction). 100 µl of 5% (v/v) TX-114 in T-PIM was added to S and P fractions and incubated at 4°C during 1 h. Then, samples were incubated at 37°C for 3 min and centrifuged at 13000 g. The aqueous upper phase (A) and the detergent-enriched lower phase (D) were separated and extracted again with detergent and aqueous solutions, respectively. The resulting samples were adjusted to equal volumes and detergent content, and proteins were precipitated with chloroform/methanol (1∶4 v/v) for Western blot analyses.Cells grown onto 60 mm dishes were washed with cold PBS , and centrifugation at 10000 g for 15 min at 4°C. Hisx6-APT-1 was then purified from the soluble lysate using a Ni2+-NTA column  according to manufacturer's instructions.The APT-1 cDNA containing a Hisx6 tag was obtained by RT-PCR using specific primers and RNA from HeLa cells. The amplified fragment was cloned into BamHI/EcoRI sites of the bacterial expression vector pRSET-A . Transformed N13GAP-43(C3S), S and P fractions were obtained from N13GAP-43(C3S) transfected and nontransfected CHO-K1 cells, respectively, by ultracentrifugation as described above. S0 fractions were cleared and P0 fractions were washed by ultracentrifugation. Membrane binding of soluble N13GAP-43(C3S) protein was done by incubation of S0 fractions obtained from transfected cells with P0 fractions obtained from untransfected cells for 1 h at 4°C. The resultant mixture was then ultracentrifuged for 1 h at 4°C and the pellet fraction (P1) was resuspended in 400 µl APT-1 reaction buffer  containing protease inhibitors (PIM plus 1 mM PMSF) and ultracentrifuged again. The resultant P2 fraction was resuspended in 250 µl APT-1 reaction buffer containing PIM/PMSF and subjected to acyl-protein thioesterase treatment by incubation with recombinant APT-1 as previously described 2 fraction with 2 µg recombinant APT-1  or 11.1 µl of APT-1 carrier buffer  and incubated at 30°C for 1 h. Then, 300 µl of APT-1 reaction buffer was added to samples and subjected to ultracentrifugation. P fractions were then resuspended in the same reaction buffer. Proteins from resulting fractions were precipitated with 10% trichloroacetic acid as described previously C14To examine the ability of purified recombinant APT-1 to depalmitoylate Confocal images were collected using an Olympus FluoView FV1000 confocal microscope  equipped with a multi-line Argon laser , two Helium Neon lasers . CFP was detected by using laser excitation at 458 nm; a 458/514 nm excitation dichroic mirror and a 470–500 nm bandpass emission filter. YFP was acquired by using laser excitation at 514 nm, a 458/514 nm excitation dichroic mirror and a 530–570 nm bandpass emission filter. Cherry protein was acquired with a laser excitation at 543 nm, a 488/543/633 nm excitation dichroic mirror, and a 560 nm long pass emission filter. For CFP/YFP or CFP/YFP/Cherry acquisition, images were sequentially acquired in line mode. This minimizes the bleed-through between channels, mainly due to overlapping emission spectra of these fluorochromes.For subcellular distribution analyses and colocalization of GAP-43 proteins with organelle markers, live cell experiments were performed at 35±2°C  and using a 100× UplanSApo oil immersion/1.4 NA  objective . Images were taken using 3 x digital zoom, and an appropriated pinhole to obtain 1 Airy unit for the fluorochrome of shortest wavelength excitation/emission properties . Images of different cells for each dish were taken in a period no longer than 30 min. Images are representative of at least three independent experiments.N13GAP-43(C3S)-YFP and N27GalNAcT-CFP, a TGN marker, or N13GAP-43(C3S)-YFP, N27GalNAcT-CFP and Cherry-APT-1 or Cherry-APT-2, were used. Live cell experiments were performed at 20°C on the Olympus FluoView FV1000 confocal microscope to minimize vesicular trafficking of GAP-43 N27GalNAc-T-CFP, N13GAP-43(C3S) and cherry-APT-1 or cherry-APT-2), images were acquired in the CFP and YFP channels for deacilation kinetic measurements. A 40 x/1.3 NA PlanApo objective oil immersion (Olympus) was used with a 5 x digital zoom. The image resolution was 320x320 pixels, the scan speed was 10 µs/pixel and the pinhole adjusted to obtain an optical slice of 3 µm. Single 2D images were taken at a frequency of 1 min-1, for 30 minutes. These acquisition conditions were optimized to acquire the Golgi compartment, to minimize bleaching during acquisition and to sample as fast as possible the amount of N13GAP-43(C3S) at the TGN.For deacylation measurements, cells coexpressing All image analysis and quantification procedures were done using ImageJ software. Significant fluorescent pixels were isolated from raw images of each channel (CFP and YFP channels) by applying the following equation:sd was obtained from a 50×50 pixel region from an area empty of cells. Negative elements resulting from the above equation are defined as 0 and consequently the elements of the Where ij in the raw image with significant intensity values were selected by A mask i.e, by multiplying raw and mask images using the image calculator tool.N27GalNAcT-CFP images) the background level in the CFP channel was determined around the organelle and not from an area empty of cells since in our conditions a fraction of N27GalNAc-T-CFP was present in the endoplasmic reticulum. After creating each TGN mask, we measured the area of TGN in pixel number units as:For TGN masks -YFP data) by the corresponding of By multiplying each ij pixel of the and the integrated YFP fluorescence intensity inside the organelle is obtained byN13GAP-43(C3S)-YFP concentration is determined by dividing Where the ROI is the same that was employed to determine TGN area. The i as follow:Because we were interested how GAP-43 is distributed between TGN and cytoplasm and the above parameters scaling up with protein expression, we measured the TGN to cytoplasm concentration ratio at each time Where TGN and Cyt refers to the TGN and cytoplasm, respectively. The concentration of GAP-43 at the cytoplasm was determined by measuring the average gray value  of GAP-43 in a small region of interest located in the cytoplasm. Typically, the cytoplasmic ROI area was comparable to that of TGN marker area. Thus, data acquisitions are independent of organelle size and initial steady state conditions. Moreover, these parameters were also independent of GAP-43 expression levels and TGN size, as previously described Kp parameter reach 1 at this time. In this sense, it was considered more appropriate to describe the acylation state measured as the amount of TGN-membrane associated GAP-43 as follow:For GAP-43 deacylation, we considered that after an infinite period of acylation inhibition in the presence of an active deacylation mechanism, the amount of TGN associated GAP-43 completely vanish. At this point, GAP-43 is completely dissociated from TGN and only localizes at the cytosol as a soluble protein. For this reason, we expect that the N13GAP-43(C3S) at time i and 0, respectively.Where Animal handling was performed according to the standards stated in the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care and approved by the local Institution .For hamster tissue mRNA purification and analyses, hamsters were anesthetized and different organs extracted. Then, the tissues were homogenized with an Ultraturrax tissue disintegrator and lysates were passed through a Dounce homogenizer until homogeneity. Then, the cleared lysates were centrifuged at 13000 g and mRNA or genomic DNA were extracted using mRNA and genomic DNA extraction kits from GE healthcare and Sigma-Aldrich, respectively.For tissue cultured cells, 24 h after seeding or differentiation, cells were washed with cold phosphate buffered saline (PBS) and harvested by scrapping in PBS. Extracts were centrifugated at 13000 g for 15 min and mRNA or genomic DNA purified using the commercial extraction kits, according to manufacture instructions. After purification, reverse transcriptase-PCR was performed using 2 µg of purified mRNA as template. Alternatively, PCR was performed using 50 ng of genomic DNA. Primers are described in supplemental http://pfam.sanger.ac.uk/) [#PF02230 (APT-1) and #PF02089 (PPT-1)] First, we performed a Blast search for nonredundant human protein sequences (nonredundant human database) which align to PPT-1 (NP_000301.1) and/or APT-1 (NP_006321.1) protein sequences Figure S1full(C3S).In vivo deacylation of single acylated GAP-43A) CHO-K1 cells transiently expressing GAP-43full(C3S)-YFP [GAP-43full(C3S)] were treated with 50 µM 2-BP (2-BP) or vehicle (Control) for 2 h. Then, cells were lysed, ultracentrifuged and the S (soluble) and P (pellet) fractions isolated. Buffer containing 1% v/v TX-114 was added to samples and the phase separation was induced at 37°C. Proteins from the A (aqueous) and D (detergent) phases were Western blotted with an antibody to GFP. The lowest panels show the Western blot using antibody to α-tubulin. B) Quantification of Western blot showed in A (see in A see . Kp = K1(TIF)Click here for additional data file.Figure S2APT-1 expression analysis in COS-7 and CHO-K1 cells. PCR analysis of APT-1 expression using the following templates: first-strand cDNA obtained from RT of CHO-K1 cells purified mRNA (RT-PCR CHO-K1); the reaction mixture [Ctrl (-)]; total mRNA from CHO-K1 cells ; first-strand cDNA obtained from RT of COS-7 cells purified mRNA (RT-PCR COS-7); genomic DNA from CHO-K1 cells (gDNA CHO-K1) and from COS-7 cells (gDNA COS-7). Genomic DNA and mRNA were extracted from CHO-K1 and COS-7 cells 2 day after seeding in 100 mm Petri dishes.(TIF)Click here for additional data file.Figure S3N13GAP-43 and GAP-43full.APT-1 overexpression did not significantly modify membrane association and deacylation kinetic of diacylated  60 h after transient transfection, CHO-K1 cells coexpressing GAP-43full and APT-1 (A) or N13GAP-43 and APT-1 (B) were treated with 50 µM 2-BP (2-BP) or vehicle (Ctrl) in the presence of CHX and protein degradation inhibitors for 0, 3 and 6 h and the GAP-43 subcellular distribution was analyzed by live cell confocal microscopy. Each panel shows image from YFP fluorescence (pseudocolored gray). The insets show the cells expressing APT-1 . Scale bar: 5 µm.(TIF)Click here for additional data file.Figure S4Analysis of acyl-protein thioesterase expression in SH-SY5Y and SK-Mel 28 cells.A) PCR screening of acyl-protein thioesterase gene expression in SH-SY5Y, treated (RA) or not (Ctrl) with retinoic acid , and SK-Mel 28 human cells (lower panels). For each indicated gene (see gene accession number at the bottom), PCRs were performed using specific primers and the following templates: reaction mixture [Ctrl (-)]; SH-SY5Y or SK-Mel 28 genomic DNA (gDNA) and first-strand cDNA obtained from RT of SH-SY5Y or SK-Mel 28 cells purified mRNA (RT-PCR). B) PCR analysis of isoform expression coded by gene # 11313. For each isoform indicated at the right (with the corresponding accession number), PCRs were performed using specific primers and the following templates: first-strand cDNA obtained from RT of SH-SY5Y [treated (RA) or not (Ctrl) with retinoic acid] or SK-Mel 28 cells purified mRNA (RT-PCR); SH-SY5Y or SK-Mel 28 genomic DNA (gDNA).(TIF)Click here for additional data file.Table S1Bioinformatic data of genes potentially coding acyl-protein thioesterases and PCR primer sequences.(DOC)Click here for additional data file.Table S2Aminoacid and nucleotide sequence alignments of APT-2 from CHO-K1 and Hela cells.(DOC)Click here for additional data file.


Their sporadic distribution has raised a debate as to whether these mobile elements have been vertically or horizontally transmitted among their hosts. The first sponge found to possess a mitochondrial intron was a spirophorid sponge from the Tetillidae family. To better understand the mode of transmission of mitochondrial introns in sponges, we studied cox 1 intron distribution among representatives of this family.Animal mitochondrial introns are rare. In sponges and cnidarians they have been found in the cox 1 sequences were examined. Among these sequences only six were found to possess group I introns. Remarkably, three different forms of introns were found, named introns 714, 723 and 870 based on their different positions in the cox 1 alignment. These introns had distinct secondary structures and encoded LAGLIDADG ORFs belonging to three different lineages. Interestingly, sponges harboring the same intron form did not always form monophyletic groups, suggesting that their introns might have been transferred horizontally. To evaluate whether the introns were vertically or horizontally transmitted in sponges and cnidarians we used a host parasite approach. We tested for co-speciation between introns 723 (the introns with the highest number of sponge representatives) and their nesting cox 1 sequences. Reciprocal AU tests indicated that the intron and cox 1 tree are significantly different, while a likelihood ratio test was not significant. A global test of co-phylogeny had significant results; however, when cnidarian sequences were analyzed separately the results were not significant.Seventeen tetillid cox 1 topologies. In addition, our results suggest that three different intron forms independently colonized the cox 1 gene of tetillids. Among sponges, the Tetillidae family seems to be experiencing an unusual number of intron insertions.The co-speciation analyses thus suggest that a vertical transmission of introns in the ancestor of sponges and cnidarians, followed by numerous independent losses, cannot solely explain the current distribution of metazoan group I introns. An alternative scenario that includes horizontal gene transfer events appears to be more suitable to explain the incongruence between the intron 723 and the  Tetilla sp. SP25456  and Plaiculatus , D. Lavriculatus -17. A retinensis . To confC. levantinensis cox 1 intron was found to be 1,138 bp long [cox 1 sequence of C. levantinensis is not complete. Using the complete cox 1 sequence of Amphimedon queenslandica [C. levantinensis intron was found to be inserted after position 723. This intron encodes a putative LAGLIDADG protein. In P. onkodes two group I introns were found in the cox 1 gene but their secondary structures were not provided [P. onkodes is inserted at the same position as the intron reported for C. levantinensis [C. levantinensis share 81.2% nucleotide sequence identity, have a similar secondary structure, and contain LAGLIDADG ORFs. By contrast, they share only ~43% sequence identity with the first intron of P. onkodes, which does not contain any ORF [The  bp long . Unfortuslandica  as referC. levantinensis intron was more closely related to fungi introns than to any animal intron known at that time [P. onkodes, [C. levantinensis intron and inserted at a different position, was horizontally transferred among actinarian cnidarians.We have previously shown that the hat time . Hence what time . Based ohat time  and in tonkodes,  Fukami eonkodes,  and Wangonkodes,  concludeonkodes,  showed tcox 1 gene of 15 tetillid sponges and present statistical evidence that the C. levantinensis intron was both horizontally and vertically transferred in animals.In order to better understand the origin and evolution of sponge mitochondrial introns we determined the cox 1 sequences from four genera were considered in our analysis: Cinachyrella (9 specimens/6 species), Craniella (five specimens/4 species) Paratetilla (one specimen) and Tetilla (two specimens/two species). To avoid bias we only considered sequences that included all the insertion site positions of both the previously discovered and newly discovered tetillid introns. However, since we did not amplify complete cox 1 sequences, introns present at the beginning  or at the end of the gene (the last 615 - 1074 bp) would not be detected by our study. The complete cox 1 sequence of the sponge A. queenslandica was used as reference for numbering positions in the alignment. The use of reference sequence allowed a comparison of insertion sites between cox 1 CDSs of different lengths, as well as between partial and complete sequences. Among the 15 new sequences only five were found to possess a longer cox 1 sequence, most probably indicating the presence of introns. Only the Cinachyrella alloclada sample was found to have an 1140 bp long insertion after position 723. This insertion is similar in length and insertion site to those previously found for the intron of C. levantinensis and the second intron of P. onkodes , have a 985 bp long insertion that is inserted after position 714  and large P9 (113-116 bp) regions  in their P1 structure . The clade comprising the ORFs of introns 714, in contrast, was not found to be related to any cnidarian sequence. Another unrelated animal clade included only cnidarian sequences. The LAGLIDADG sequences of this clade originated from introns inserted at position 888. Goddard et al. [To identify the evolutionary relationships of the LAGLIDADG sequences encoded within the three intron forms, as well as their putative origin, a phylogenetic tree was reconstructed based on the LAGLIDADG protein sequences encoded within sponge introns together with similar sequences identified by a BLASTP search  see Met. The fir723 cf. ,12,13) b,13 bP. osavaglia , which hd et al.  studied None of the animal LAGLIDADG sequences were found to be closely related (i.e. similarity below 65%) to any fungi or plant sequence.cox 1 sequences  and intron sequences (the sequences included both the LAGLIDADG and the non-coding regions involved in the intron secondary structure) nesting within the cox 1 genes. Such methods are usually used to explore co-speciation between two organisms . In the case of cox 1 sequences and their introns it is indeed possible to consider the introns as parasites of the cox 1 genes. Since in Tetillidae we have three unrelated "parasitic" introns , the history of each intron should be considered separately. Because only a few species were found to possess an intron 714 or an intron 870, our co-speciation analyses were only based on introns 723. The cox 1 and intron 723 sequences of 20 corals and three sponges possessing such introns were considered in these analyses.In order to examine whether the sponge introns were transmitted vertically, we checked for co-evolution between cox 1 ML topology gene .Following Goddard et al. , we firse Figure . Both thcox 1 and LAGLIDADG genes. However, in this test both data sets are not considered separately. The test computes the difference between the likelihood scores obtained when both data sets have different topologies ('two trees model') and the likelihood scores obtained when both data sets have the same topology ('one tree model'). This difference is then compared to a null distribution generated by non-parametric bootstrapping (see Methods). Unlike the AU tests, the likelihood ratio test does not reject the H0 hypothesis that the one-tree hypothesis (both genes have the same topology) is favored over the two-tree model (each gene has a different topology), although marginal significance is observed , albeit low . Only 3 out of 23 pairwise co-speciation links examined were significant, and again the link values were low . These three significant links represented the relationships between each of the sponges sampled and their intron sequences. When these three taxa were removed from the analysis, the global test indicated no co-speciation . The ParaFit result thus suggests that intron 723 could have been vertically transferred in tetillid and homoscleromorph sponges. However, this result is most probably an artifact due to the fact that only three sponges, belonging to different distant classes, were considered in this analysis. In support of such an idea, it is worth noting that the two tetillid LAGLIDADG proteins are closer to the cnidarian sequences than to the homoscleromorph sequence  were found to possess mitochondrial introns. This indicates an unusually high number of introns within this family. More surprisingly, our findings pointed to the existence of three different introns within the tetillid family. Indeed, each intron form has a different insertion site and secondary structure, and the LAGLIDADG they encode belong to unrelated clades.There are two characteristics that might explain the presence of introns in tetillid sponges. First, sponges, similar to cnidarians, have a slower mitochondrial evolutionary rate than bilaterians ,27. Thiscox 1 gene in sponges [cox 1 sequence of C. alloclada using those primers, whereas we successfully amplified this species using different primers. This suggests that "standard" barcoding primers might not be adapted for the sponge and cnidarian species that share introns 714 and 870. It is therefore likely that other sponge lineages might contain mitochondrial introns, in particular those with a budding ability, for example, members the genus Tethya [Our results suggest that the family Tetillidae is a hot spot for the presence of group I introns in animals. However, we cannot exclude the possibility that introns might be overlooked in other sponge lineages. Indeed, two of the discovered introns are located within the reverse primer that has been recommended for the amplification of the barcoding region of the  sponges . As a ca sponges  failed ts Tethya ,37.cox 1 sequence , thecox 1 and intron topologies are significantly different rejecting the hypothesis of co-phylogeny. Based on the ParaFit results none of the host-parasite link is significant within Faviina corals, supporting the absence of co-phylogeny. The ParaFit results are not affected by either the node supports or the topology of the studied genes. It is possible that our ParaFit results are affected by differences in molecular-evolutionary rates between the two genes. Indeed, the cox 1 sequences of the coral Blastomussa wellsi and Physogyra lichtensteini appear to have evolved at a faster rate than other coral species , fou, foucox  Pal2Nal . The und Pal2Nal  with the Pal2Nal  was thennsertion . The coxcox 1 host sequence, could only be performed for intron 723. Other introns did not include enough representatives, except intron 888, which was already studied by Goddard et al. [Three different introns were found in sponges (see Results section). However, co-speciation tests, between intron sequences and their d et al. .cox 1 and intron data sets were respectively 630 and 1078 bp long , with the log-likelihood 1 Lobtained under the hypothesis that each data set evolved independently, resulting in different trees and sets of parameters. Computations were performed with PAUP* under the GTR + Γ + I model for both gene. The log-likelihood ratio statistic (d) is defined as:Three co-speciation tests were conducted. Following Goddard et al.  the firsd was obtained using non-parametric bootstrap. Five hundred bootstrap files were generated separately for the cox 1 and the intron data sets using Mesquite 2.72 [cox 1 bootstrap file was concatenated with one LAGLIDADG bootstrap file. The cox 1 and intron sequences were then randomized within each concatenated file using a Perl script designed for this purpose. Three ML trees were constructed for each data set. The first was based on the first 630 positions of the concatenated and randomized matrix, the second on the last 1078 positions and the third on the total matrix length (1708 bp). The tree reconstructions were performed with PAUP* using rounds of heuristic searches starting with a neighbor-joining (NJ) tree and using tree bisection-reconnection (TBR) branch-swapping. The initial model parameter values were those estimated by Modeltest. After a first round of heuristic search the parameters were estimated on the resulting tree and then used for the subsequent round of heuristic search. The process was repeated until all parameters converged. Ln 0 Lwas computed by assuming that the first 630 bp and the last 1078 bp shared the ML tree obtained using the whole data set . Ln 1 Lwas computed by assuming that the first 630 bp and the last 1078 bp had different trees and different models of evolution. The third approach applied the program ParaFit [cox 1 - intron link. Following the recommendation of the ParaFit manual, the only parameter considered was ParaFitLink1. This parameter is indeed more adapted when the global test is significant but the local tests show a mixed trend. The principle coordinates (PCOs) of the patristic matrices were calculated with DistPCoA [The null distribution of ite 2.72 . Each co ParaFit  to perfoDistPCoA  using thDH conceived the study and supervised the sequencing and analyses. MI collected and identified the biological specimens. AS and CR carried out the sequencing. AS conducted the phylogenetic analysis. AS and DH drafted the manuscript. All other authors assisted in writing the manuscript. All authors read and approved the final manuscript.Tetilla sp. from Israel and Cinachyrella levantinensis from Lebanon18S rRNA sequences of . DNA sequences alignment in nexus format.Click here for filed)The null distribution of the log-likelihood ratio statistic (. The null distribution of the log-likelihood ratio statistic (d) generated by the LRT analysis of co-evolution.Click here for fileOrigin of the tetillid samples used in this study. A table describing the voucher number, GenBank accession number, geographical origin and contributor of each sample sequenced in this study.Click here for filePCR primers used in this study. The sequences of PCR primers and their utilization in this study, specified for each specimen, are described in this file.Click here for fileLAGLIDADG protein alignment. Protein sequence alignment (in Nexus format) used to reconstruct the phylogenetic tree present in Figure Click here for fileCox 1 DNA alignment. DNA sequence alignment (in Nexus format) used to reconstruct the phylogenetic tree present in Figure Click here for filecox 1 sequences from taxa possessing introns 723DNA alignment of the . Cox 1 sequence alignment in Nexus format used in the co-evolution analyses.Click here for fileDNA alignment of the sponge and cnidarian introns 723. Intron sequence alignment in Nexus format used in co-evolution analyses.Click here for file


Gastrointestinal (GI) sarcoidosis is a very rare disease, which clinically presents along with systemic disease or as an isolated finding. Gastric sarcoidosis is the most common form of GI sarcoidosis. Symptomatic gastric sarcoidosis is rare and only few case reports have been described in the literature with well-documented histological evidence of noncaseating granulomas. We present an interesting case of gastric sarcoidosis in a 39-year-old Caucasian man with symptoms of epigastric pain and profound weight loss. His endoscopic gastric mucosal biopsies revealed noncaseating granulomas consistent with gastric sarcoidosis. Treatment with oral steroids alleviated his symptoms with no recurrence in 2 years. Gastric sarcoidosis should be considered in patients with history of sarcoidosis and GI symptoms. Sarcoidosis is a chronic noncaseating granulomatous systemic inflammatory disease. Even though it was first described by Sir Jonathan Hutchinson 140 years ago, to date the etiology is unclear . SymptomHelicobacter pylori, and Mycobacterium (AFB stain) and fungal organisms were all negative. Other laboratory workup to rule out the differential causes also came negative. With history of sarcoidosis, symptoms, and lab workup along with the histological findings the diagnosis was confirmed as gastric sarcoidosis. Prednisone 60 mg per day was started and he had alleviation of symptoms within four days. He was placed on a tapering dose of prednisone for a period of 6 months with no recurrence of symptoms in 2 years.A 39-year-old Caucasian man who is a race car driver by profession presented to the emergency department with a six-month history of nausea, vomiting, and profound weight loss along with one-month history of progressively increasing epigastric pain. His past history included incidental diagnosis of sarcoidosis in spleen and gall bladder one year ago when he met with a motor vehicle accident and underwent elective cholecystectomy and splenectomy. Physical examination was remarkable for mild tenderness in the epigastric region. Rest of the examination was unremarkable. Complete blood counts, comprehensive metabolic panel, and chest X-ray were normal. Computed tomography scan of the abdomen and pelvis revealed extensive adenopathy in the mesentery and retroperitoneum . EsophagMost gastric sarcoidosis cases are asymptomatic. It mainly affects the antrum of the stomach and symptoms can be related to the ulceration of the gastric mucosa or due to the diffuse infiltration and fibrosis of the mucosa leading to the narrowing of the gastric lumen. Epigastric pain 75%) is the most common symptom. Other symptoms are early satiety, nausea, vomiting, hematemesis, melena, and weight loss [% is the Gastrointestinal sarcoidosis can present either as an ulcer or as diffuse involvement resembling linitis plastica. Endoscopy along with biopsies is critical in the diagnosis of the gastric sarcoidosis. Depending on the pathology, endoscopic findings can differ. With diffuse infiltration of the mucosa it can appear as linitis plastica (leather bottle) as in our case. In other pathologies we can see mucosal ulcers with or without erythema and polypoid/nodular lesions (due to granulomas). In asymptomatic patients the gastric mucosa can be normal and can be easily overlooked.Mycobacterium, syphilis, histoplasmosis, gastric cancer, lymphoma, Langerhans cell histiocytosis, foreign body reaction, and Crohn's and Whipple's diseases.Gastric sarcoidosis is unique as it can mimic other GI diseases in presentation and its diagnosis requires proper interpretation of the biopsies as many other etiologies can present with noncaseating granulomas. In the differential diagnosis we need to consider peptic ulcer disease, Ménétrier disease, hypertrophic gastritis, Treatment depends on the symptoms. Asymptomatic patients do not need any specific therapy. Steroids are the initial treatment of choice in symptomatic patients with or without proton pump inhibitors depending on the presence of ulcers. In a recent report 66% of patients had alleviation of symptoms with steroids [Gastric sarcoidosis should be considered in patients with history of sarcoidosis and complaints of epigastric pain and unexplained weight loss.


Environmentally transmitted pathogens face ecological interactions  in the outside-host environment and host immune system during infection. Despite the ubiquitousness of environmental opportunist pathogens, traditional epidemiology focuses on obligatory pathogens incapable of environmental growth. Here we ask how competitive interactions in the outside-host environment affect the dynamics of an opportunist pathogen. We present a model coupling the classical SI and Lotka–Volterra competition models. In this model we compare a linear infectivity response and a sigmoidal infectivity response. An important assumption is that pathogen virulence is traded off with competitive ability in the environment. Removing this trade-off easily results in host extinction. The sigmoidal response is associated with catastrophic appearances of disease outbreaks when outside-host species richness, or overall competition pressure, decreases. This indicates that alleviating outside-host competition with antibacterial substances that also target the competitors can have unexpected outcomes by providing benefits for opportunist pathogens. These findings may help in developing alternative ways of controlling environmental opportunist pathogens. Traditionally, ecological and eco-evolutionary epidemiological models describe the dynamics of infectious diseases by considering susceptible, infected and recovered hosts with host-to-host, or host-environment-host transmission en masse to prevent environmental infections to humans and cultivated animals. This is likely to cause changes in the composition of environmental communities and have an impact on ecosystem functioning, health and disease The role of environmental transmission in disease dynamics and the evolution of virulence has attracted increasing interest Vibrio cholerastreptococciStaphylococcus aureusFlavobacterium columnareInterspecific interactions, such as competition, mutualism, predation, and parasitism constitute the core of ecological research. An important implication of these interactions is that the dynamics and stability of individual populations within ecological networks  can strongly depend on the composition of these networks and the details of between-species interactions We explore the dynamics of a model that combines environmental opportunist pathogen–host dynamics to community dynamics outside the host. By the term environmental opportunist pathogen we mean an organism that is both (i) able to grow in the environment in the absence of hosts and (ii) infect susceptible hosts. Whether the pathogen can infect one or several host species is not important in this simple model in which we consider all (susceptible) hosts similar from the pathogens perspective. The model contains susceptible and infected hosts as in a classical SI-model and a competitive community in the outside-host environment. One of the competitors is a pathogen that can return from a dead host to the environment and thus the host does not represent an ecological or evolutionary dead end for the pathogen. This is opposite to the common assumption of the theory of “co-incidental virulence” A striking feature of the model is that reducing competitor species richness in the outside-host environment can lead to an abrupt emergence of disease outbreaks. If the infectivity response of the pathogen is a sigmoidal function of pathogen density in the environment, epidemiological dynamics are sensitive to the intensity of competitive suppression by the outside-host community. With linear infectivity response and pathogen growth in the environment no disease outbreaks are observed, i.e. population dynamics remain stable, and pathogen and host densities are relatively insensitive to manipulation of diversity. Under sigmoidal infectivity, reduction of competitive pressure on the pathogen, either due to loss of diversity from the outside-host community , or increased loss rate of all species in the outside-host community  can lead to catastrophic disease outbreaks.t are denoted by S(t) and I(t), respectively. The pathogen, P(t), has n competitors, with densities denoted by iB(t), i.e., community size is N = n +1. The population densities vary according to the following differential equations:The dynamical model is a combination of the classical SI-disease dynamics hr and a carrying capacity hK (eqn. 1.1). In the presence of an infective pathogen infected host individuals are formed. The infected individuals compete for resources with the susceptible individuals, but do not contribute to host reproduction . Susceptible hosts are infected with a rate βSf(P) depending on the infectivity response f(P). Two alternative infectivity response functions were explored. Most previous theoretical work has assumed a linear infectivity response. However, we argue that a more realistic assumption for many circumstances is a sigmoidal dose-dependent response that is supported by empirical data f(P), because it is a simple way to conceive this type of dose-dependence for infections ID50 and κ affects the shape of the response. f(P) = P was used for comparison. In either case, the infectivity rate is scaled by parameter β in eqn (1.1). In eqn. (1.2) infected hosts die from the disease with rate ν and recover from the infection with rate δ. With the parameter set in In the absence of an infection, susceptible hosts follow a logistic growth model with a growth rate P) and the competing non-pathogenic strains (iB) grow logistically with rates pr and br. Carrying capacities are pK and bK in a Lotka–Volterra competition setting, respectively . The competition in the outside-host community was modeled with a very general diffuse competition model where the competitive ability of non-pathogens against the pathogen was varied. The intraspecific competition coefficients were set to 1, whereas between species interaction strength is given by The pathogen . If hosts are sparse, intense outside-host competition in addition to out-flow mortality η can drive the pathogen extinct while waiting for the next infection. The effect competition has on the pathogen species could be realised either through reduction in pathogen competitive ability (α) or increasing the number of competitors present in the outside host community (n). The equilibrium pathogen density reaches zero with α = 1/(2n) in the absence of the host, due to competition. The survival of the pathogen beyond this level of competition is still possible via coupling to host dynamics due to fitness benefits gained by inside-hosts growth. On the other hand if the pathogen is very infective and lethal, it may kill all the suitable hosts and thereafter become extinct by competitive exclusion. This outcome resembles that of the classical trade-off between virulence and transmission, albeit via an entirely different mechanism. Equilibrium densities for the pathogen and non-pathogenic species in the absence of hosts are:r is the common growth rate for all species. Note that P* is only positive when r>η.A mortality term with rate The model assumes that a burst of pathogens is released to the environment with the death of an infected host. This identifies to saprotrophy where the dead host body is consumed to some degree. As the dead host typically represents an extremely rich resource in comparison to the typical outside-host environment The pathogen growth rate of two divisions per day represents the lower end of bacterial growth rates. The infected hosts remain infected for a relatively long period since both infection kill rate and recovery rate are low. This is plausible e.g. for an untreated bacterial disease many of which are very persistent. The host growth rate is suitable for a fish host 6 leads to a scenario where the unit of area could be a cubic metre of water with 100 small fish hosts and 1010 bacterial cells. The infectivity parameter values used here allow for effective infecting without driving the host extinct too easily. Extensive simulations with randomly selected parameter values indicate that the results presented here are qualitatively robust  behaviour was analysed numerically using a Runge-Kutta fourth order routine. Means, maxima, and standard deviations of population densities were recorded from the final 100 time units from each simulation in order to assess the type of the dynamics (e.g. outbreaks). The analysis focused on the asymptotic behavior of the model and excluded the initial transients. Trajectories were simulated for 200 time units that was a sufficiently long time interval for analysing the asymptotic behaviour of the model in all cases. The initial state in all simulations was The choice of the infectivity response function is crucial to the behaviour of the model. Therefore the following results are presented according to this dichotomy between linear and sigmoidal infectivity responses.f(P) = P, pr< η) and that there was no significant recovery of infected hosts. Linear response stabilises the dynamics because it prevents host supply re-growth by making the pathogen efficient in infecting also when the pathogen density is low. While pathogen infections reduce the number of susceptible hosts well below their carrying capacity  or competitive disadvantage (α) decreases the equilibrium density of the pathogen (eqn. 3). Under sigmoidal transmission this means that when the pathogen is unable to survive in the outside-host environment without fitness gains from host infection, the presence of susceptible hosts can be associated with two alternative attractors, with pathogens either present or absent. When competition is intensive enough, pathogens are unable to reach sufficiently high densities for infections to arise, inevitably leading to pathogen extinction.Similar patterns to those shown in eter set . This isη) in outside-host environment reduces the competitive pressure from the rest of the outside-host community on the pathogen. When this density-independent mortality is intermediate, the pathogen can thrive in environments where competition would otherwise drive it to extinction  The shape of the infectivity response has a strong impact on the dynamical behavior of the system; (2) Under a sigmoidal dose-dependent pathogen infectivity response reducing species richness of non-pathogenic competitors in the outside-host environment provides a novel mechanism for disease outbreaks; (3) With the sigmoidal infectivity too high pathogen virulence can lead to host extinction, which leads to virulence becoming useless for the pathogen.The importance of biodiversity on the stability and functioning of ecological communities continues to motivate ecological research These results are in agreement with numerous empirical observations promoting the importance of biodiversity for disease dynamics. For example, biodiversity loss has been associated with both increases and decreases in disease transmission Our results indicate that the way increasing competitive pressure on the pathogen in the outside-host community affects pathogen dynamics depends crucially on the shape of the pathogen’s infectivity response  , S1. A cSimilarly to the direct host-to-host transmission of obligatory pathogens studied by Regoes et al. As indicated above, the shape of the infectivity response at low pathogen densities has a profound impact on the dynamics. If infectivity is low (and increases slowly) at low pathogen densities, the pathogen needs to reach relatively high densities in the environment to become infective. This can in turn be prevented by, e.g., ecological interactions in the outside-host environment that reduce pathogen densities . The importance of the shape of infectivity response in environmental transmission has been recognized by Boldin & Kisdi Outside-host growth of pathogens is widespread in nature. Models of environmentally transmitted diseases typically allow only exponential decay of pathogens Under a linear infectivity response density-dependent pathogen growth in the outside-host environment effectively filters the inflow pathogens from infected hosts, resulting in stable population dynamics Flavobacterium columnare, a saprotrophic fish pathogen that causes considerable economical losses in fisheries Serratia marcescens and Pseudomonas sp. both capable of infecting a wide range of hosts.Multi-cellular hosts are high resource environments with a potentially deadly immune system for parasites. Parasites often have to allocate considerable amount of resources for infecting and overcoming the immune system, and sacrifice part of their competitive ability in the outside host environment Opportunism coupled with the ability to grow in the free-living environment, may be an important step in the evolution of virulence for bacteria. This naturally requires that pathogens are released from the infected host to the environment, i.e., the host is not an ecological or evolutionary dead end. Free-living bacteria may develop infectivity but lack the means for effective host-to-host transmission. Thus they would benefit from infectivity only if they are virulent enough to gain reproductive output from the host and survive in the environment until encountering a new host. The waiting time before a new infection may be long and the costs of maintaining infectivity traits are likely to restrict the growth rate of the pathogen. If the resources are sparse and there is competition for them, the non-pathogenic competitors are likely to out-compete the pathogen, and this may lead to local pathogen extinction.Applying a non-specific mortality factor upon the outside-host community can reduce competitive pressure on the pathogen. While the density of all species, including the pathogen, is reduced equally in the outside-host environment, the pathogen gets an indirect advantage through within-host reproduction . This suFigure S1Equilibrium pathogen and susceptible host densities as a function of competitive disadvantage of the pathogen (α). Black dots represent mean densities. At cyclic ranges open circles represent an alternative attractor and filled symbols indicate minima and maxima. Here the number of competitors is fixed to n = 7 and linear mortality parameter η to 1.0. Linear infectivity response with rate constant β/2ID50 was used in panels (A) and (B), and sigmoidal response (response  in panel(TIF)Click here for additional data file.Figure S2Mean pathogen densities versus competitive disadvantages of the pathogen (α) from sensitivity analysis replicates. In panel (A) with linear infectivity response red dots represent individual simulation outcomes. In panel (B) with sigmoidal infectivity response cyan dots represent outcomes from simulations resulting in stable dynamics (s.d.(P) <10). Red and blue dots represent outcomes from simulations resulting in cyclic dynamics (s.d.(P) >10). To distinguish between alternative outcomes, values above 1000 are coloured red and those below 1000 are blue. Grey dots represent standard deviations (s.d.(P)) above and below the mean value. The replicates resulting in host extinction (mean(S) <5) have been excluded from both panels. Panel (A) has 50720 points and panel (B) has 86615 coloured points of which 14159 are cyclic and 72456 are stable. Total number of simulations is 100000 in both cases.(TIF)Click here for additional data file.Figure S3S) >5)Posterior parameter distributions from sensitivity analysis replicates not resulting in host extinction , host growth rate (hr), host recovery rate (δ), infected death rate (ν), and pathogen release (k) were picked from uniform random distributions. Selecting the cases with host persistence resulted in 50720 replicates in the upper panels (linear infectivity) and 86615 replicates in the lower panels  from a total of 100000 replicates for each infectivity response.(TIF)Click here for additional data file.Materials S1Sensitivity analysis.(DOCX)Click here for additional data file.


This homing intron encodes an endonuclease thought to promote the intron's promiscuous behavior. A promising experimental approach to study endonuclease activity and intron transmission involves somatic cell hybridization, which in plants leads to mitochondrial fusion and genome recombination. However, the cox1 intron has not yet been found in the ideal group for plant somatic genetics - the Solanaceae. We therefore undertook an extensive survey of this family to find members with the intron and to learn more about the evolutionary history of this exceptionally mobile genetic element.The most frequent case of horizontal transfer in plants involves a group I intron in the mitochondrial gene cox1 intron, it is uniformly present in three phylogenetically disjunct clades. Despite strong overall incongruence of cox1 intron phylogeny with angiosperm phylogeny, two of these clades possess nearly identical intron sequences and are monophyletic in intron phylogeny. These two clades, and possibly the third also, contain a co-conversion tract (CCT) downstream of the intron that is extended relative to all previously recognized CCTs in angiosperm cox1. Re-examination of all published cox1 genes uncovered additional cases of extended co-conversion and identified a rare case of putative intron loss, accompanied by full retention of the CCT.Although 409 of the 426 species of Solanaceae examined lack the cox1 intron was separately and recently acquired by at least three different lineages of Solanaceae. The striking identity of the intron and CCT from two of these lineages suggests that one of these three intron captures may have occurred by a within-family transfer event. This is consistent with previous evidence that horizontal transfer in plants is biased towards phylogenetically local events. The discovery of extended co-conversion suggests that other cox1 conversions may be longer than realized but obscured by the exceptional conservation of plant mitochondrial sequences. Our findings provide further support for the rampant-transfer model of cox1 intron evolution and recommend the Solanaceae as a model system for the experimental analysis of cox1 intron transfer in plants.We infer that the  All relevant studies . Finally, with respect to the probability of intron gain, the cox1 intron is so clearly a highly mobile intron that to postulate one additional horizontal transfer (two gains within Solanoideae rather than one), when more than 80 such events have already been documented [We strongly favor the latter model. First, consider the probability of loss versus gain of the lanaceae   that cox1 is otherwise virtually identical across the Solanoideae ..per se, cox1 that A) lacked the intron, B) lacked the CCT, and C) was retained in many descendant lineages throughout at minimum the first 15 million years of early Solanoideae diversification [cox1 were protected from homing-mediated intron spread by being sheltered in another compartment or organism should it persist in an intron-less state. "Another compartment" basically means the nucleus , but a  Figures  given thBrunfelsia jamaicensis acquired its cox1 intron independently of Mandragora and Hyoscyameae, and it is likely that these latter two lineages acquired their introns via independent horizontal transfer events, in which case the intron has been acquired at least three times during Solanaceae evolution  and, imcox1 intron evolution postulates over 100 losses across angiosperms of the multi-character CCT, with each CCT loss accompanied by intron loss. To account for these many concomitant losses, Cusimano et al. [cox1 coconversion tract is usually lost during the intron excision process...most likely...by reverse transcription-mRNA-mediated coconversion." There is, however, no published evidence that any reverse transcriptases engage in co-conversion and, even if they did, the cox1 mRNAs that would mediate this putative co-conversion would still possess the CCT and therefore the CCT region would be unaffected. Furthermore, although plant mitochondrial intron loss is indeed an RNA-mediated process, known as "retroprocessing" [cox1 data, both across angiosperms and within the Solanaceae. For instance, the many lineages of intron-lacking Solanaceae  contain C at the closest RNA edit site to the intron  CCT augments two previously reported cases of intron loss unaccompanied by CCT loss [et al. [et al.'s proposed model for CCT loss is both mechanistically implausible and fails to fit any of the observed cox1 data.We will, however, confront more explicitly the issue of CCT evolution, because it is so fundamental to interpretation of the gain/loss history of the intron in the Solanoideae. Cusimano et al.'s all-loss model of o et al.  proposedcessing" ,46, thisCCT loss  and furt [et al.  that retcox1 genes that harbor the homing group I intron in question [cox1 genes sometimes extends considerably further than this, at least 35-81 bp downstream of the intron in four different intron clades, and raises the possibility that 5' co-conversion might also occur. In a sense, these results are not surprising, given experimental studies in such diverse systems as yeast mitochondria (including the cognate cox1 intron), Chlamydomonas chloroplasts, and phage T4, which have shown that CCTs are commonly hundreds and sometimes thousands of bp in length, and are often found on both sides of a newly arrived intron [appear to be so short in angiosperm cox1 genes. Appearances may be deceiving here: the combination of exceptionally low mutation rates in most plant mitochondrial genomes [cox1 sequence evolution [cox1 sequences, even across angiosperms, that CCTs of dozens to hundreds of bp in length could easily go undetected, and probably often do.Previous studies recognized a short  3' CCT motif, and no 5' CCT, in angiosperm question ,10,12,15d intron ,18-20. M genomes -36,44 anvolution  results That the great majority of intron-containing angiosperms show no evidence of 5' co-conversion and only 18-21 bp of 3' co-conversion may be largely a consequence of the crucial horizontal transfer event that first introduced this intron into angiosperms. Assuming the donor in this event was a fungus ,14, thenMandragora and Hyoscyameae, whose current distributions are restricted largely to Eurasia, with a few species found in northern Africa [Mandragora is restricted to the Mediterranean-Turanian region, while the intron-containing clade of Hyoscyameae has a broader distribution, with two subclades also restricted to the Mediterranean-Turanian region but other lineages found in various parts of Asia. Given this, and the very close relationship between the introns of these two clades . If so, then there is no basis for favoring transfer in one direction vs. the other. This is because current estimates of divergence times for the two groups [Mandragora and Hyoscyameae leave open the possibility that intron transfer between the two groups occurred via a shared mycorrhizal associate or pollinator, or by illegitimate pollination.The center of diversity (and most likely the place of origin) of the Solanaceae is in the New World, with a minimum of 8 dispersal events to the Old World inferred from phylogenetic studies and overall distribution . Among tn Africa . The int Figures  and 5, io groups  fail to Mandragora intron type is unclear based on intron phylogeny  and also both extant species of Cynomorium among over 200 examined intron-containing angiosperms representing an estimated 80+ intron acquisitions. We therefore predict that any angiosperms found to contain a more closely related intron to the Hyoscyameae/Mandragora type will also have the same, extended CCT. The association with Cynomorium is intriguing, given the frequent transfer, in both directions, of mitochondrial genes between parasitic plants and their hosts [Mandragora and one or both species of Cynomorium.The non-Solanaceae donor of the Hyoscyameae/y Figure . HoweverT Figure  with onlir hosts ,49,50. ABrunfelsia may have acquired the intron quite recently, as B. jamaicensis is the only one of 7 species examined in the genus found to possess it. However, in the absence of any solid estimates of phylogeny and divergence times for the genus and of comprehensive sampling of the 40-50 species in the genus, the timing and location of transfer and the phylogenetic distribution of the intron within the genus are uncertain. It will be interesting to determine the relationship of B. jamaicensis to the 5 other species of Brunfelsia endemic to the Caribbean island of Jamaica, none of them yet sampled, and whether any of them also possess the intron.cox1 intron was acquired by horizontal transfer on at least 3 separate occasions during the evolution of the Solanaceae. One lineage of intron-containing Solanaceae may have acquired its intron from another lineage in the family, consistent with previous evidence that horizontal transfer in plants is biased towards phylogenetically local events. Discovery of these transfers was dependent on extensive sampling of the family. This underscores the importance of greatly expanded sampling of angiosperms in general in order to gain a deeper understanding of the intron's evolutionary history, including not only an accurate estimate of the number and timing of its many transfers but also to untangle to the extent possible mechanisms of transfer and donor-recipient relationships for specific transfer events.Multiple lines of evidence lead us to conclude that the cox1 intron, which encodes a homing endonuclease, is an exceptionally mobile genetic element in angiosperms. These results, together with the discovery of a rare case of likely loss of this intron accompanied by retention of the CCT, provide still further support for the long-standing, rampant-transfer model for the evolution of this intron in angiosperms [Our findings strongly reinforce the idea that the iosperms -12 and riosperms  even morcox1 co-conversions may be longer than realized but obscured by the exceptional conservation of plant mitochondrial sequences. This is also consistent with the hypothesis that the intron's founding arrival in angiosperms, probably from a fungal donor, was aided by unusually short co-conversion, thereby minimizing the potentially deleterious effects of creating a chimeric, fungal/plant form of the key respiratory protein encoded by cox1. The discovery of the cox1 intron in 3 distinct lineages of the Solanaceae opens the door to experimental, somatic-cell genetic studies on the transmission and co-conversion properties of this intron in plants. Cybrids have been reported between tobacco, which lacks the intron, and two species in the intron-containing Hyoscyameae clade [The identification of exonic co-conversion tracks substantially longer than those previously recognized for this intron in angiosperms implies that other ae clade ,51 and mPlant materials were collected by different researchers from around the world. Seeds of various species were obtained from the Nijmegen Botanical Garden. Plant DNAs were either extracted from fresh or dried leaves using a cetyl-trimethyl-ammonium-bromide DNA-extraction protocol  or obtaicox1, a PCR/gel sizing assay was performed using two primers - cox1-3 (5'-CATCTCTTTYTGTTCTTCGGT-3') and cox1-6 (5'-AGCTGGAAGTTCTCCAAAAGT-3') - that amplify most of exon 2 and a small portion of exon 1, yielding products of either 800 bp (if the intron is absent) or 1.8 kb (if the intron is present). For selected species, additional amplifications were done with primers cox1-1 (5'-AYGAMAAATCYGGTYGATGG-3') and cox1-4 (5'-ACCGRATCCAGGCAGAATGRG-3'), which amplify most of exon 1 and a small portion of exon 2, yielding products of either 750 bp or 1735 bp. Selected PCR products were sequenced using an ABI 3730 (Applied Biosystems). Sequencing primers included PCR primers and two additional primers, both located within the intron: cox1-10 (5'-TGACTACTATCAAAGTAGA-3') and cox1-8 (5'-GTAGAGTCTTATAAGGTAGT-3'). GenBank accession numbers of sequences determined in this study are listed in Additional File To survey the presence/absence of the group I intron in Sequences were aligned manually with MacClade 4.0 . Editingcox1 intron sequences and 108 cox1 exon sequences, all from angiosperms. GenBank accession numbers of cox1 sequences obtained from NCBI are listed in Additional File cox1 exon character matrix. Maximum likelihood analyses of the intron and exon data sets were performed with Garli 0.951 [Phylogenetic analyses were performed on data sets of 71 li 0.951  under thli 0.951 . Ten indMandragora, Melia and Cynomorium, and B) monophyly of the introns from Brunfelsia jamaicensis, Hyoscyameae, and Mandragora. The most likely tree under each constraint was determined by searching for the best tree compatible with that constraint using PAUP* [The approximately unbiased (AU) test was used to test whether a particular intron-based topology is significantly better than a specified (constrained) alternative topology. The CONSEL package  was usedng PAUP* . The sitAU: approximately unbiased; CCT: co-conversion tract; HGT: horizontal gene transfer; ORF: open reading frameThe authors declare that they have no competing interests.MVSP co-designed the study, generated some of the data, analyzed the data, designed the figures, and co-wrote the manuscript. CCA and SZ obtained most of the DNA sequences generated for the study. ET, LB and RGO provided almost all of the DNAs used in the study, performed most of the PCR survey, and commented on the manuscript. JDP co-designed the study and co-wrote the manuscript. All of the authors read and approved the final manuscript.List of taxa from the family Solanaceae examined in this study. Taxonomic information, geographic origin or source (if known), collection number (voucher herbarium), and GenBank accession numbers of taxa from the family Solanaceae examined in this study.Click here for filecox1 gene alignmentThe . Nucleotide alignment of the cox1 gene (including its intron sequence) for all taxa included in the phylogenetic analysis shown in Figure 4. Sites of predicted RNA editing are in red in the reference sequence, while the putative endonuclease ORF is in green.Click here for fileTaxonomic information and GenBank accession numbers. Taxonomic information and GenBank accession numbers of all taxa included in the analyses shown in Figures 3 and 4.Click here for fileBrunfelsia sppPhylogenetic tree of . based on chloroplast data. Maximum likelihood phylogeny of 7 species of Brunfelsia based on analysis of chloroplast ndhF and trnLF. Numbers above branches are bootstrap support values > 50%. GenBank numbers for sequences generated here are shown in boldface. Primers used for sequence amplification are from Olmstead et al [ad et al .Click here for file


In the third sentence of the second paragraph of the Methods, Korean characters were inadvertently changed to questions marks during the typesetting process. The publisher apologizes for the error. The correct sentence is: "Using the survey results, the definition of ILI and meetings of the authors, we picked 12 queries: new influenza (신종인플루엔자 in Korean), influenza (인플루엔자), new flu (신종플루), flu (플루), swine flu (돼지독감), bird flu (조류독감), H1N1 (H1N1), bad cold (독감), Tamiflu (타미플루), fever (열), cough (기침), and sore throat (인후통)."


Phosphorylated and truncated TAR DNA-binding protein-43 (TDP-43) is a major component of ubiquitinated cytoplasmic inclusions in neuronal and glial cells of two TDP-43 proteinopathies, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Modifications of TDP-43 are thus considered to play an important role in the pathogenesis of TDP-43 proteinopathies. However, both the initial cause of these abnormal modifications and the TDP-43 region responsible for its aggregation remain uncertain. Here we report that the 32 kDa C-terminal fragment of TDP-43, which lacks the RNP2 motif of RNA binding motif 1 (RRM1), formed aggregates in cultured cells, and that similar phenotypes were obtained when the RNP2 motif was either deleted from or mutated in full-length TDP-43. These aggregations were ubiquitinated, phosphorylated and truncated, and sequestered the 25 kDa C-terminal TDP-43 fragment seen in the neurons of TDP-43 proteinopathy patients. In addition, incubation with RNase decreased the solubility of TDP-43 in cell lysates. These findings suggest that the RNP2 motif of RRM1 plays a substantial role in pathological TDP-43 modifications and that it is possible that disruption of RNA binding may underlie the process of TDP-43 aggregation. TARDBP, the gene encoding TDP-43, in familial and sporadic ALS and/or FTLD patients further confirmed the importance of this molecule in the pathogenesis of TDP-43 proteinopathies Amyotrophic lateral sclerosis (ALS) and certain forms of frontotemporal lobar degeneration (FTLD) with ubiquitin-positive but tau-negative inclusions have been considered to be a single disease spectrum of TAR DNA-binding protein 43 (TDP-43) proteinopathy since 2006, when this protein was reported to be a major component of ubiquitin-positive inclusions in the affected neuronal and glial cells of both ALS and FTLD Although TDP-43 normally localizes to the nucleus, it is distributed from nucleus to cytoplasm or neurite and forms aggregates that mainly consist of C-terminal fragments (CTFs) in the affected neurons of TDP-43 proteinopathy patients. In addition, aberrantly aggregated TDP-43 is hyperphosphorylated at multiple C-terminal sites Although it does not fully recapitulate the pathological features of TDP-43 proteinopathies, artificial axonal damage induces transient cytoplasmic distribution of TDP-43 in mouse motor neurons These findings provide us with a clue for elucidating the mechanism of these modifications. Nevertheless, both the initial cause of these abnormal modifications and the region of TDP-43 responsible for its aggregation remain unknown. Here we report that RNP2 in RNA binding motif 1 (RRM1) plays a substantial role in the pathological TDP-43 modifications that are seen in TDP-43 proteinopathies.Mouse NSC34 motor neuron-like cells  Human wild-type TDP-43 (WT-TDP-43) (accession number NM 007375) cDNA was amplified by PCR from human spinal cord cDNA as previously described g for 15 min. To prevent carryover, the pellets were washed with RIPA buffer, followed by sonication and centrifugation. RIPA-insoluble pellets were lysed in 100 µl urea buffer , sonicated, and centrifuged at 100,000 g for 15 min.We used NE-PER Nuclear Cytoplasmic Reagents (Thermo Fisher Scientific) for the analysis of the cytoplasmic/nuclear ratio. For analysis of protein solubility, cells cultured in 10-cm plates were lysed in 1 ml RIPA buffer (Thermo Fisher Scientific). Lysates were sonicated and centrifuged at 100,000 2PO4 and blocked with 4% BSA. Secondary antibody probing and detection were performed using ECL Plus detection reagents . Chemiluminescence signals were digitized  and band intensities were quantified using Multi Gauge software .After the denaturation, 5 µl of each sample was separated by SDS-PAGE (5%–20% gradient gel) and the proteins were then transferred to Hybond-P membranes (Amersham Pharmacia Biotech). The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 and incubated with the intended primary antibodies. The primary antibodies used were as follows: anti-TDP-43 rabbit polyclonal antibody ; anti-pTDP-43 (phospho Ser409/410) rabbit polyclonal antibody ; anti-ubiquitin mouse monoclonal antibody (MBL); anti-histone H1 mouse monoclonal antibody ; anti-GAPDH mouse monoclonal antibody ; anti-GFP mouse monoclonal antibody ; and anti-V5 mouse monoclonal antibody . For the anti-ubiquitin antibody, the membranes were fixed with 0.05% glutaraldehyde/0.1M KHNSC34 cells were fixed in 4% paraformaldehyde, incubated in PBS containing 0.3% Triton X-100 for 5 min, blocked with Image-iT FX signal enhancer (Invitrogen), and incubated overnight at 4°C with anti-TDP-43 rabbit polyclonal antibody , anti-pTDP-43 (phospho Ser409/410) rabbit polyclonal antibody , anti-TIAR mouse monoclonal antibody , anti-ubiquitin mouse monoclonal antibody , anti-V5 rabbit polyclonal antibody  or anti-V5 mouse monoclonal antibody (Invitrogen). After washing, samples were incubated with Alexa-488-conjugated goat anti-mouse IgG and Alexa-555-conjugated goat anti-rabbit IgG  for 60 min, mounted with Prolong Gold antifade reagent with DAPI (Invitrogen), and then imaged with a confocal microscope .For the counting of inclusion-bearing cells, we randomly selected 100 transfected cells from three separate experiments. The colocalization coefficient, which reflects the fraction of green pixels that are also positive for red pixels, was calculated using the Zeiss LSM software. We calculated the colocalization coefficient by randomly selected 10 fields from three separate experiments. To obtain images for calculating the colocalization coefficient, the settings of the confocal microscopy and the threshold of positive/negative fluorescence was fixed within each experiment.Transfected HEK293 cells were washed with PBS and lysed in immunoprecipitation buffer (Thermo Fisher Scientific). After sonication on ice, the samples were agitated for 30 min at 4°C. The samples were centrifuged and supernatants were incubated with magnetic beads: anti-V5 magnetic beads (MBL), anti-GFP magnetic beads (MBL), and anti-ubiquitin magnetic beads (MBL). Samples were rotated overnight at 4°C. Immunoprecipitates were separated by SDS-PAGE (5%–20% gradient gel). Western blotting was performed using anti-V5-HRP antibody (MBL) and anti-GFP-HRP antibody (MBL).ACCAACCAGTTGAGTTCCAGAT (forward) and GAATGATTCACATTGCCGTAGA (reverse).Ribonucleoprotein (RNP) immunoprecipitation was performed using a RIP assay kit (MBL), according to the manufacturer's instructions. RNA concentrations were measured with a Nanodrop (Thermo Fisher Scientific). Electrophoresis of precipitated RNA was performed with a Bioanalyzer (Agilent Technologies) according to the manufacturer's instructions. For analysis of neurofilament light chain (hNFL) mRNA 3′UTR content, RNA obtained from immunoprecipitates was reverse transcribed into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and a PCR was performed with the following primers: g for 15 min. To prevent carryover, the pellets were washed with TS buffer, followed by sonication and centrifugation. TS-insoluble pellets were lysed in 1 ml of Triton X-100 (TX) buffer (TS buffer containing 1% Triton X-100), sonicated, and centrifuged at 100,000 g for 15 min. The pellets were washed with TX buffer, followed by sonication and centrifugation. TX-insoluble pellets were lysed in 500 µl of Sarkosyl (Sar) buffer (TS buffer containing 1% Sarkosyl), sonicated and centrifuged at 100,000 g for 15 min. Sar-insoluble pellets were lysed in 100 µl of urea buffer.For analysis of protein solubility with or without RNase, HEK293 cells cultured in 10-cm plates were lysed in 1 ml of Tris-saline (TS) buffer . Lysates were sonicated and then divided into two samples. RNase A (10 µg/ml) was added to one of the samples. Samples incubated for 0 and 15 h at 4°C were centrifuged at 100,000 t-test or one-factor factorial ANOVA followed by Tukey post hoc tests. A p value of 0.05 or less was considered to be statistically significant.Statistical analyses were performed using GraphPad Prism software (GraphPad software inc.). Biochemical data were statistically analyzed using a Student's To identify the region of TDP-43 that is responsible for the pathological modification of this protein, we created various TDP-43 mutants and investigated their intracellular localizations. In particular, given that the CTFs of TDP-43 form aggregates in the cytoplasm of affected neurons, we focused on the mutant TDP-43 in which the NLS is disrupted (dNLS) and on TDP-43 CTFs: 35 kDa (TDP35), 32 kDa (TDP32), and 25 kDa (TDP25) fragments . We examAlthough wild-type TDP-43 showed a nuclear-dominant distribution, the dNLS mutant localized to the cytosol more preferentially than wild-type TDP-43 . The CTFThe relatively low steady-state levels of TDP32 and TDP25 prompted us to explore the possibility that these CTFs form aggregates. Given that the expression levels of TDP25 are substantially lower than other forms of TDP-43, we first focused on dNLS, TDP35, and TDP32. In immunocytochemical analysis using anti-ubiquitin and -V5 antibodies, wild-type TDP-43 chiefly localized to the nucleus, but a substantial amount of dNLS and TDP35 distributed to the cytosol . TDP32 aIn contrast, ubiquitin-positive aggregates were virtually undetectable in the cells expressing TDP35 . Since Tp<0.001; To quantitatively analyze the relationship between ubiquitin and TDP-43 CTFs, we calculated the colocalization coefficient of ubiquitin and V5 immunofluorescence. We found that the colocalization coefficient of ubiquitin and V5 was significantly higher in TDP32-expressing cells than in those expressing TDP35 p<0.001; . We alsop<0.01; p<0.001; Like ubiquitination, insolubilization and phosphorylation are characteristics of TDP-43 proteinopathies. Therefore, we investigated TDP-43 CTF solubility and phosphorylation, finding that although the cytosolic aggregates of TDP32 were well stained with anti-phosphorylated TDP-43 (pTDP-43) antibody, the other forms showed no detectable pTDP-43-containing aggregates . The quaThe phenotype of TDP32 was distinctly different from that of TDP35, although both of which lack the NLS, span amino acids 85–414 and 112–414, respectively . Althougp<0.001; To investigate the role of the RNP2 motif in the modification of TDP-43, we created two defective mutants, ΔRNP2 and mtRNP2 . While Δp<0.05; Immunoprecipitation analyses showed that mtRNP2 inclusions were strongly ubiquitinated in comparison with those of wild-type TDP-43 . InclusiSmall CTFs of 18–26 kDa accumulate in the cytosol of affected neurons of TDP-43 proteinopathies Next we investigated whether the 18–26 kDa TDP-43 fragments are included in the aggregates of the CTFs we created. Following prolonged exposure of the immunoblots of TDP-43 mutants, we found that 25 kDa and shorter fragments were detected in the insoluble fraction of TDP32 and mtRNP2, but not TDP35, using pan-TDP-43 antibody . These fSince 25 kDa and shorter CTFs were detected in the insoluble fraction of mtRNP2, we assumed that mtRNP2 binds to, and thereby sequesters, the 25 kDa CTF. To test this hypothesis, experiments using the 25 kDa and 14 kDa CTFs of TDP-43 were performed . We co-tNext we performed immunoprecipitation to examine the binding of small CTFs to mtRNP2. The results showed that TDP25 binds to V5-mtRNP2, but not to the V5-mtRNP2 (1–273) that lacks the C-terminal domain of TDP-43 . Since TThe RNP2 motif of RRM1 is important for its RNA binding ability Based on the observation that the disruption of the RNP2 motif increases the aggregation of TDP-43, we hypothesized that the decreased binding to RNA leads to the formation of insoluble aggregates of TDP-43. Therefore, we investigated the effects of RNase on the properties of the endogenous TDP-43. To increase the detection sensitivity of insolubilized TDP-43, we used a mild buffer and four-step fractionation. The results demonstrated that the amount of urea-insoluble endogenous TDP-43 was increased from that of the start sample (0 h) by a 16-h incubation with RNase .The ubiquitin-positive, phosphorylated inclusion of TDP-43in neuronal cytoplasm is a pathological hallmark of TDP-43 proteinopathy. Since this inclusion contains 18–26 kDa CTFs of TDP-43 that do not have the RRM1 We first focused on the physical features of two types of TDP-43 CTFs, TDP35 and TDP32. The cells expressing TDP32 only exhibited ubiquitin-positive and phosphorylated aggregations. In addition, the immunoblots also showed that TDP32, but not TDP35, was insoluble and phosphorylated, suggesting that the RNP2 motif in the RRM1 is responsible for the process of TDP-43 aggregation. Although the cells expressing TDP35 occasionally appeared to form cytoplasmic aggregates, those were the components of stress granules and were neither insoluble nor ubiquitinated. On the other hand, the cells expressing TDP25 did not form aggregates in our experimental conditions, although the TDP25 did not contain the RNP2 motif. This discrepancy might be explained by our observation that the disrupted RNP2 motif and the remaining RRM1, but not RRM2, are both required for the aggregations of TDP-43. In addition, the finding that ΔRRM1 TDP-43 does not form aggregates also supports this hypothesis.Since the RNP2 motif in the RRM1 is responsible for the ability of TDP-43 to bind RNAs with specific sequences We have discussed the fact that the CTFs did not, except for TDP32, aggregate in normal conditions, whereas full-length TDP-43 could aggregate when RNA binding is disrupted. However, a question is raised as the major components of aggregated TDP-43 in TDP-43 proteinopathy patients are CTFs, such as TDP25. Although TDP25 did not by itself aggregate in our system, the cells expressing mtRNP2 or TDP32 contained ∼25 kDa phosphorylated TDP-43 in their insoluble fractions. In addition, in the cells co-expressing mtRNP2 and TDP25, TDP25 formed aggregates and colocalized with mtRNP2, suggesting that mtRNP2 sequesters TDP25 in the aggregations.On the other hand, mtRNP2 (1–273), which lacks the C-terminal domain, did not have the ability to sequester TDP25. The C-terminal domain of TDP-43, in which most of the disease mutations are located, contains a glutamine/asparagine-rich (Q/N-rich) domain; also referred to as the prion-like domain, it is involved in the self-assembly of misfolded CTFs and the sequestration of TDP-43 into polyglutamine aggregates In summary, we demonstrated that the RNP2 motif in RRM1 plays a substantial role in pathological TDP-43 modifications and that disruption of RNA binding may underlie the process of TDP-43 aggregation.Figure S1Immunocytochemistry of NSC34 cells expressing TDP35 or TDP32. Cells were stained with anti-V5 (red) and anti-TIAR (green) antibodies. The aggregates with TDP35, but not TDP32, colocalized with TIAR. Scale bar = 5 µm.(TIF)Click here for additional data file.Figure S2The colocalization coefficient of the ubiquitin and V5 signals. Cells were stained with anti-V5 (green) and anti-ubiquitin (red) antibodies. Colocalization with ubiquitin was significantly higher in the cells expressing TDP32 than in those bearing TDP35 (p<0.001). Scale bar = 5 µm. Error bars indicate SEM (n = 3).(TIF)Click here for additional data file.Figure S3The colocalization coefficient of the pTDP-43 and V5 signals. Cells were stained with anti-V5 (green) and anti-pTDP-43 (red) antibodies. Colocalization with pTDP-43 was significantly higher in the cells bearing TDP32 than in those expressing TDP35 (p<0.001). Scale bar = 5 µm. Error bars indicate SEM (n = 3).(TIF)Click here for additional data file.Figure S4Immunocytochemistry of NSC34 cells expressing ΔRNP2 or mtRNP2. Cells were stained with anti-V5 (red) and anti-TIAR (green) antibodies. TIAR did not colocalize with the aggregates of ΔRNP2 or mtRNP2. Scale bar = 5 µm.(TIF)Click here for additional data file.Figure S5The colocalization coefficient of the ubiquitin and V5 signals. Cells were stained with anti-V5 (green) and anti-ubiquitin (red) antibodies. Colocalization with ubiquitin was significantly higher in the cells expressing mtRNP2 than in those bearing wild-type TDP-43 (p<0.001). Scale bar = 5 µm. Error bars indicate SEM (n = 3).(TIF)Click here for additional data file.Figure S6The colocalization coefficient of the pTDP-43 and V5 signals. Cells were stained with anti-V5 (green) and anti-pTDP-43 (red) antibodies. Colocalization with pTDP-43 was significantly higher in the cells expressing mtRNP2 than in those with wild-type of TDP-43 (p<0.05). Scale bar = 5 µm. Error bars indicate SEM (n = 3).(TIF)Click here for additional data file.Figure S7Biological features of non-tagged mtRNP2 and V5-tagged mtRNP1. (A) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing non-tagged wild-type and mtRNP2 TDP-43. (B) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing V5-tagged wild-type and mtRNP1 TDP-43.(TIF)Click here for additional data file.Figure S8Intracellular localizations of N-terminal fragments of TDP-43 with mutated RNP2 and TDP-43 lacking RRM1. (A) Structures of mtRNP2, mtRNP2 (1–273), mtRNP2 (1–185), TDP (1–105), and ΔRRM1 TDP-43. (B) Images of NSC34 cells expressing V5-mtRNP2 (1–273), mtRNP2 (1–185), TDP (1–105), and ΔRRM1 TDP-43. The cells bearing mtRNP2 (1–273) and mtRNP2 (1–185), but not mtRNP2 (1–105) or ΔRRM1, formed aggregates. Scale bar = 10 µm. (C) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing wild-type and ΔRRM1.(TIF)Click here for additional data file.Figure S9Effect of tag on TDP-43 insolubilization. (A) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing GFP-tagged wild-type and CTFs of TDP-43. (B) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing non-tagged wild-type and CTFs of TDP-43.(TIF)Click here for additional data file.Figure S10Lack of interaction between TDP-43 mutants and IgG/beads. Immunoprecipitaions with mouse IgG from cells expressing wild-type and mutations of TDP-43.(TIF)Click here for additional data file.Table S1Primers for TOPO cloning.(DOCX)Click here for additional data file.Table S2Primers for mutagenesis.(DOCX)Click here for additional data file.