P = 0.018) was significantly associated with less contralateral lung metastasis in multivariate analysis. Micropapillary predominant  was significantly associated with brain metastasis. Acinar predominant  was a significant prognostic factor for better contralateral lung metastasis-free survival in multivariate analysis. Micropapillary predominant  and solid predominant  were significant prognostic factors for worse brain metastasis-free survival and liver metastasis free-survival, respectively. There are significant differences in metastatic behavior between predominant pathological subtypes of lung adenocarcinoma. This information is important for patient follow-up strategy and identification of organ-specific distant metastasis. Prospective multi-institutional studies are mandatory for further validation.The aim of the study is to demonstrate the relationship between clinicopathological variables and organ sites of metastasis in resected lung adenocarcinoma. The clinicopathological characteristics of 748 patients of resected lung adenocarcinoma at Taipei Veterans General Hospital between 2004 and 2012 were retrospectively reviewed. The prognostic value of clinicopathological variables for specific organ site metastasis-free survival was demonstrated. Among the 182 patients with distant metastasis, 93 (51.1%) patients developed contralateral lung metastasis, 81 (44.5%) had brain metastasis, 71 (39.0%) had bone metastasis, and 18 (8.9%) had liver metastasis during follow-up. Acinar predominant (Hazard ratio [HR], 0.468; 95% confidence interval [CI]: 0.250 to 0.877;  Lung cancer is the main cause of cancer-related death worldwide . SurgicaOrgan tropism, also known as the seed-and-soil hypothesis, was first proposed by Stephen Paget in 1889 . Solid tIn our previous studies , 23, we The median follow-up time for all the 748 patients was 33.6 months . The median number of mediastinal lymph node dissection/sampling was 18.0 . The characteristics of these patients were listed in Table 2 test and the paired independent sample t-test to investigate the relationship between specific organ sites of distant metastasis and clinicopathological variables (Table P = 0.005) was associated with less contralateral lung metastasis. Micropapillary (P = 0.002) and solid (P = 0.018) subtype predominant were associated with more contralateral lung metastasis. Micropapillary (P < 0.001) and solid (P = 0.024) subtype predominant were associated with brain metastasis. Micropapillary (P = 0.012) and solid (P < 0.001) subtype predominant were also associated with bone metastasis. Solid subtype predominant (P < 0.001) was associated with liver metastasis.We first performed χP = 0.006), N2 (vs. N0 or N1) (P = 0.046), and stage II or III (vs. stage I) (P = 0.002) were significantly associated with more contralateral lung metastasis in multivariate analysis. Acinar subtype predominant  was significantly associated with less contralateral lung metastasis. Greater tumor size (P = 0.017), stage II or III (vs. stage I) (P = 0.014), angiolymphatic invasion (P = 0.037), and micropapillary subtype predominant  were significantly associated with more brain metastasis in multivariate analysis. Greater tumor size (P = 0.002), N2 status (vs. N0 or N1) (P = 0.011), and angiolymphatic invasion (P = 0.003) were significantly associated with more bone metastasis in multivariate analysis. Angiolymphatic invasion (P = 0.031) was significantly associated with more liver metastasis in multivariate analysis. Solid subtype predominant (P = 0.059) showed a trend toward being significantly associated with more liver metastasis.We further performed univariate and multivariate logistic regression analyses for specific organ sites of distant metastasis Table . GreaterP = 0.011) was a significant prognostic factor for worse CLMFS, while acinar subtype predominant  was a significantly prognostic factor for better CLMFS in multivariate analysis (Table P = 0.002) and micropapillary subtype predominant  were significantly worse prognostic factors in multivariate analysis . Greater tumor size  and angiolymphatic invasion  were significantly worse prognostic factors in multivariate analysis. For LMFS, angiolymphatic invasion  and solid subtype predominant  was a significantly worse prognostic factor in multivariate analysis  were divided into two groups according to number of positive risk predictors: none positive or one positive (group 1) (n = 541), and two positive (group 2) (n = 15). Kaplan-Meier analysis showed that patients with both micropapillary subtype predominant and angiolymphatic invasion had significantly worse BrMFS than others (P < 0.001)  were divided into two groups according to number of positive risk predictors: none positive or one positive (group 1) (n = 540), and two positive (group 2) (n = 16). Kaplan-Meier analysis showed that patients with both solid subtype predominant and angiolymphatic invasion had significantly worse LMFS than others (P < 0.001) . For all these patients, the 3-year overall survival and disease-free survival rates were 96.0% and 94.0%, respectively. Twenty-two (5.0%) of the 438 patients developed distant metastasis during follow-up. Among the 22 patients with distant metastasis, there were 6 (27.3%) patients with acinar predominant, 2 (9.1%) with papillary predominant, 4 (18.2%) with micropapillary predominant, and 10 (45.5%) with solid predominant adenocarcinoma. The median time to recurrence for the 22 patients was 12.0 months . Among the 22 patients, 9 (40.9%) patients developed contralateral lung metastasis, 7 (31.8%) had brain metastasis, 8 (36.4%) had bone metastasis, and 3 (13.6%) had liver metastasis during follow-up. Eleven (50.0%) of the 22 patients had local recurrence.P < 0.001) was significantly associated with more contralateral lung metastasis in multivariate analysis. N2 (vs. N0 or N1) (P = 0.003) and micropapillary subtype predominant  were significantly associated with more brain metastasis. Stage II or III (vs. stage I) (P = 0.014) was significantly associated with more bone metastasis. Solid subtype predominant (P = 0.025) was significantly associated with more liver metastasis in univariate analysis. Multivariate analysis was not performed for liver metastasis due to the small number of patients with liver metastasis (n = 3).We first performed univariate logistic regression analyses for specific organ sites of distant metastasis in the validation cohort . The bacThis study demonstrated that pathological subtypes of lung adenocarcinoma are associated with organ-specific metastasis in patients of resected lung adenocarcinoma with distant metastasis. Acinar, micropapillary, and solid predominant adenocarcinomas are significantly associated with contralateral lung metastasis, brain metastasis, and liver metastasis, respectively. Acinar, micropapillary and solid predominant adenocarcinomas are also significant prognostic factors for CLMFS, BrMFS and LMFS, respectively. By combination of risk factors, stage I patients with micropapillary subtype predominant and angiolymphatic invasion have significant worse BrMFS. Those with solid subtype predominant and angiolymphatic invasion have significant worse LMFS.The new classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011 was a significant prognostic factor for survival and recurrence in lung adenocarcinoma –21. In oThe lung, brain and bone are the most common organ sites of metastasis in resected NSCLC , 15, 16.Although liver was not a common organ site of metastasis in lung cancer, liver metastasis was not rare during follow-up after surgery. Approximately 9% of patients with distant metastasis developed liver metastasis in our study. The predictor of liver metastasis from lung cancer has not been reported. In the study, we have demonstrated that solid predominant adenocarcinoma tended to be significantly associated with a higher frequency of liver metastasis. Solid predominant adenocarcinoma was a significant prognostic factor for worse LMFS. For stage I lung adenocarcinoma, patients with both solid subtype predominant and angiolymphatic invasion had significantly worse LMFS. In the validation cohort, solid subtype predominant adenocarcinoma was significantly associated with more liver metastasis in univariate analysis. Although multivariate analysis was not performed due to the small number of patients with liver metastasis, the association between solid predominant adenocarcinoma and liver metastasis was worthy of further investigation in the future. The results are important because patients with solid predominant adenocarcinoma may undergo more frequently liver imaging study during follow-up for early diagnosis of liver metastasis.The associations between age, sex, tumor size, N status, tumor stage, visceral pleural invasion, angiolymphatic invasion, or adjuvant therapy and organ site of metastasis from lung cancer have not been well demonstrated. In the current study, female is a significant prognostic factor for better BoMFS. Greater tumor size was associated with more contralateral lung, brain, and bone metastasis. Greater tumor size was also a significant prognostic factor for worse CLMFS and BoMFS. N2 status (vs. N0 or N1) was associated with more contralateral lung and bone metastasis. Stage II or III (vs. stage I) was associated with more contralateral lung and brain metastasis. Angiolymphatic invasion was associated with more brain, bone, and liver metastasis. Angiolymphatic invasion was also a significant prognostic factor for worse BrMFS and BoMFS. No significant association between other conventional clinicopathological variables and organ sites of metastasis was identified.There are some limitations and biases of this study that should be mentioned. As a retrospective single institute study, patient selection bias and time trend bias were inevitable. Another limitation is the diagnostic bias that conventional imaging would not necessarily detect all metastatic disease. Subclinical metastases may be missed simply because imaging not performed. There was also bias in defining a new primary lung cancer from a recurrent NSCLC. The major strength of this study is that a full and detailed follow-up of organ-specific metastasis made the analyses for specific organ site metastasis-free survival possible. Furthermore, we have conducted a validation cohort for internal validation. However, prospective multi-institutional studies are mandatory to further validate the prognostic value of the predominant pathological subtypes of lung adenocarcinoma on organ-specific metastasis after surgical resection.In conclusion, this study demonstrates significant differences in metastatic behavior between predominant pathological subtypes of lung adenocarcinoma. Acinar predominant adenocarcinoma is associated with less contralateral lung metastasis. Micropapillary and solid predominant adenocarcinomas are significantly associated with brain and bone metastasis, respectively. This information is important for patient follow-up strategy and further study of molecular mechanisms leading to organ-specific metastasis in lung adenocarcinoma.This study has been approved by the Institutional Review Board of Taipei Veterans General Hospital. From January 2004 to December 2012, all patients underwent completely resection for lung adenocarcinomas at Taipei Veterans General Hospital were retrospectively reviewed. Patients undergoing neoadjuvant chemotherapy or with stage IV disease were excluded. Patients with incomplete clinical information and follow-up were also excluded. A total of 801 patients were eligible for the study. Among them, 566 (70.7%) patients were free of tumor recurrence and 235 29.3%) patients developed recurrence during follow-up. Only two patients with recurrence were diagnosed as lepidic predominant adenocarcinoma. The number of patients of lepidic predominant adenocarcinoma developing recurrence was small as compared with the other four subtypes of adenocarcinoma. Therefore, the 2 patients with lepidic predominant adenocarcinoma were excluded. Fifty-one (21.9%) of the remained 233 patients developed local only recurrence during follow-up, and was excluded for analysis. The remained 182 patients developed distant metastasis during follow-up. The 182 patients with distant metastasis and the 566 patients without tumor recurrence were included for analysis in the current study. The preoperative staging work-up was routinely performed as previously described , 23. Med.3% patieAll resected specimens were formalin fixed and stained with hematoxylin and eosin, and were evaluated microscopically as previously described . Each tuAll patients were followed-up at our outpatient department quarterly in the first 2 years after resection and semi-annually thereafter. The modalities and protocols during follow-up were used as previously described , 22, 23.To investigate their impact on specific organ site metastasis-free survival, clinicopathologic factors were examined in univariate and multivariate analyses. Local recurrence was defined as tumor recurrence in contiguous anatomical sites, including the ipsilateral hemithorax and mediastinum after surgical resection. Distant metastasis was defined as tumor recurrence in the contralateral lung or outside the hemithorax and mediastinum after surgical resection. Local only recurrence was defined as only local recurrence identified from initial operation to death or last follow-up. Distant only metastasis was defined as only distant metastasis discovered from initial operation to death or last follow-up. Secondary primary lung cancer was differentiated from recurrent NSCLC in patients undergoing surgical resection or biopsy according to the criteria proposed by Girard et al . For thoTo confirm the findings of the study, we conducted a validation cohort for further verification. From January 2013 to December 2014, all patients underwent completely resection for lung adenocarcinomas at Taipei Veterans General Hospital were retrospectively reviewed. The same exclusion criteria used in the original cohort were applied in the validation cohort. A total of 438 patients were eligible. Among them, 416 (95.0%) patients were free of tumor recurrence and 22 (5.0%) patients developed distant metastasis during follow-up.2 test and the paired independent sample t-test were used as appropriate. To investigate their association with specific organ sites of metastasis, clinicopathological factors were analyzed in univariate and multivariate logistic regression. For specific organ site metastasis-free survival, univariate and multivariate analyses were performed by means of the Cox proportional hazards model using SPSS software . All variables with P < 0.1 in univariate analysis were entered into multivariate analysis. Age and sex were also entered for mutual adjustment despite P > 0.1. Statistical significance was defined as P < 0.05.The specific organ site metastasis-free survival was calculated by the Kaplan-Meier method . The log


Bemisia tabaci Q biotype is an emerging global invasive species that has caused extensive damages to field crops as well as ornamental plants.ABC transporter superfamily is one of the largest and ubiquitous groups of proteins. Because of their role in detoxification, insect ABC transporters have gained more attention in recent years. In this study, we annotated ABC transporters from a newly sequenced sweetpotato whitefly genome. B. tabaci Q genome, including 8 ABCAs, 3 ABCBs, 6 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 23 ABCGs and 9 ABCHs. In comparison to other species, subfamilies G and H in both phloem- and blood-sucking arthropods are expanded. The temporal expression profiles of these 55 ABC transporters throughout B. tabaci developmental stages and their responses to imidacloprid, a neonicotinoid insecticide, were investigated using RNA-seq analysis. Furthermore, the mRNA expression of 24 ABC transporters  representing all eight subfamilies was confirmed by the quantitative real-time PCR (RT-qPCR). Furthermore, mRNA expression levels estimated by RT-qPCR and RNA-seq analyses were significantly correlated .A total of 55 ABC transporters containing all eight described subfamilies (A to H) were identified in the B. tabaci. The identification of these ABC transporters, their temporal expression profiles during B. tabaci development, and their response to a neonicotinoid insecticide lay the foundation for functional genomic understanding of their contribution to the invasiveness of B. tabaci.It is the first genome-wide analysis of the entire repertoire of ABC transporters in The online version of this article (doi:10.1186/s12864-017-3706-6) contains supplementary material, which is available to authorized users. Drosophila melanogaster, exists in arthropods and zebrafish but not in mammals, plants and fungi [ATP-binding cassette (ABC) transporters constitute one of the largest protein superfamily and exists in all living organisms –3. Thesend fungi –12.Most ABC transporters encodes membrane-bound proteins that carry a wide range of molecules  across membranes . ABC traD. melanogaster [Daphnia pulex [Bombyx mori [Tribolium castaneum [Tetranychus urticae [et al. suggested that ABC transporters were involved in the permethrin resistance in a malaria vector, Anopheles stephensi [Helicoverpa armigera [.The advent of Genomics Era has greatly improved our understanding of the diversity and function of invertebrate ABC transporters, including 56 in the fruit fly nogaster , 64 in tia pulex , 51 in tbyx mori , 73 in tastaneum  and 103 tephensi . Similararmigera .Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a phloem-feeding insect pest that cause substantial damages through feeding on over 600 host plants as well as by transmitting over 100 plant viruses [B. tabaci is generally considered as a species complex consisting of many biotypes or putative species with different biological and genetic characteristics [B. tabaci biotypes are the Q  and B  [B. tabaci has demonstrated a remarkable ability to develop resistance to insecticides in the field, including organophosphates, carbamates, pyrethroids, neonicotinoids and juvenile hormone mimics [B. tabaci biotype replacement events in China  [B. tabaci. To archive this goal, we 1) provided the first genome-wide analysis of the entire repertoire of ABC transporters in the whitefly B. tabaci Q; 2) investigated the temporal expression profiles of these ABC transporters in different developmental stages; and 3) documented their transcriptomic response to imidacloprid, a neonicotinoid insecticide.The sweetpotato whitefly,  viruses . As one eristics . The two, MEAM1) . B. tabae mimics –24. Insein 2009) . The oveB. tabaci Q genome  in the blood-sucking Cimex lectularius. The percentage reduces to 21% (18% + 3%) in the red flour beetle, T. castaneum. The expansion of ABCG and ABCH subfamilies in these polyphagous and phloem-sucking arthropods, including the whitefly B. tabaci, the wheat aphid D. noxia and the spider mite T. urticae, suggest the potential contribution of ABC transporters to their adaptability and invasiveness. Phylogenetic analysis categorized the 55 B. tabaci ABC transporters into eight subfamilies   form a sister-group with a high bootstrap support. This sister-group clustered with D. noxia DnXP_015366480.1 (ABCA3), T. japonicas TjABCA3 and seven T. urticae genes. These genes then clustered with D. melanogaster DmCG31731 with a moderate bootstrap support. DmCG31731 was down-regulated in the salivary glands of a D. melanogaster E93 mutant [D. melanogaster development [T. castaneum [The 3 mutant . As an eelopment . InjectiB. tabaci Q, we identified three ABCB half-transporters but no full-transporters  belong to three clades, indicating that these genes are homologous to ABCB genes in human, fruit fly and other insects , T. urticae (Tu17g02000), T. japonicas (TjABCB8) and H. sapiens (hABCB8).In rs Table . These BH. sapiens, hABCB6, hABCB7 and hABCB8/MABC1 are mitochondrial transporters with roles in iron metabolism and transportation of Fe/S protein precursors [Drosophila melanogaster DmCG4225 (ABCB6) was tolerant to cadmium [C. elegans ABTM- 1, a homologue of ABCB7, induced oxidative stress and premature cell death [H. sapiens ABCB7 in Aedes aegypti was up-regulated in an insecticide resistant strain [D. pulex, B. tabaci Q do not have homologous genes of human ABCB half-transporters that were associated with antigen processing  [In ecursors , 31. Dro cadmium . Disruptll death . Furthert strain . Disruptt strain . Similar hABCB9) .B. tabaci Q, we identified six ABCC full-transporters, including Btabq019529.2, Btabq000311.1, Btabq008004.1, Btabq026695.3, Btabq004618.1 and Btabq003933.1. ABCC subfamily proteins are full-transporters with diverse functions, such as cell-surface receptor, ion transport and translocation of a broad range of substrates including drugs and endogenous compounds [H. sapiens, “long” MRPs like ABCC1, 2, 3, 6 and 10  have a TMD0, the third N-terminal transmembrane-spanning domain, while “short” MRPs like ABCC4, 5, 11 and 12  lack TMD0 [Insects typically have different number of ABCC transporters Table . In B. tompounds –39. Due ompounds . In H. sack TMD0 .B. tabaci ABCC1 genes, Btabq019529.2 and Btabq000311.1, clustered with D. noxia DnXP_015366208.1, DnXP_015366209.1, D. melanogaster DmCG6214, T. japonicus ABCC1 and a group of H. sapiens “long” MRPs   channels [D. melanogaster epidermis, where chitin disruption was observed [D. melanogaster suggested that chitin synthesis was independent of SUR [T. castaneum did not result in a phenotype [Manduca sexta but also participate in host plant selection in T. urticae [Btabq004618.1 clustered with or drugs . In addichannels , 49. Altchannels , it did observed . Moreovet of SUR . Similarhenotype . ABCC suBemisia tabaci Q has two ABCD transporters, Btabq026746.1 and Btabq017051.1 . The ABCG transporters in metazoans are half-transporters and can be a functional transporter only after dimerization. In plants and fungi, however, ABCGs are full-transporters, also called pleiotropic drug resistance proteins (PDRs) , 62. BecB. tabaci  [Pediculus humanus humanus) [Drosophila [A. mellifera [et al. [D. pulex and D. melanogaster genomes was resulted from extensive lineage specific gene duplications.ABCG proteins represent the largest ABC subfamily 23 members) in ci Table  and alsoD. noxia , An. gam gambiae , bed bugularius) , human bhumanus) , Drosophosophila  and A. mellifera   , a 20-hydroxyecdysone (20E)-induced ABC transporter, regulated metamorphosis, probably by removing 20E from cells [T. castaneum [Btabq001288.1 and Btabq001290.1 clustered with DnXP_01579755.1, DnXP_015379710.1, DmCG11069, DmCG31121, Tu01g16280, TjABCG5, hABCG5 and hABCG8. hABCG5 and hABCG8 mediated the intestinal and biliary efflux of cholesterol and sterols . A clearom cells . SimilarDrosophila as pigment precursor transporters  [B. tabaci ABCG proteins, Btabq014028.1, Btabq015484.1, Btabq002474.1 and Btabq022510.1, showed homologous relationships with these pigment precursor transporters in D. noxia, An. gambiae and D. melanogaster. Besides transporting pigment precursors, these transporters also function in courtship behavior [Pxwhite gene was tightly linked to Cry1Ac resistance in Plutella xylostella [Among invertebrates, the functions of ABCGs were first characterized in e genes) , 72. In behavior , transpobehavior . RNAi-melostella .B. tabaci, suggesting the involvement of these genes in insecticide resistance [T. urticae but also reduced the chemical defense from host plants [B. mori, at least five ABCG genes acted as 20-hydroxyecdysone (20E)-response genes and participated in hormonal regulation [B. tabaci Q, however, contains predominantly G, but few B and C family members  linked ABC transporters with insecticides-resistance . Memberssistance . In addit plants . In B. mD. melanogaster and have only annotated in arthropods and zebrafish [C. elegans, plants and mammals [As inverse half-transporters, ABCH proteins share the same domain architecture as ABCG transporters. ABCH transporters were first discovered in ebrafish , 13, 17. mammals , 54, 62. mammals .B. tabaci. Phylogenetic analysis showed that ABCH subfamily members were species-specific. Bemisia tabaci ABCH clustered with genes in D. noxia with a high bootstrap value , was the most up-regulated ABC transporter [TcABCH-9C, an ABCH in T. castaneum, resulted in 100% larval mortality and a significant reduction of fecundity and hatching rate. In addition, T. castaneum larvae injected with TcABCH-9C dsRNA showed a lack of lipids in their epicuticle, indicating that TcABCH-9C may be required for the formation of a waterproof barrier in the epicuticle [Helicoverpa armigera and Manduca sexta, the expression of ABCH subfamily was highly induced after larvae fed with secondary metabolites [A total of nine ABCH transporters were identified in s lethal , 81. In nsporter . Knockdoicuticle . In the abolites , 53.B. tabaci life stages , showed high expression level throughout the life cycle [TcABCE-3A transcripts were abundant in all developmental stages of T. castaneum [T. japonicas, and sea lamprey, Petromyzon marinus, showed similar trends as well [Btabq014578.1 (ABCF2) had the highest expression level throughout the life cycle. ABCE1, ABCF1 and ABCF2 were highly expressed in all developmental stages of B. tabaci. ABCF3, also ubiquitously expressed, exhibited a lower expression level. These gene expression patterns were consistent with the fundamental functions in ribosome biogenesis and translation regulation [B. tabaci.Based on RNA-seq data, all 55 ABC transporters were expressed with a RPKM (Reads Per Kilobases per Million reads) >1 in at least one of the ges Fig. . In the fe cycle . Moreoveastaneum . The exp as well , 84. Btagulation , 85, 86.B. tabaci were relatively low with the exception of Btabq003232.1 (ABCA3), which was highly expressed in all life stages  expressed in all developmental stages, especially in the fourth nymph stage and adult. Btabq013065.1 (ABCB8) expressed in all stages but at a lower level than other ABCB genes. Homo sapiens ABCB half-transporters, ABCB6, ABCB7, ABCB8 and ABCB10, were localized at mitochondria and functioned in iron metabolism [The expression levels for ABCA transporters in tabolism .B. tabaci Q, and the expression patterns were different among these genes .The expression profiles of 24 selected ABC transporters were confirmed by the RT-qPCR Fig. . Within B. tabaci  and Phase II , ABC transporters represent a major class of Phase III enzymes.H. armigera defense against secondary metabolites such as nicotine and tomatine [H. armigera was the results of nicotine ingestion [An. stephensi was up-regulated in response to pyrethroid insecticide [C. lectularius [B. tabaci metabolic resistance.ABC transporters, especially those in the ABCG subfamily, facilitated tomatine . The higngestion . In addiecticide , 69. Thetularius . Then kntularius . ABC tratularius . OverallB. tabaci Q genome. ABC transporter subfamilies G and H are expanded in B. tabaci Q genome. The expression profiles of these ABC transporters were validated using both RT-qPCR and RNA-seq analyses. Furthermore, 80% of the significantly up-regulated ABC transporters in imidacloprid-challenged B. tabaci were ABCGs. These combined results imply the potential contribution of ABCGs to the adaptability of this emerging global invasive pest.In this study, we identified 55 ABC transporters in the sweetpotato whitefly Bemisia tabaci Q biotype  population maintained on cotton plant at 27 ± 1 °C, with a photoperiod of 16 h light: 8 h darkness and 70 ± 10% relative humidity. Every 3–5 generations, the purity of the strain was monitored using a mitochondrial cytochrome oxidase I (mtCOI) marker [) marker .B. tabaci Q on cotton were collected to be treat with imidacloprid following adult leaf-dip bioassay method [B. tabaci adults were added to each tube. After 48 h, adults were collected, snap frozen in liquid nitrogen, and transferred to −80 °C for the long-term storage. Three biological replications for all B. tabaci samples were carried out independently. A total of 100 individuals were collected for each biological replication, and these samples were subjected to RNA-seq analysis.The different developmental stage samples  were collected from the Q biotype population described above. Newly emerged (within 5 days) adults of y method , 93. In B. tabaci Q genome [D. melanogaster as queries to search against the updated GLEAN gene in B. tabaci genome with an E-value threshold of 10−5 to identify ABC transporters. Each putative ABC transporter gene was confirmed by subjected it to BLASTX analysis with the non-redundant protein sequence (NR) at GenBank (http://www.ncbi.nlm.nih.gov/).To identify open reading frames (ORFs) encoding putative ABC transporters, we carried out tblastn searches against the Q genome  and tranhttp://smart.embl-heidelberg.de/) [The exon/intron boundary and start/stop codons of each ABC gene were confirm from the genome and transcriptome. The conserved domains of these identified ABC proteins were predicted using SMART   and conferg.de/) .B. tabaci, we collected the readily available ABC transporters from the fruit fly, D. melanogaster, wheat aphid, D. noxia, mosquito, An. gambiae, spider mite, T. urticae, intertidal copepod, T. japonicus, human, H. sapiens, nematode, Caenorhabditis elegans, and yeast, Saccharomyces cerevisiae. NBDs of B. tabaci ABC transporters were used to resolve their phylogenetic relationships at the subfamily level. Within each ABC subfamily, we used the full-length protein sequences to construct their respective phylogeny trees [To analyze the evolutionary placement of ABC transporters in ny trees , 10. Addny trees . The phyB. tabaci transcript and each sample were derived and were normalized to Reads Per Kilobase of transcript per Million mapped reads (RPKM). Differentially expressed genes  were identified by the DESeq package [Expression profiling of ABC transporters was assessed using transcriptome data of different developmental stages and imidacloprid-treated whitefly as described earlier. ABC genes were also verified by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions . RNA was quantified using a Nanodrop 2000  meanwhile purity was checked on 1% agarose gels. RNA-seq libraries were constructed as previously described  and sequ package –103.B. tabaci Q biotype were confirmed using RT-qPCR analysis as well. Based on the RPKM value generated from the RNA-seq data, we selected 24 transcripts representing all eight subfamilies of the B. tabaci Q biotype ABC transporters for the RT-qPCR validation study  and TAF10 RNA polymerase II (TAF) were selected as reference genes [−ΔΔCt method [Besides transcriptomic validation, mRNA expressions of ABC transporters in ce genes , 105. Tht method . SPSS 19Additional file 1: Figure S1.Bemisia tabaci and other organisms. (PDF 23 kb)Comparison of gene numbers in each subfamily of ABC transporters between Additional file 2: Figure S2.Bemisia tabaci ABCA subfamily with other organisms. Full-length ABC transporters were aligned using ClustalW and subjected to a maximum likelihood analysis by MEGA5 [B. tabaci (red); Dn, D. noxia (blue); AGA, A. gambiae (green); Dm, D. melanogaster (purple); Tu, T. urticae (light blue); Tj, T. japonicus (black); h, H. sapiens (gray); Ce, C. elegans (brick-red); Sc, Saccharomyces cervisiae (orange). (PDF 28 kb)Phylogenetic relationship of by MEGA5 . NumbersAdditional file 3: Figure S3.Bemisia tabaci ABCB subfamily with other organisms. See Figure S2 legend for details. (PDF 14 kb)Phylogenetic relationship of Additional file 4: Figure S4.Bemisia tabaci ABCC subfamily with other organisms. See Figure S2 legend for details. (PDF 15 kb)Phylogenetic relationship of Additional file 5: Figure S5.Bemisia tabaci ABCD subfamily with other organisms. See Figure S2 legend for details. (PDF 19 kb)Phylogenetic relationship of Additional file 6: Figure S6.Bemisia tabaci ABCE subfamily with other organisms. See Figure S2 legend for details. (PDF 37 kb)Phylogenetic relationship of Additional file 7: Figure S7.Bemisia tabaci ABCF subfamily with other organisms. See Figure S2 legend for details. (PDF 89 kb)Phylogenetic relationship of Additional file 8: Figure S8.Bemisia tabaci ABCG subfamily with other organisms. See Figure S2 legend for details. (PDF 16 kb)Phylogenetic relationship of Additional file 9: Figure S9.Bemisia tabaci ABCH subfamily with other organisms. See Figure S2 legend for details. (PDF 134 kb)Phylogenetic relationship of Additional file 10: Table S1.B. tabaci Q ABC transporters at different developmental stages (DOCX 13 kb)RPKM values of Additional file 11: Table S4.B. tabaci Q exposure to imidacloprid (DOCX 8 kb)Fold-change of gene expression following Additional file 12: Table S5.Models used for phylogenetic analysis (DOCX 15 kb)Additional file 13: Table S2.B. tabaci Q ABC transporters for the RT-qPCR validation study (DOCX 143 kb)Selected Additional file 14: Table S3.Primers used for the RT-qPCR analysis (DOCX 89 kb)


RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods. In an ever-growing world of new classes of RNAs, the need to reveal their function and structure is expanding . This neX-ray crystallography produces high-resolution data but relies on RNA samples that are rigid enough to crystallize. It is often not limited by the size of the sample Fig.  and is tFluorescence/Förster resonance energy transfer (FRET) is based on two fluorophore tags, which can transfer energy on the basis of their relative proximity. The distance between the tags can be assessed depending on the efficiency of the FRET exchange, and this information can then be used to calculate low-resolution structures. FRET structure calculation requires the tags be chemically bonded to the molecule, potentially inhibiting relevant interactions. Most often, more than one donor–acceptor pair is necessary, requiring several samples. However, this is a relatively fast method and usually requires a small amount of sample. FRET is most often used on ribosomes, but will likely have large use in the localization of regulatory RNA on genomic DNA or other RNA interactions .Electron paramagnetic resonance (EPR) can be used to measure crude long-distance interactions on the basis of a spin label covalently bonded to the RNA of interest. The method is in its infancy but can provide information for challenging systems, where other structural methods fail. In analogy to FRET, it requires positioning of artificial spin labels that could potentially interfere with the native structure .15N and 13C isotopic labeling of the sample is a prerequisite when high-resolution structural data need to be inferred from NMR. With increasing size, partial or total deuteration of the sample is required.NMR methods provide atomic resolution information, allow secondary and tertiary structure determination, and make possible the characterization of molecular motion on a wide range of timescales , 30. A bSmall-angle X-ray scattering/neutron scattering measures the diffraction properties of the atoms in the sample and retrieves information on the envelope of the molecule. Purified samples can be measured in native solutions. Small-angle X-ray scattering/neutron scattering is often used as a complementary source of information for integrated structural biology studies as it can easily access the relative position of the components of large multimolecular complexes . For smaSecondary structure chemical probing using enzymes or metal ions has been used for decades, but has recently seen a revival with small molecules such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probing to reveal the RNA structure –35. StruBiophysical methods, such as UV spectroscopy, circular dichroism spectroscopy, and isothermal titration calorimetry, are routinely used as first characterization steps in structural biology approaches. They are normally performed fast and require a low amount of purified sample , howeverRNA puzzle, where different methods compete with and validate each other every 2 years, providing a great display of which software is available and how the different types perform methyl methyl  groups ay]methyl  and 2′-ty]methyl  , allowing the RNA to remain soluble while the DNA undergoes repartition at the interface. A detailed and reedited version of the original protocol was published , and mang (approximately 105g) on top of a sucrose cushion to form a pellet of the required ribosomes [2+ content of the sucrose gradient used for purification, one can obtain complete ribosomes or individual subunits. A lower magnesium ion concentration (1 mM) will aid in separating the individual subunits, whereas a higher concentration will promote isolation of complete ribosomes [translatome, also use the ultracentrifugation technique [DNA origami, where ultracentrifugation onto glycerol gradients has been used as a good complement to more conventional and established agarose gel electrophoresis purification methods [Ultracentrifugation is the standard way of purifying large RNA machines, such as ribosomes and ribosomal subunits, as well as other macromolecules of biological origin in the same size range. Ultracentrifugation with a gradient of a solute such as sucrose has been used for more than half a century . For theibosomes . The matibosomes . After sibosomes . Althougechnique , 99. Ult methods .For decades, polyacrylamide gel electrophoresis (PAGE) was the standard method to purify large amounts of RNA  with single-nucleotide resolution as it can be easily applied to a wide range of RNA sizes and requires a minimal setup with cost-effective reagents. Like all electrophoretic techniques, PAGE uses a polymeric mesh to separate molecules by size, and/or conformation, as charged macromolecules migrate through an electric field. The mesh is prepared by polymerization of an acrylamide/bisacrylamide solution. The concentrations of acrylamide/bisacrylamide monomers can be varied to obtain the desired pore size and resolving power in the final mesh. Typical monomer concentrations range from 5% to 20% for RNA gels with 1:19 acrylamide/bisacrylamide relative ratio. In this range, short to intermediate length (5–500-nucleotide) RNAs can be resolved . The acrLiquid chromatography techniques are well established for the purification and analysis of nucleic acids and oligonucleotides spanning a wide size range. On the basis of the concept of allowing solutes in a solvent (mobile phase) to run through a column containing a solid material (stationary phase) that interacts with the solutes to different extent, separation is achieved . Here weE. coli [This technique is based on the use of lipophilic cations; quaternary ammonium compounds that ion-pair with the negatively charged sugar–phosphate backbone of the oligonucleotide are commonly used. These ion-paired complexes then become lipophilic and can interact with the stationary phase of a reversed-phase chromatography column Fig. . The lipE. coli . In thisE. coli . This wotrans-acting hammerhead ribozymes  [d-biotin or dextran [As stated before, the affinity chromatograph technique falls within the category of sorptive techniques; that is, the compound to be separated interacts chemically with the stationary phase Fig. . The maj (SELEX) . One app dextran . In anot dextran .As the name implies, SEC separation is based on the difference in size or hydrodynamic radius of the molecules. When a porous stationary phase is used, large molecules cannot enter the pores and they pass through the stationary phase, whereas smaller molecules are retained Fig. . On the Sample preparation is currently a bottleneck in the structural characterization of RNA, impeded by RNases and a lack of development for large-scale preparative methods. However, new developments are opening up new avenues that can be combined to answer many new, challenging, and interesting questions in RNA structural biology. Here we reviewed recent progress in well-established production and purification methods that allows preparation of large amounts of RNA commonly needed for structural characterization. Several new exciting methods that are emerging, such as circular RNAs, PLOR, and polymerase chain transcription, have great potential to reduce the amount of work and time needed for RNA sample production. On the other side, obtaining a sample that is pure and homogeneous is usually the most challenging step in structural studies, and new, fast, and innovative methods for sample preparation are currently lacking. However, a combination of typically two different purification methods will most often result in a reliable, easy to work with sample that will save time in the long run.


Caenorhabditis elegans expresses both DAT (DAT-1) and Syntaxin-1A (UNC-64), we used this model system to investigate the functional and behavioral effects caused by lack of expression of unc-64 in cultured dopaminergic neurons and in living animals. Using an inheritable RNA silencing technique, we were able to knockdown unc-64 specifically in the dopaminergic neurons. This cell-specific knockdown approach avoids the pleiotropic phenotypes caused by knockout mutations of unc-64 and ensures the transmission of dopaminergic specific unc-64 silencing to the progeny. We found that, similarly to dat-1 knockouts and dat-1 silenced lines, animals with reduced unc-64 expression in the dopaminergic neurons did not respond to Amph treatment when tested for locomotor behaviors. Our in vitro data demonstrated that in neuronal cultures derived from animals silenced for unc-64, the DA uptake was reduced by 30% when compared to controls, and this reduction was similar to that measured in neurons isolated from animals silenced for dat-1 (40%). Moreover, reduced expression of unc-64 in the dopaminergic neurons significantly reduced the DA release elicited by Amph. Because in C. elegans DAT-1 is the only protein capable to reuptake DA, these data show that reduced expression of unc-64 in the dopaminergic neurons decreases the capability of DAT in re-accumulating synaptic DA. Moreover, these results demonstrate that decreased expression of unc-64 in the dopaminergic neurons abrogates the locomotor behavior induced by Amph. Taken together these data suggest that Syntaxin-1A plays an important role in both functional and behavioral effects caused by Amph.The dopamine transporter (DAT) is a cell membrane protein whose main function is to reuptake the dopamine (DA) released in the synaptic cleft back into the dopaminergic neurons. Previous studies suggested that the activity of DAT is regulated by allosteric proteins such as Syntaxin-1A and is altered by drugs of abuse such as amphetamine (Amph). Because  The DAT is a plasma membrane protein which reuptakes the DA released in the synaptic cleft back into the neurons. By so doing, DAT plays a central role in controlling the extracellular content of DA and regulating the amplitude of the dopaminergic signaling. DAT has been involved in the etiology and treatment of various neurologic disorders including schizophrenia, attention deficit hyperactivity disorder (ADHD) and Parkinson’s disease . InteresN-ethylmaleimide-sensitive factor attachment protein receptor) proteins complex involved in the process of membrane vesicle fusion which leads to exocytosis and thus neurotransmitter release  and regulates the electrogenic properties of DAT-1 . We found that the pdat-1::unc-64 sas animals show a slight but statistically significant reduction in body bends when tested in plates without bacteria but they swim normally. However, when treated with Amph, the percentage of animals exhibiting SWIP was significantly reduced with respect to wild type animals. These behavioral results were supported by in vitro data showing that cultured neurons isolated from pdat-1::unc-64 sas animals exhibit reduced DA uptake and reduced DA release induced by Amph. Taken together these data suggest that Syntaxin-1A is required to mediate the physiological and behavioral effects caused by Amph in C. elegans.Previously, we showed that, like in mammals, the of DAT-1 . Here weogenesis , we creaC. elegans strains were cultivated under standard conditions, in non-crowded conditions, at 20°C on NGM plates seeded with the OP50 or NA22 Escherichia coli strains (E. coli strain HB101. The N2 wild type (Bristol variety), RM2702 dat-1(ok157) III, CB1112 cat-2(e1112) II, OH7547 otIs199 , and CB246 unc-64(e246) III strains were provided by the Caenorhabditis Genetics Center . BY200 vtIs1  V strain was obtained from Dr. Randy Blakely at the Brain Institute, Florida Atlantic University. To knockdown GFP encoding gene (gfp) in the dopaminergic neurons under the promoter of cat-2 (pcat-2), the following transgene was used: gbEx525 ; whereas, under the promoter of dat-1 (pdat-1) we used: gbEx572 . To knockdown the dat-1 gene under pdat-1, the following transgenes were used: gbEx584  and gbEx624 . To knockdown the cat-2 gene: gbEx574 . To knockdown the kal-1 gene: gbEx599 . To knockdown the unc-64 gene: gbEx585  and gbEx613 .All  strains , except C. elegans genomic DNA, unless otherwise noted. The promoter regions of cat-2 or dat-1 genes were chosen based on the cis-regulatory modules (CRM) necessary to drive expression specifically in dopaminergic neurons . For the cat-2 promoter a 600 bp fragment, upstream of the ATG, was amplified using the following primers: Pf cat-2: ataataaaactgcgtggcgtg; Pr cat-2: ctcttccaatttttcaagggg. The nested primer used for the second step of fusion was: Pf∗2 cat-2: cgtgttgttaagaacgtgcttgatcg. For the dat-1 promoter a 795 bp fragment, upstream of the ATG, was amplified using the following primers: Pf dat-1: aaagtctttctgcccacacaa; Pr dat-1: agtaaaccgtagcgggatcag. The nested primer used for the second step was: Pf∗dat-1: cgacctcatacactttctctcg. To amplify the C. elegans target genes to be silenced, we amplified the same exon rich regions that have been used for RNAi by feeding experiments  plasmids pPD95.75 and L4417, respectively. Primers used to fuse the gfp fragment to dat-1 promoter for expression were: Tf pdat-1::gfp sas: ctgatcccgctacggtttactTCACTATAGGGAGACCGGCA; Tr pdat-1::gfp sas: ctgatcccgctacggtttactTCACTATAGGGCGAATTGGG. Primers used to fuse the gfp fragment to cat-2 promoter for expression were: Tf pcat-2::gfp sas: ccccttgaaaaattggaagagTCACTATAGGGAGACCGGCA; Tr pcat-2::gfp sas: ccccttgaaaaattggaagagTCACTATAGGGCGAATTGGG. Primers used to amplify the gfp region (890 bp) for knockdown were: Tfa gfp sas: gttgtaaaacgacggccagt; Tfs gfp sas: GGCCGATTCATTAATGCAG; Tf∗gfp sas: tcactataGGGAGACCGGCA; Tr∗gfp sas: tcactatagggcgaattggg. Primers used to amplify the cat-2 region (1092 bp) for knockdown were: Tfa cat-2 sas: caagctcttgtgatccgtga; Tfs cat-2 sas: acaatctgctgaacgccttt Tf∗cat-2 sas: GAAATTCTCGATTTTCGCCA; Tr∗cat-1 sas: CTTCTTTGCACAACCCGAAT. Primers used to fuse the cat-2 fragment to dat-1 promoter for knockdown were: Tf pdat-1::cat-2 sas: ctgatcccgctacggtttactGAAATTCTCGATTTTCGCCA; Tr pdat-1::cat-2 sas: ctgatcccgctacggtttactCTTCTTTGCACAACCCGAAT. Primers used to amplify the dat-1 region (1190 bp) for knockdown were: Tfs dat-1 sas: TTCGAACCTGATCTCAACCC; Tfa dat-1 sas: TGCAGTTGGTGCCTACAGG; Tf∗dat-1 sas: AAGCAAATGCACCGAACTCT; Tr∗dat-1 sas: AGCTCCAGCAAAACTTCCAA. Primers used to fuse the dat-1 fragment to dat-1 promoter for knockdown were: Tf pdat-1::dat-1 sas: ctgatcccgctacggtttactAAGCAAATGCACCGAACTCT; Tr pdat-1::dat-1 sas: ctgatcccgctacggtttactAGCTCCAGCAAAACTTCCAA. Primers used to amplify the unc-64 region (1999 bp) for knockdown were: Tfa1 unc-64 sas: cttttcgtgtcgagacctgtc; Tfs unc-64 sas: AATGCCAGGAATATACTGAATGAG; Tr∗unc-64 (1) sas: CTCAATTCGATCAACCATCTCTC; Tf∗unc-64 (1) sas: AGAGATTCGTGGAAGTGTGGATA. Primers used to fuse unc-64 fragments to dat-1 promoter for knockdown were: Tf pdat-1::unc-64 sas: ctgatcccgctacggtttactAGAGATTCGTGGAAGTGTGGATA; Tr pdat-1::unc-64 sas: ctgatcccgctacggtttactCTCAATTCGATCAACCATCTCTC. All the Tf and Tr primers had at their 5′-end 20/21 additional nucleotides complementary to 3′ end of the promoter used to drive the knockdown. A mixture of sense and anti-sense PCR fusion product at the concentration of 50 ng/μL was microinjected together with co-transformation markers into the gonad of animals using standard microinjection technique  silencing construct was injected in otIs199 [pcat-2::GFP] transgenic strain and vice versa, pcat-2::gfp (RNAi sas) silencing construct was injected in BY200 ]) transgenic strain to avoid any disturbance of the same promoter on gene knockdown and on gene expression. To follow dopaminergic neurons in cultures some of the transgenic lines  were obtained adding to the injection mix also GBF325 pdat-1::GFP fusion construct at 1 ng/μL. At least two lines for each genotype were analyzed in all cases and data pooled together.The construction of transgenes for neuron-specific knockdown was made by PCR fusion as previously described . Genomic neurons . The proeriments . For gfpechnique . The folgfp knockdown in dopaminergic neurons was performed using Zeiss Axioskop microscopes .Animals were mounted and anesthetized with 0.01% tetramisole hydrochloride  on 4% agar pads. The analysis of GFP expression and Basal slowing response assay was performed as previously described . Young aAnimals were grown in agar plates seeded with NA22 bacteria and SWIP assays were performed as previously described . Briefly6 cells/well) were pre-loaded with 5 nM [3H]DA (NEN) for 30 min at room temperature. Cells were washed five times with bath solution containing 145 mM NaCl or NMDG+, 5 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 10 mM HEPES, and 20 mM D-glucose (pH 7.2 and 350 osmolarity) and 100 μM Amph or bath solution were then applied for 1 min to induce DA release. Samples were collected and counted for radioactivity. For uptake experiments 2 × 106 cells were plated per well. Two days after seeded, cells were washed with bath solution and incubated with 50 nM [3H]DA for 5 min at room temperature. Uptake was terminated by washing the cells three times with ice-cold bath solution. Cells were quickly lysed using 1% SDS and samples were collected in vials to count radioactivity.We prepared embryonal cultures from animals grown on NA22 bacteria, as previously described . Brieflyt-tests. The SWIP data passed the Shapiro–Wilk normality test (α = 0.05). Data are reported as averages of multiple observations ± Standard error.GraphPad Prism software  software was used for statistical analyses. The statistical significance was determined using one-way ANOVA with Bonferroni post-test, Kruskal–Wallis and Student’s unc-64 null mutants exhibit larval lethality, locomotion abnormalities, pharyngeal pumping and defecation defects  , we tested their efficiency in silencing the GFP in transgenic lines where GFP is constitutively expressed in all dopaminergic neurons . The pdat-1 promoter was largely more efficient in silencing the gfp (strain pdat-1::gfp sas), with only 15% of all dopaminergic neurons detectable by fluorescence microscopy (p < 0.001 vs control), while when using pcat-2 (strain pcat-2::gfp sas), 90% GFP-positive neurons were still detectable, a percentage very similar to controls (98%). These results showed that the transgene driven neuron-specific silencing technique (RNAi sas) can be successfully applied to the dopaminergic neurons, that pdat-1 is the appropriate promoter for this approach and we therefore used only this promoter in all the experiments. We then tested the efficiency of the dat-1 promoter in silencing a gene known to play a function in the dopaminergic neurons. Thus, we evaluated the ability of pdat-1 to knockdown cat-2 gene by performing BSR experiments. This behavioral assay tests the ability of wild type animals to slow down their rate of locomotion when they encounter a bacterial lawn. This behavior is mediated by DA; in fact, cat-2 null mutants do not exhibit BSR . We observed that, similar to cat-2(e1112) null mutants, the transgenic animals silenced for cat-2 (pdat-1::cat-2 sas) exhibited a defective BSR with respect to control animals (pdat-1::gfp sas). By representing BSR as the ratio between the locomotion rate off food and on food  we found that, similar to the cat-2(e1112) null mutants, the pdat-1::cat-2 sas animals exhibited a reduction in BSR, 6 and 15%, respectively. On the other hand, both the pdat-1::gfp sas animals (controls) and the wild type animals exhibited normal BSR values (45%). These results demonstrate that the RNAi sas technique is efficient, gene specific, and allows altering a DA-mediated behavior. defects . Knockdo defects . These dNAi sas) . We initd pdat-1 . The catA levels . In C. e neurons . After cibit BSR . We usedFigure 1B encouraged us to apply the RNAi sas technique to specifically knockdown unc-64 in the dopaminergic neurons. Therefore using the dat-1 promoter, we created the pdat-1::unc-64 sas mutants. Contrary to what observed in unc-64 null or hypomorphic mutants or after systemic RNAi , which cannot synthesize DA, the pdat-1::unc-64 sas lines, which cannot release DA via vesicle fusion, failed to show BSR, 6.4 and 3%, respectively . For these experiments we created, as negative control, transgenic animals that were knocked down for the kal-1 gene using pdat-1 . We reasoned that kal-1 silencing represents the proper control because, while gfp is exogenously injected in worms to create transgenic animals, kal-1 is natively expressed in most C. elegans neurons . In absence of food, we observed a slight but statistically significant 23% reduction (#p < 0.001) of body bends in the pdat-1::unc-64 sas animals with respect to controls . These data suggest that the pdat-1::unc-64 sas animals have a modest reduction in locomotion. To better understand the extent of this reduction, we tested the hypomorphic unc-64(e246) mutant. Although non-lethal, the unc-64(e246) mutation produces a point mutation in the unc-64 gene (unc-64(e246) animals besides lacking the BSR phenotype, also showed a strong reduction of body bends (4 ± 0.4) in absence of food with respect to wild type animals . Since the number of body bends in absence of food of the pdat-1::unc-64 sas animals was significantly higher (10 ± 0.4) then those observed in unc-64(e246) mutants, this result might suggest that in the pdat-1::unc-64 sas mutants the unc-64 silencing occurs in a restricted number of cells, most likely the dopaminergic neurons, rather than malfunctioning in every cells, as in unc-64(e246) animals. On the other hand, the number of body bends of the pdat-1::unc-64 sas tested on food was significantly higher than those measured in the pdat-1::kal-1 sas animals . This result shows that pdat-1::unc-64 sas mutants are impaired in the BSR behavior compared to the control animals .The results shown in mic RNAi , the pda neurons . As expe-64 gene  which capdat-1::unc-64 sas animals, we utilized another DA-dependent behavior: SWIP. Previously, we showed that when immersed in liquid solutions, wild type worms swim vigorously for hours; however, dat-1 null mutants exhibit SWIP, i.e., animals sink to the bottom of the well and do not move , the animals would not show SWIP because DA cannot be released (pdat-1::unc-64 sas). Thus, using the pdat-1, we knocked down unc-64 in the dat-1 null mutants and create the dat-1 ko;pdat-1::unc-64 sas double mutants. We found that the double mutants did not exhibited SWIP , confirming therefore that the RNAi sas technique we used, causes genetic ablation of unc-64 in the dopaminergic neurons and, as a consequence, it prevents DA release. No SWIP was observed in control animals  or pdat-1::unc-64 sas mutants . Taken together, these results suggest that the pdat-1::unc-64 sas mutants have reduced ability to release DA most likely because of the reduced expression of unc-64 in the dopaminergic neurons.To further investigate the lack of function of UNC-64 in the dopaminergic neurons in the not move . Since wnot move , 2010, wC. elegans (unc-64 in the dopaminergic neurons (pdat-1::unc-64 sas) would alter Amph-induced SWIP. We found that three independent transgenic lines of pdat-1::unc-64 sas animals treated with Amph exhibited significantly reduced SWIP with respect to wild type animals . We also measured SWIP in transgenic animals that were silenced for the dat-1 (pdat-1::dat-1 sas) or kal-1  genes, used as positive and negative controls, respectively. Interestingly, the reduction observed in the pdat-1::unc-64 sas animals was comparable to that observed in the two independent transgenic lines of pdat-1::dat-1 sas animals . And, as expected, the two independent transgenic lines of pdat-1::kal-1 sas animals did not show significant change in Amph-induced SWIP with respect to wild type animals . These results suggest that unc-64 might play a role in the mechanism of action of Amph that ultimately generates SWIP.Previously, we demonstrated that Amph, a drug that increases extracellular DA , causes  elegans . Here wepdat-1::unc-64 sas animals suggested that UNC-64, like its mammalian homolog Syntaxin-1A, is a protein required by Amph to induce DA efflux .The strong reduction of Amph-induced SWIP measured in the A efflux . To testcultures . After p3H]DA uptake experiments . We found a significant reduction (31 ± 5%) in the uptake of the pdat-1::unc-64 sas cells with respect to the controls . Interestingly, this reduction was comparable to that obtained in pdat-1::dat-1 sas cells derived from animals in which dat-1 gene was silenced using the RNAi sas technique (42 ± 5%). Because DA is accumulated inside the neurons by DAT, these results suggest that reduced expression of UNC-64 in the dopaminergic neurons causes a reduction of DAT activity or, as 3H]DA release observed in the pdat-1::unc-64 sas cells  may result from less [3H]DA moving inside the neurons as seen in our uptake results .Previous studies showed that Syntaxin-1A interacts and regulates the activity of DAT . For exaC. elegans homolog, unc-64, in the dopaminergic neurons. We used C. elegans because this model is amenable to genetic manipulations and conserves a high homology with the human dopaminergic-signaling pathway. To overcome the limitations presented by genetic null/hypomorphic mutants or systemic RNAi technique, we applied an alternative RNAi strategy, named RNAi sense and antisense (RNAi sas), to generate transgenic animals in which the function of unc-64 gene was knocked down only in the dopaminergic neurons. This strategy, originally developed in our laboratory . The dat-1 promoter sequence we chose contains three CRM or DA motifs, which are required and sufficient to drive expression in all dopaminergic neurons; on the other hand, the cat-2 promoter we used had only one CRM motif . Two distinct behavioral assays, BSR and SWIP, were used to test whether we effectively silenced the expression of unc-64 in the dopaminergic neurons of pdat-1::unc-64 sas mutants. Both assays depend on the ability of the dopaminergic neurons to release DA, which ultimately makes the animals to slow down when they encounter food, BSR . Moreover, the SWIP behavior observed in the dat-1 ko mutants was recovered in the dat-1 ko;pdat-1::unc-64 sas double mutants, i.e., in animals that cannot release DA. Taken together, these results suggest that the RNAi sas strategy we used effectively silences unc-64 in the dopaminergic neurons. We cannot exclude that the pdat-1::unc-64 sas mutants are deficient of unc-64 in additional cells other than the dopaminergic neurons; however, we can speculate that the lack of obvious developmental defects seen in these animals makes this possibility quite unlikely. This hypothesis is also supported by our behavioral data . In fact, while the hypomorphic unc-64 mutants (unc-64(e246)) move very slowly in absence of food (four body bends/20 s), the pdat-1::unc-64 sas mutants move almost three times faster (10 body bends/20 s) than the unc-64 hypomorphic mutants, and only slightly slower than the wild type animals (13 body bends/20 s).Several studies have recognized the SNARE protein Syntaxin-1A as a key player of neurotransmitter release  and one comotion . Moreoveood, BSR , or to sood, BSR . The pdapdat-1::unc-64 sas transgenic lines obtained with the RNAi sas technique , exhibited a strong reduction in Amph-induced SWIP. Because the increase of extracellular DA is one of the causes generating Amph-induced SWIP  diminished the DA uptake . These results are in accordance with previous published data showing that Syntaxin-1A decreases DA uptake by reducing the amount of DAT on the cell membrane , then we can speculate that less Amph is taken up inside these neurons since Amph, like DA, is a DAT substrate. This, in turn, would be the cause of the reduced DA release we measured in the pdat-1::unc-64 sas neurons . In fact, with less Amph moved inside, we get less DA released out. Regardless of these speculations and interpretations, we may conclude here that in C. elegans, Syntaxin-1A besides controlling the basal release of DA also moderates the behavioral effects generated by Amph by reducing the amount of DA in the synaptic cleft.Three ced SWIP , 2014, tchanisms , one veschanisms  and one chanisms . Becausemembrane . If we a3H]DA release and uptake experiments. IG did the initial molecular genetic manipulations and initial characterization. GZ participated in the genetic manipulations. SRK performed the BSR assays. LC and EDS conceived, designed, coordinated the study and wrote the manuscript. All authors read and approved the final manuscript.AL carried out the molecular genetic manipulations, microscope analysis, and BSR assays. BDS performed SWIP assays, the [The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows the characteristic properties of a bona fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells.Muscle development requires the coordinated activities of specific protein folding and degradation factors. UFD-2, a U-box ubiquitin ligase, has been reported to play a central role in this orchestra regulating the myosin chaperone UNC-45. Here, we apply an integrative in vitro and in vivo approach to delineate the substrate-targeting mechanism of UFD-2 and elucidate its distinct mechanistic features as an E3/E4 enzyme. Using  C. elegans myosin chaperone UNC-45 as an adaptor protein.The U-box ubiquitin ligase UFD-2 is one of the most abundant components of the ubiquitin proteasome system in muscle cells. Here the authors perform in vitro and in vivo experiments and show that UFD-2 has E3 ligase activity and that it ubiquitinates unfolded myosin using the  The importance of UNC-45 for muscle development was first demonstrated in the nematode Caenorhabditis elegans (C. elegans), where a number of UNC-45 temperature-sensitive (ts) mutants have been identified6. When grown at the restrictive temperature, ts worms display a reduced number of thick filaments in their body wall muscles and exhibit an uncoordinated (unc) phenotype1. Further studies in Drosophila and zebrafish established that UNC-45 (Unc45b in vertebrates) is important for skeletal and cardiac muscle development7. Specifically, UNC-45 was shown to promote the folding of the motor domain of myosin molecules10. In this process, UNC-45 acts as a co-chaperone together with the general chaperones Hsp70 and Hsp9012.Forming a functional muscle requires the activity of dedicated assembly factors that organize structural and motor proteins into the highly ordered structure of the sarcomere, the basic contractile unit of the muscle. A major player in this process is the UCS (UNC-45/Cro1/She4p) protein UNC-45, a myosin chaperone critical for the assembly of thick filaments14. To regulate UNC-45 function, higher eukaryotes developed a tailored degradation system relying on a highly specialized ubiquitin ligase, UFD-216. UFD-2 was originally identified as part of the ubiquitin fusion degradation (UFD) network in Saccharomyces cerevisiae17. Although UFD-2 could not initiate the ubiquitination of target proteins itself, the ligase can extend short ubiquitin chains previously added by its partner E3 enzyme UFD418. To describe this unique ubiquitination activity, the term “E4 ligase” was introduced18, with UFD-2 serving as a model for similarly acting enzymes19. Further studies revealed the collaboration of the UFD-2 E4 ligase with other E3 enzymes, for example in the ERAD pathway21, where UFD-2 is associated with the DOA10 E3 ligase and the CDC48 ATPase, ubiquitinating and remodeling damaged proteins of the endoplasmic reticulum. The activity of UFD-2 has also been studied in context of the regulated degradation of selected substrates such as Mps1, a cell-cycle kinase, and proteins involved in DNA damage response24. In addition, UFD-2 can bind via its N-terminal domain to the UBL (ubiquitin-like) domain of Rad23 and Dsk2, substrate receptors of the 26S proteasome26. In conclusion, these studies suggest that UFD-2 acts downstream of E3 ligases extending the pre-assembled ubiquitin chains and preparing substrates for proteasomal degradation.Despite its critical role for muscle biogenesis and function, the activity of UNC-45 needs to be carefully controlled, as elevated levels of the myosin chaperone are linked to severe muscle distortion15. Together with CHN-1  UFD-2 forms an E3/E4 ligase complex poly-ubiquitinating UNC-45. Importantly, the isolated UFD-2 and CHN-1 ligases were found to only attach single ubiquitin moieties to UNC-45; however, when applied together in vitro, UFD-2 develops its E4 activity promoting the polyubiquitination of the substrate15. Both enzymes, CHN-1 and UFD-2, contain a so-called U-box domain, a structural and functional homolog of the RING domain, that enables them to interact with ubiquitin-conjugating E2 enzymes and to transfer ubiquitin from the E2 to the substrate protein28. While CHIP proteins such as CHN-1 function as general quality-control factors marking misfolded, chaperone-bound proteins for proteasomal degradation29, UFD-2 appears to be a more specific ubiquitination enzyme in higher eukaryotes important for UNC-45 regulation and muscle function. Consistent with such a specific role, the mammalian Ufd2a exhibits a remarkably distinctive expression pattern, showing the highest protein levels in skeletal muscle tissue30. Moreover, ufd2a knockout studies in mice demonstrated that the protein is essential for myocardial development31.In contrast to the numerous E4 substrates of UFD-2, the myosin chaperone UNC-45 is the only protein described to be directly ubiquitinated by the ligaseC. elegans UFD-2 with CHN-1 in modifying UNC-45. Our in vitro and in vivo data reveal that UFD-2 is a bona fide E3 ligase capable of poly-ubiquitinating unfolded protein segments and point to a role of the UFD-2/UNC-45 pair in myosin proteostasis.Despite its important biological role and unique ubiquitination activity, the molecular mechanisms underlying UFD-2 ligase activity are largely unknown. To better understand how UFD-2 combines its general quality-control function in the ERAD and UFD pathways with its specific role in the proteolysis of the myosin chaperone UNC-45, we addressed its substrate-targeting mechanism. To this end, we analyzed the interplay of 19. The so far best-characterized E4 reaction is the polyubiquitination of UNC-45, mediated by the concerted activities of UFD-2 and CHN-1. To better understand the interplay of the E3/E4 ubiquitin ligases UFD-2 and CHN-1 in this process, we reconstituted the ubiquitination reaction in vitro using recombinantly expressed and purified components of the C. elegans system including the relevant E2 enzyme, UBC-215. Upon incubating the substrate (UNC-45) with the isolated E3 ligases (UFD-2 or CHN-1) or the E4 complex (UFD-2/CHN-1 mixed in equimolar amounts), we followed the ubiquitination of UNC-45 by western blot analysis. Consistent with the previous results, CHN-1 alone could attach single ubiquitin moieties to UNC-45, but failed to generate poly-ubiquitin chains on the substrate ). To determine the contribution of the two ubiquitin ligases in promoting the degradation of UNC-45, we analyzed worms lacking the functional CHN-1 (a strain carrying the chn-1(by155) allele) or UFD-2 (a strain carrying the ufd-2(tm1380) allele) ligases. Western blot analysis of the worm lysates confirmed that UNC-45 levels were markedly increased in the unc-45(OE) strain  transgene into the ufd-2(tm1380) or chn-1(by155) mutant background did not further increase the UNC-45 levels  and is later downregulated to a lower steady-state level  and ufd-2(tm1380) worms. As the endogenous UNC-45 protein levels were almost indistinguishable from wild type  worms additionally displayed marked behavioral changes, including an increase in reversals and omega turns (two modes of changing direction in the arena) compared to wild-type animals , which originally revealed the functional connection between UFD-2 and UNC-45. According to Janiesch et al.16, the absence of UFD-2 partially rescues the unc phenotype of the ts-mutant worms, most likely by stabilizing the corresponding UNC-45E781K mutant at the restrictive temperature. When analyzing the motility of unc-45(m94) worms in crawling assays, we could confirm that the lack of UFD-2 partially rescues the temperature-dependent unc phenotype. Moreover, our detailed, time-resolved analysis revealed that the lack of UFD-2 delays the onset of the unc phenotype in unc-45(m94) worms, but cannot prevent it over time  ts-mutant strain , unc-45, and unc-45, pointing to a common compensatory mechanism  ts-mutant strain13, we observed a strong decrease in myosin protein levels in unc-45(m94) worms. However, the levels of muscle myosin are not stabilized in the ufd-2 deletion background  ufd-2(tm1380) worms, as suggested by their partially restored motility  or the Hsp70/Hsp90-binding TPR domain (UNC-45∆TPR). In addition, we generated a GST-fusion construct harboring the otherwise unstable UCS domain (UNC-45GST-UCS). When we incubated UNC-45∆UCS with the UFD-2 E3 ligase, we could only observe residual ubiquitination activity, as reflected by the lack of any high-molecular weight-bands from poly-ubiquitinated proteins  analysis. This analysis revealed a specific ubiquitination pattern with 8 from 10 identified sites residing in the UCS domain, the myosin-binding domain (Supplementary Table canyon) mutant, while the K637R and K938/943 R mutants were not as strongly poly-ubiquitinated as wild-type UNC-45  and analyzed the resultant mutants in ubiquitination assays. Unexpectedly, mutating lysine residues lining the myosin-binding canyon yielded a substrate that was better poly-ubiquitinated than wild-type UNC-45 Fig. . Quantit-45 Fig. . These f717  was determined at 3.8 Å resolution  is less efficiently cleaved by the protease  revealed a clear correlation between protein stability and the efficiency in being ubiquitinated by UFD-2. The most efficiently targeted UNC-45 KRcanyon mutant proteins were also the most destabilized variants as indicated by a markedly lowered Tm value (33 °C compared to 35 °C of wild type). In addition, we noted that deletion of the UCS domain generated a truncated protein having a higher Tm value (38 °C) than the wild-type protein. These data indicate that the UCS domain represents the most unstable portion of the UNC-45 molecule. Altogether, the limited proteolysis experiments are consistent with a bipartite architecture of the UCS domain. Mutations of residues that are close to the molecular hinge connecting N-terminal and C-terminal half result in the most pronounced destabilization of the UCS domain. In particular, mutations of lysine residues located within the H3 helix of ARM repeat 12 or directly within the loop region preceding H2 of ARM repeat 13 reduced the stability of the domain  measurements. The recorded melting curve of wild-type UNC-45 revealed an unfolding step at about 35 °C and a further transition occurring at 60–70 °C due to aggregation of the protein   Fig. . The UNCEC) Fig. . Our SECWe next used the purified UNC-45:myosin complex as a substrate in the ubiquitination assay and could demonstrate that UFD-2 is able to poly-ubiquitinate myosin bound to UNC-45 Fig. . To test∆TPR and UNC-45∆UCS, impaired in their interaction with UFD-2 and myosin, respectively, were mixed with MHC-B and tested in the ubiquitination assay, the formation of poly-ubiquitinated myosin was largely abolished. The same effect was observed for the UNC-45E781Kts-mutant protein, suggesting that the mutant is not able to present its UCS domain  to target misfolded proteins44, while CHIP interacts with the general chaperone Hsp70 to forward aberrant client proteins for proteasomal degradation29. These data illustrate that pairs of chaperones and E3 ligases constitute central nodes in the quality-control network.To target a specific subset of substrate proteins, E3 ligases often make use of the so-called adaptor proteins. Prominent examples of such modular ubiquitination enzymes are the cullin-RING ligases and the CHIP ligase45. Among the many E3 enzymes, UFD-2 is highly abundant in muscle tissues. Its important role during development31 was previously linked to the degradation of the myosin chaperone UNC-4516. Our in vitro and in vivo findings disagree with this model. Our data rather suggest that UFD-2 functions as an E3 ligase that collaborates with the UNC-45 chaperone in removing unfolded myosin. Of note, the myosin quality-control system comprises several other members including MuRF E3 ligases46, Atrogin-147, Ozz E3 ligase48, and p9749. As we could not detect a stabilization of myosin in the ufd-2 deletion strain, the distinct pathways ensuring myosin functionality appear to work in parallel compensating for each other. Such a redundant set of quality-control factors is a well-documented characteristic of cellular proteostasis, as seen for example in yeast, where the E3 ligases Ubr1 and San1 work in parallel, targeting unfolded proteins for degradation44. Similarly, substrates of the CHIP/Hsp70 system can be targeted by other E3 ligases when CHIP is absent50. Given this functional redundancy, it will be important to delineate further factors that operate in the same pathway as UNC-45 and UFD-2, keeping muscle proteins in shape.The ubiquitin proteasome system is critical for ensuring the proper assembly of muscle sarcomeres and maintaining their integrity, for example during stress situations or agingΔUCS (1–521) and UNC-45GST-UCS (525–961) were cloned with a C-terminal STREP-tag or His6-tag from cDNA and a synthesized UNC-45 gene . The sequence of HsUNC-45b was synthesized and codon-optimized (Supplementary Table E. coli (Life Technologies) and cloned into pET21a to generate a C-terminally His6-tagged construct. Luciferase was cloned from pGEM®-luc (Promega) with a C-terminal STREP-tag into pET21a using accordingly designed DNA primers. CHN-1 and UFD-2 were cloned from C. elegans cDNA into the pET SUMO vector and pET28a to generate N-terminally His6-SUMO-tagged and His6-tagged construct, respectively. C-terminally Strep-tagged UFD-2, C-terminally Strep-tagged UNC-45E781K, and C-terminally Strep-tagged and His6-tagged UNC-45ΔTPR(135–961) were cloned into pCoofy. hUba1 in pET22b was a kind gift from Andrea Pichler. Primers are listed in Supplementary Table E. coli BL21-DE3 cells (Invitrogen) for HsUNC-45b-His6, luciferase-Strep, and His6-SUMO-CHN-1 and in BL21-DE3-RIL (Stratagene) cells for His6-UFD-2, UFD-2-Strep, His6-SUMO-UFD-2, UNC-45ΔUCS-Strep/His6 and UNC-45GST-UCS-Strep/His6, UNC-45ΔTPR-Strep/His6, UNC-45-Strep/His6 full-length, and UNC-45-Strep/His6 point mutants. Protein expression was induced by adding IPTG to a final concentration of 250 µM, and incubating the cells for 5 h at 25 °C , for 12 h at 25 °C (luciferase-Strep) or for 12 h at 18 °C . Expression of UNC-45 full-length and UNC-45 point mutants was induced with a final concentration of 100 µM IPTG for 12 h at 18 °C. The cells were harvested by centrifugation and lysed by sonication in 50 mM of Na2HPO4, pH 8.0, and 300 mM of NaCl (His6-tagged and GST-tagged proteins) or 20 mM of Tris, pH 8.0, and 300 mM NaCl (Strep-tagged proteins). The tagged proteins were first purified by NiNTA, GST-Trap , or Streptactin (IBA) affinity chromatography, with the final elution buffer containing 150 mM imidazole, 10 mM glutathione, or 2.5 mM d-desthiobiotin, respectively. The His6-SUMO-CHN-1 and His6-SUMO-UFD-2 fusion proteins were incubated with His6-SUMO-protease, and subsequently untagged CHN-1/UFD-2 was isolated in the flow-through fraction of an additional round of NiNTA affinity chromatography. All proteins were subjected to SEC using a Superdex 200 column  equilibrated with 20 mM Tris, pH 7.5 (pH 8.0 for UNC-45 full-length proteins), 150 mM NaCl.UNC-45UCS 525–91 were clC. elegans MHC-B motor domain (1–790), referred to as myosin, was cloned from cDNA into pACEBac1 with a C-terminal fusion to a His6-tag. UNC-45 was cloned into a pIDC derivative encoding an N-terminal Twin-Strep-tag (former One-Strep-tag) followed by a 3C protease cleavage site. The MultiBac was generated by Cre recombination (Geneva Biotech) and bacmids were generated by transposition of E. coli DH10EMBacY. Baculovirus was produced by transfection of SF9 cells (Expression Systems) with FuGENE HD (Promega). For expression, High Five cells (Expression Systems) at a density of 1 × 106 ml−1 were infected with baculovirus (P1 amplification). The cells were incubated post infection for 96 h at 21 °C. The UNC-45:myosin complex was purified from infected cells by a two-step affinity purification procedure, first utilizing the His6-tag on myosin and then the Twin-Strep-tag on UNC-45. The cells were collected by centrifugation, lysed by freeze–thaw, and resuspended in 50 mM Tris, pH 7.5, 300 mM NaCl (buffer A), containing 20 mM imidazole. The cleared cell lysate was loaded onto a NiNTA column  equilibrated with buffer A. After washing the column, myosin and associated UNC-45 were eluted with buffer A,Data collection and refinement statistics are containing 150 mM imidazole. The elution was concentrated and loaded onto a Streptactin column (IBA), equilibrated with buffer A. Upon washing the column, the protein complex was eluted with 20 mM Tris, pH 8.0, 150 mM NaCl, and 2.5 mM d-desthiobiotin. Finally, the UNC-45:myosin complex was loaded onto a Superdex 200 column equilibrated with 20 mM Tris, pH 8.0, 150 mM NaCl. To obtain folded myosin, the wash fractions from the Streptactin purification step, containing myosin, were applied to a Superdex 200 column equilibrated with 20 mM Tris, pH 8.0, 150 mM NaCl. To obtain a SEC standard for an unfolded myosin, the folded protein (5 µM) was incubated for 60 min at 4 °C or 27 °C and applied to a Superdex 200 column in the same buffer.The 11. Compared to the initially characterized crystals (UNC-45815), the new crystal form UNC-45717 had a shortened c-axis, as reflected by the different unit-cell parameters of the P6122 space group . Diffraction data were collected at the ESRF beamline ID 23-1 (wavelength: 0.979 Ǻ), integrated with DENZO and scaled with SCALEPACK51. The structure of the UNC-45717 crystal form was solved by molecular replacement, which was carried out with PHASER using the core region, i.e., TPR, central, and neck domains of UNC-45815 (PDB code: 4i2z) as search model52. Refinement of the partial model with CNS yielded an improved electron density map that allowed incorporation of large parts of the UCS domain54. Data collection and refinement statistics are summarized in Supplementary Table R-factor of 29.8% 55. Structure figures and distance measurements were generated with PYMOL56. Ramachandran statistics were computed with PHENIX .UNC-45 crystals were obtained by the sitting-drop vapor-diffusion method, upon mixing 2 μl of protein (200 μM) with 0.4 μl of additive (0.5 M NaF) and 0.5 μl reservoir solution . Crystals grown at 19 °C were mounted in 90°-bent loops, shortly incubated in the crystallization solution containing 25% ethylene glycol, and flash-frozen in liquid nitrogen−1 protein concentration in 20 mM Tris pH 8.0, 150 mM NaCl using a Chirascan plus CD spectrometer (Applied Photophysics) connected to a temperature control unit . Melting temperatures were determined by analyzing the data with the Global3 evaluation software.CD spectra were recorded at 0.7 mg mlUNC-45 proteins were incubated with trypsin (Roche) at a 1/100 (w/w) ratio for the indicated times. Reactions were stopped by mixing with SDS-PAGE loading buffer and resolved on SDS-PAGE gels. N-terminal sequencing of selected protein bands was performed by Alphalyse.E781K, UNC-45ΔUCS, UNC-45GST-UCS, UNC-45ΔTPR HsUNC-45b, and luciferase or MHC-B head domain) unless otherwise indicated in figure legends. For Fig. 2 to a final concentration of 10 mM and stopped by adding SDS-PAGE loading buffer. Reaction mixes were resolved on SDS-PAGE gels and stained with Coomassie or transferred to a PVDF membrane. Ubiquitination products were detected by performing western blot analysis using substrate-specific antibodies. anti-UNC-45b , anti-Strep , anti-Penta-His , and anti-CeUNC-45  were used in this study. Secondary antibodies conjugated to horseradish peroxidase  were used in conjunction with Amersham ECL Western Blotting Detection Reagent . Protein bands were visualized using the ChemiDoc MP Imaging system (Bio-Rad), taking care to remain within the linear dynamic range of the detector.Ubiquitination assays contained 60 μM ubiquitin or methylated ubiquitin (BostonBiochem), 100 nM E1 (hUba1), 0.6 μM E2 (UBC-2), 1 μM E3 (UFD-2 or CHN-1), and 1 μM substrate protein , lane profiles of the western blots were generated for each reaction. A line was drawn at the center of the lane and the profile was plotted in Fiji57. Overlays of the lane profiles were generated in Prism (GraphPad). In case of an offset between different lanes, the lane position (X-axis of the graph) of different profiles was adjusted using the maximum of the monomer peak observed in all samples as a reference point. Ubiquitinated UNC-45 , the signal derived from lanes of control reactions without ATP, covering the same area, was subtracted from all measurements. Finally, the signal of the ubiquitinated species at different time points was plotted in Prism (GraphPad).Quantification of in vitro ubiquitination reactions was performed on western blots developed using the ChemiDoc MP Imaging system (BioRad), which is designed for quantitative analysis of chemiluminescent signal. To additionally ensure that the quantified western blot signal is within the linear range of detection, we generated standard curves using a dilution series of the respective ubiquitination reaction, which was incubated for 180 min at 30 °C. The signal was quantified in ImageLab (Biorad) by drawing a box of the same size at the center of each lane showing ubiquitinated protein. The same size box was used to measure the background signal that was subtracted. The standard curve was generated in Prism (GraphPad) using the linear regression curve fitting model. The ubiquitination reactions in Supplementary Figs 6-tagged UNC-45 proteins with or without myosin were pre-incubated at 27 °C for 60 min. Strep-UFD-2 was added, and the three proteins incubated at 30 °C for another 60 min to mimic the ubiquitination reaction conditions. Control reactions ; LGII, ufd-2; and LGIII, unc-45, unc-45, unc-45, and unc-45. All mutants were outcrossed at least four times to wild type prior to analysis/introduction of markers. For the generation of worms overexpressing UNC-45, the unc-45 cDNA containing a 3′ FLAG-tag sequence was subcloned into pPD30.38  and 20 ng/µl of the resulting construct pPD30.38- Punc-54::unc-45FLAG was co-injected with 20 ng/µl of the marker plasmid pPD114.108 (mec-7::GFP).The 2 to prevent them from leaving a 56 mm × 56 mm center area. A number of 60–100 adult animals , which had been removed from food 12 min prior to examination, were used in a single experiment. Each experiment was carried out three times.Worms were transferred without food to 14 cm NGM assay plates containing a cut out arena of Whatman filter paper soaked in 20 mM CuCl58. The resulting trajectories were smoothed and used to calculate the instantaneous speed of the worm’s centroid (1 s binning). Forward speed was obtained by excluding periods of backward locomotion  and turns. Deep omega turns and reversals were detected based on characteristic changes in object eccentricity and angular speed59, and their frequencies were calculated in 15-s bins. Traces of behavioral time courses show mean and shaded SEM of all animals of all experiments. For quantification in Fig. Freely behaving animals were illuminated with flat red LED lights and recorded for 10 min at 3 fps on a 4 megapixel CCD camera (Jai) using Streampix software (Norpix). Movies were analyzed by customized MatLab-based image processing and tracking scripts based on software previously describedunc-45(m94), and unc-45(m94) ufd-2(tm1380) worms were grown at 16 °C and transferred to 23 °C for 16, 22, or 30 h before the crawling assay. Individual young adult worms were placed onto NGM agar plates seeded with E. coli OP50 and tracks were analyzed after 1 h as described in Hoppe et al15. A minimum of 10 worms were tested for each strain.Wild-type, C. elegans extracts, the worms were synchronized at the L1 stage by treating mixed cultures with bleach and hatching embryos in the absence of food, and then grown in liquid culture at 20 °C until harvesting at 22 h (L3), 45 h (L4), and 70 h (adult). UNC-45 overexpressing worms were picked manually to ensure the presence of the extrachromosomal array. Worm lysates were subjected to western blot analysis using UNC-45, UNC-54 (mAb 28.2)60, and PGK-1 (Invitrogen)-specific antibodies.For preparing staged C. elegans His6-UFD-2 and UNC-45-His6 were generated by injecting recombinant protein into rabbits and collecting sera after several rounds of antigen boosting (performed by Gramsch laboratories). Antibodies were purified by applying sera onto an NHS-coupled resin  charged with untagged UFD-2 or UNC-45-Strep. After extensive washing with PBS, the antibodies were eluted with 0.1 M Glycine pH 2.6, neutralized with 1 M Tris pH 8 and dialyzed into PBS containing 10% glycerol.Polyclonal antibodies against Ubiquitination reactions were performed as described above, applied to SDS-PAGE gels and then were stained with Coomassie. The four replicates shown in Supplementary Table C. elegans lysate, prepared by grinding frozen worms and subsequent centrifugation, was applied to the antibody-coupled beads. After incubation for 1 h at 4 °C the beads were washed with 20 mM HEPES, pH 7.5, 300 mM KCl, 1 mM EDTA, 10% glycerol, 0.05% NP-40, and IP buffer. Finally, antibody-bound protein complexes were eluted with 0.2 M glycine pH 2.0, eluates neutralized with 1 M Tris pH 9.0, and subjected to MS analysis.Synchronous L1 worms were grown in liquid culture for 45 h at 20 °C. Upon reaching the L4 stage, the worms were grown for an additional 2.5 h at 34 °C and recovered for 2 h at 16 °C before harvesting. Worm pellets were washed and finally resuspended in 20 mM HEPES, pH 7.5, 150 mM KCl, 1 mM EDTA, 10% glycerol (IP buffer) supplemented with COMPLETE protease inhibitors (Roche). UNC-45 and UFD-2 antibodies as well as an unspecific anti-rabbit antibody were chemically cross-linked to ProteinA-Dynabeads (Life technologies) using DMP (dimethyl pimelimidate). Cleared Elution fractions from IP experiments were reduced with DTT (dithiothreitol), alkylated with IAA (iodoacetamide), and enzymatically cleaved with trypsin (Promega Gold) o/n at 37 °C. The peptide solution was acidified with TFA to stop the enzymatic reaction and stored at −80 °C prior to the nanoLC-MS analysis.MS analysis of ubiquitination sites and the coIP protein sample were conducted on different systems, as described below.TM Ion Source , and coupled to a Q Exactive mass spectrometer . First, tryptic peptides were concentrated by a trap column  that was operated at a temperature of 30°C and a flow rate of 25 μl min-1, using 0.1% TFA as mobile phase. After 10 min, the trap column was switched in line with the analytical column . A 165 min gradient from buffer A  to B  was applied to elute peptides. The LTQ Orbitrap Velos was operated in data-dependent mode, using a full scan in the Orbitrap  followed by MS/MS scans of the 12 most abundant ions in the linear ion trap. MS/MS spectra  were acquired, and subsequent activation was performed through multistage activation. The neutral loss mass list was set to −98, −49, and −32.6 m/z, respectively. Dynamic exclusion was set to 60 s, and only charge states 2 and higher were accepted. For peptide identification, the RAW files were loaded into Proteome Discoverer . All hereby created MS/MS spectra were searched using Mascot 2.2.07  against the Uniprot protein sequence database , using the taxonomy C. elegans . The following search parameters were used: beta-methylthiolation on cysteine was set as a fixed modification, oxidation on methionine, acetylation on lysine and protein-N-terminus, phosphorylation on serine, threonine, and tyrosine, and ubiquitination on lysine were set as variable modifications. Monoisotopic masses were searched within unrestricted protein masses for tryptic peptides. The peptide mass tolerance was set to ± 5 ppm and the fragment mass tolerance to ± 30 mmu. The maximal number of missed cleavages was set to 2. The result was filtered to 1% FDR using Percolator algorithm integrated in Proteome Discoverer. The localization of the phosphorylation sites within the peptides was performed with the tool phosphoRS61.For the IP experiment shown in Supplementary Table -1 using 0.1% TFA as mobile phase. After 15 min, the trap column was switched in line with the analytical column . Peptides were eluted using a flow rate of 275 nl min-1, and a ternary gradient, as described62 was used, with the following mobile phases: A , B , and C . The total run time was 120 min. The LTQ FT was operated in data-dependent mode using a full scan in the ICR cell  followed by MS/MS scans of the five most abundant ions in the linear ion trap. MS/MS spectra  were acquired in the multistage activation mode, where subsequent activation was performed on fragment ions resulting from the neutral loss of −98, −49, or −32.6 m/z. Precursor ions selected for fragmentation (charge state 2 and higher) were put on a dynamic exclusion list for 180 s. Monoisotopic precursor selection was enabled. Additionally, singly charged parent ions were excluded from selection for MS/MS experiments and the monoisotopic precursor selection feature was enabled. For peptide identification, all MS/MS spectra were searched using Mascot 2.2.04  against a custom-made protein sequence database containing the proteins of interest and contaminants. The generation of dta-files for Mascot was performed using the Extract MSn program . The following search parameters were used: carbamidomethylation on cysteine was set as a fixed modification, oxidation on methionine, and ubiquitination on lysine were set as variable modifications. The peptide mass tolerance was set to ± 5 ppm and the fragment mass tolerance to ± 0.5 Da. The maximal number of missed cleavages was set to 2. Monoisotopic masses were searched within unrestricted protein masses for tryptic enzymatic specificity. Post-translationally modified peptides were filtered by a minimum Mascot Ions Score of 15 and validated by manual inspection.For the mapping of Ub sites shown in Supplementary Table m/z range 370–1000, nominal resolution of 15,000, target value 3E6) and the maximum of 6 following PRM scans . Based on the shotgun analysis data, the unique peptides of both proteins of interest were manually selected. Supplementary Table m/z, peptide sequence, charge, scheduling window, and NCE. Acquired PRM data were processed in Skyline software 63 and manually verified. Final protein areas were calculated as the sum of individual peptide areas, which consist of areas of all unambiguously detected peptide fragments. Peptide fragments selected for the calculation co-eluted at the same retention time and they did not have any interfering signal in 5 min around the peak apex.The nano HPLC system used was an UltiMate 3000 RSLC nano system (Thermo Fisher Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) equipped with a Proxeon nanospray source (Thermo Fisher Scientific). The nano HPLC system operated under same condition as in described above. The Q Exactive HF mass spectrometer was operating in scheduled PRM mode using low-resolution Full scan (717 have been deposited in the Protein Data Bank under accession code 5mzu. Other data are available from the corresponding authors upon reasonable request.Coordinates and structure factors of UNC-45Supplementary InformationPeer Review File