Bombyx germ cell line and mouse testes. The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the  Mature piRNAs are produced from long single-stranded precursor RNAs. Here the authors show that RNase κ generates 2′,3′-cyclic phosphate-containing piRNA precursors and promotes piRNA production. The precursor RNAs are bound by the RNA helicase Armitage 6 and are processed by the mitochondria-associated endonuclease Zucchini  and by PIWI/piRNA complexes10. The resulting RNAs with mature 5′-ends, called pre-piRNAs, are loaded onto the PIWI proteins, and their 3′-terminal extended regions are trimmed by a PARN-like 3′- to 5′-exonuclease Trimmer (PNLDC1 in mice)14. The Tudor domain-containing protein Papi (TDRKH in mice) interacts with the symmetrical dimethylarginines (sDMAs) of PIWI proteins at the mitochondrial surface and recruits Trimmer to the PIWI/pre-piRNA complexes17. The piRNA maturation process is accomplished by 2′-O-methylation, which is catalyzed by Hen1 methyltransferase, to form mature 3′-ends of piRNAs23. In contrast to the well-studied downstream pathway after pre-piRNA production, the mechanisms involved in the upstream pathway of Zuc- or PIWI/piRNA-mediated processing of single-stranded precursor RNAs remain unclear.PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) are predominantly expressed in animal germlines, where they maintain genome integrity by silencing the transposable elements and regulating gene expression24. The involvement of cP-RNAs in piRNA generation was previously implied by studies on Bombyx and Drosophila. In Bombyx BmN4 cells, cP-containing 5′-tRNA half molecules were shown to serve as direct precursors for tRNA-derived piRNAs (tdpiRNAs)25. In Drosophila ovarian somatic cells (OSCs), Armi-interacting RNAs, which are postulated as piRNA precursors, were shown to harbor cP26. However, cP-RNAs have not been well studied because cP-RNAs are not captured by the standard RNA-seq methods as the cP end cannot be ligated to the 3′-adapter (AD). We developed a cP-RNA-seq method that can specifically and efficiently sequences the cP-RNAs to evaluate this hidden component of the transcriptome28. In this study, we have comprehensively characterized the cP-RNA expression in Bombyx BmN4 cells and mouse testes using cP-RNA-seq and its advanced version P-cP-RNA-seq, and we have demonstrated extensive correlative profiles of cP-RNAs and piRNAs. Further, PIWI-loading of cP-RNAs was observed, suggesting direct utilization of cP-RNAs as piRNA precursor molecules. We identified that Bombyx RNase Kappa (BmRNase κ), a mitochondria-associated endoribonuclease, is responsible for cP-RNA production and robust piRNA generation. Our in vitro analysis demonstrated cP-end formation by BmRNase κ-mediated RNA cleavage. BmRNase κ preferably cleaved in-between cytidine and adenosine, which matches the nucleotide composition biases of cP-RNAs endogenously expressed in BmN4 cells and mouse testes. BmRNase κ-depleted Bombyx embryo exhibited enhanced transposon levels and failure of feminization, both of which are indicative of a disrupted piRNA pathway. These results collectively revealed that piRNA biogenesis is intermediated by BmRNase κ-generated cP-RNAs. The generation of cP-RNAs would enable germ cells to implement the robust piRNA production that is required for embryonic development.RNA molecules generated by ribonuclease cleavage sometimes harbor the 2′,3′-cyclic phosphate (cP) at their 3′-end, and these cP-containing RNAs (cP-RNAs) have a functional role in various biological processesBombyx ovary-derived, culturable germ cells that express Bombyx PIWI proteins, Siwi and BmAgo3, and their bound piRNAs29. To capture the cP-RNAs, 30–70-nt RNAs from BmN4 cells were subjected to cP-RNA-seq28  using the mitochondrial fraction of the BmN4 cells. We subjected 30–70-nt mitochondrial RNAs to “P-cP-RNA-seq” in which 5′-AD ligation was first performed to exclusively capture the P-cP-RNAs  of the tRNA-derived reads in the cP-RNA library  tRNAAspGUC was the major tRNA source for 5′-tRNA halves and tdpiRNAs cP-RNAs. Both Siwi- and BmAgo3-bound piRNAs, as well as total piRNAs, were highly mapped to the cP-RNAs and P-cP-RNAs  than that between piRNAs and cP-RNAs. Together with a higher rate of piRNA-mapped P-cP-RNA species  were selectively sequenced and their abundances were compared. BmN4 30–70-nt RNAs were mixed with spike-in control cP-/OH-RNAs and subjected to cP-RNA-seq and OH-RNA-seq, which specifically sequence cP-RNAs and OH-RNAs, respectively , treating the total RNAs with T4 PNK markedly enhanced the amplification signal Fig. , whereassed Fig. , possiblsed Fig. . Along w33, were subjected to cP-RNA-seq and piRNA-seq. Both methods successfully amplified cDNAs with expected sizes , in which Mili-bound pachytene piRNAs are abundantly expressedzes Fig. . MoreoveR2 = 0.99) to those of Mili-bound piRNAs in each pachytene piRNA cluster  was predicted to also be localized at the mitochondrial outer membrane. Indeed, the fractionation of BmN4 cell lysate and western blots using anti-BmRNase κ antibody  leads to the generation of mature piR-I that subsequently generates piR-J from Importin-5 mRNA via an artificial one-to-one ping-pong amplification loop35. As shown in Fig. BmRNase κ KD, which confirmed the contribution of BmRNase κ to de novo piRNA production. As a result of the reduced piRNA production, we observed increased transposon levels upon BmRNase κ KD in BmN4 cells  of  KD Fig. . StrikinNAs Fig. , whereasNAs Fig. . Both piBmPapi, BmSpindle-E (BmSpn-E), BmVasa, and Trimmer36. The KDs were confirmed by the reduced levels of targeted mRNAs   for BmRNase κ mRNA were designed and injected into the Bombyx embryos (24 each). After 72 h, the levels of three transposons were measured. Although variabilities were observed between individual embryos, some embryos in the BmRNase κ KD group exhibited elevated levels of transposons, which was not observed in the control group  was shown to be the crucial feminizing factor in the Bombyx embryos39, we further examined the effect of BmRNase κ KD on sex determination. At 120-h post-injection of a control siRNA or BmRNase κ siRNA #1, female embryos were selected by analyzing the expression of Fem RNAs, which is transcribed from the Fem gene located on the female-specific W chromosome40. The selected female embryos were then subjected to RT-PCR quantification of male-type Imp (ImpM) mRNA42 to analyze potential female-to-male conversion. As expected, the control female embryos did not exhibit ImpM mRNA expression. Strikingly, 4 of the 8 female embryos injected with BmRNase κ siRNA #1 expressed the male-specific mRNA , a member of the RNase A superfamily, generates cP-RNAs53 and preferably cleavages 5′-CA-3′ sequence54. Despite similar nucleotide preferences for cP-RNA generation with ANG, RNase κ does not contain the catalytic residues that are predictable by comparing ANG or other endoribonucleases. Although our study suggested K9 as a potential catalytic residue for RNase κ cleavage activity, further characterization is required to clarify how such a small protein with two transmembrane regions exerts endoribonuclease activity. Unlike many piRNA biogenesis factors, RNase κ is not a germline-specific factor. RNase κ is ubiquitously expressed in various tissues and cells55. Analyses of the endogenous cP-RNA species generated by RNase κ and their biological functions in somatic cells, as well as germ cells, might unveil previously uncharacterized noncoding RNA pathways.The catalytic mechanism underlying RNase κ-mediated RNA cleavage is unknown. The RNase A superfamily, which is the most well-studied ribonuclease group, contains a conserved catalytic triad15.The BmN4 cells were cultured at 27 °C in an Insect-Xpress medium (Lonza). The mitochondrial fraction of the cells were prepared as described previously56 under the highly stringent condition used in the HITS-CLIP method as described previously33.Mouse experiments were conducted in compliance with the standards and guidelines of the National Institutes of Health (NIH) and were approved by the Institutional Animal Care and Use Committee at Thomas Jefferson University. C57BL/6J (B6) mice were housed in ventilated cages at a humidity of 30–70%, at a controlled temperature of 20–22 °C on a 12-h dark–light cycle with ad libitum food and water. Testes were obtained from p14–20 male mice. After subjecting the testes to detunication and UV cross-linking, Mili immunoprecipitation was performed using 17.8 anti-Mili antibody4 on ice for 40 min in the dark, followed by ethanol precipitation. Directional ligation of 3′- and 5′-ADs, cDNA synthesis, and PCR amplification were performed using the TruSeq Small RNA Sample Prep Kit (Illumina), following the manufacturer’s instructions. For piRNA-seq using BmRNase κ KD cells, short RNA fractions were first collected by mirVana miRNA Isolation Kit (Thermo Fisher Scientific), followed by incubation with NaIO4 as described above. After the oxidization was quenched by adding 20% glycerol, RNAs were recovered by ethanol precipitation. The RNAs were then quantified and synthetic spike-in RNAs (5′-AUGUUGUGCCACAUAUUGAGCCAGUAGC-3′ and 5′-UGCCACAUAUUGAGCCAGUAGCGCGGUGUAUUA-3′) were added prior to adapter ligations. cP-RNA-seq was performed as described previously28. In P-cP-RNA-seq, the gel-purified ~30–70-nt RNAs or the RNAs of Mili-immunoprecipitates were first subjected to 5′-AD ligation by incubating the RNAs in a reaction mixture containing 5′-AD, T4 RNA Ligase , 1× T4 Rnl buffer, 1 mM ATP, 0.1 mg/ml bovine serum albumin (BSA), and RNase inhibitor at 28 °C for 1 h, followed by overnight incubation at 4 °C. After purification using CENTRI-SPIN 20 (Princeton Separation), the ligated RNAs were subjected to cP-RNA-seq (without 5′-AD ligation step). The amplified cDNAs were gel-purified and sequenced using NextSeq 500 (Illumina). The piRNA libraries from Mili-immunoprecipitates were obtained not by piRNA-seq but by standard small RNA-seq procedure (without NaIO4 oxidization) using the TruSeq Small RNA Sample Prep Kit (Illumina), because piRNAs are expected to be highly enriched in the fraction.The RNAs from the BmN4 cells, BmN4 mitochondrial fraction, mouse testes, and Mili-immunoprecipitates were isolated using the TRIsure reagent (Bioline). Approximately, 25–30-nt RNAs and 30–70-nt RNAs were gel-purified for piRNA-seq and (P-)cP-RNA-seq, respectively (RNAs from Mili-immunoprecipitates were used without gel-purification). In piRNA-seq, the gel-purified 25–30-nt RNAs were incubated with 10 mM NaIO57 with 5% mismatches penalized. For Bombyx sequences, the reads were mapped to Bombyx tRNAs as described previously25, followed by mapping to rRNAs and to 1811 B. mori transposons30. For mouse sequences, the reads were mapped to 471 mature cyto tRNAs obtained from GtRNAdb58, followed by mapping to rRNAs and to pachytene piRNA clusters5. Nucleotide compositions were analyzed using FastQC. The matching rates of terminal positions between piRNAs and cP-RNAs/P-cP-RNAs were calculated by comparing the terminal position of piRNA reads to their mapped cP-RNA/P-cP-RNA reads. The Integrative Genomics Viewer (IGV)59 was used for visualizing the aligned reads. Ping–pong analysis was performed by calculating distances between 5′-ends of piRNAs across opposite genomic strands as described previously34.The obtained sequence libraries are summarized in Supplementary Table 27. Briefly, total RNA was treated with T4 PNK (New England Biolabs) to dephosphorylate the cP, followed by 3′-AD ligation using T4 Rnl. The ligated products were quantified by TaqMan RT-qPCR using the One Step PrimeScript RT-PCR Kit (TaKaRa) on the StepOnePlus Real-Time PCR System (Applied Biosystems). The expression levels were normalized to those of 5 S rRNA. The sequences of the primers and TaqMan probes are shown in Supplementary Table The specific quantification of cP-RNAs was performed by TaqMan RT-qPCR as described previously34. The expression levels of miRNAs, snoRNA, and 5 S rRNA were quantified as described previously34. The expression levels were normalized to those of 5S rRNA.The expression levels of piRNAs were quantified by RT-qPCR using a stem-loop RT primer as described previouslyRp49 mRNA.After total RNA was treated with RQ1 DNase (Promega), the target sequences were reverse-transcribed using RevertAid Reverse Transcriptase (Thermo Fisher Scientific). The cDNA was subjected to real-time PCR using the SYBR Green PCR Master Mix (Applied Biosystems). The primers used are shown in Supplementary Table BmRNase κ cDNA expressed in the BmN4 cells were determined by RACE using the FirstChoice RLM-RACE kit (Life Technologies) as described previously15.The full-length sequences of Anti-BmRNase κ antibody was prepared by immunizing the rabbits with a synthetic peptide . The whole IgG was purified from the sera using Protein G Agarose (Sigma) to obtain the “BMK5263” anti-BmRNase κ antibody.34. The following antibodies were used: 17.8 anti-Mili (1:1000)56, BMK5263 anti-BmRNase κ (1:500), S213 anti-Siwi (1:1000)15, anti-BmAgo3 (1:500)29, anti-BmPapi (1:1000)11, BmVasa571 anti-BmVasa (1:500)60, anti-Hsp60 , anti-Tom20 , anti-β-Actin , anti-β-Tubulin , and anti-FLAG .The BmN4 cell lysates were subjected to western blots as described previouslyBombyx cyto tRNAAspGUC and its 5′-half as described previously25. Probes for piR-a, piR-b, and piR-RT3-1 were described previously44, and piR-t was detected by the probe 5′-GCATTCTGACGGAACTTGTAATGGTA-3′.Northern blots were performed for 29 was transfected into 1.65 × 105 BmN4 cells using 30 µl of Escort IV Transfection Reagent (Sigma). The cells were harvested at day 3 post-transfection. RNAi KD of BmRNase κ was performed as described previously25 with minor modifications. Briefly, to synthesize the templates for the in vitro production of dsRNA, BmN4 total RNA was subjected to reverse transcription using random hexamer primers. The cDNA was subjected to PCR amplification using the following primers: forward, 5′-TAATACGACTCACTATAGGGTAGTGGTCGATATTTAGTATTAAAG-3′ and reverse, 5′-TAATACGACTCACTATAGGGCTCCTTCAATATTACATACTAACTG-3′. The amplified DNAs were used as templates for T7 in vitro transcription system , and the synthesized RNAs were purified using MEGAclear (Ambion). The generated dsRNA (10 µg) was transfected into 3 × 106 BmN4 cells using 30 µl of X-tremeGENE HP DNA Transfection Reagent (Sigma) (day 1). At days 4 and 7, an identical dsRNA transfection was performed as a second and third transfection, respectively. At day 8, 6 µg of a pIZ-Siwi was transfected as described above, and the cells were harvested on day 10. RNAi KDs of BmPapi, BmSpn-E, BmVasa, and Trimmer were performed as described previously15. To synthesize dsRNA template for BmVasa KD, PCR was performed using the following primers: forward, 5′-TAATACGACTCACTATAGGGCCTATGTGCCTCCAGAACCAA-3′ and reverse, 5′-TAATACGACTCACTATAGGGTCTTACACTGCCAAATGATACACGA-3′. For induction of de novo piR-J synthesis, instead of pIZ-Siwi transection, 6 µg of the pIEX-4 vector encoding pre-piR-I or pre-piR-I-nc (negative control)35 was transfected using 30 µl of Escort IV Transfection Reagent (Sigma).To accelerate the de novo synthesis of piRNAs, 6 µg of a pIZ-based plasmid encoding FLAG-tagged SiwiGFP-fused BmRNase κ cDNA was cloned into the pIZ/V5 plasmid. To prevent dimerization of GFP, site-directed mutagenesis was performed to introduce GFP A206K mutation61. For the C-terminal GFP-fusion construct, the first methionine in the ORF of GFP was replaced with alanine to avoid initiation of translation from this site. The resultant plasmids were transfected into BmN4 cells using Escort IV Transfection Reagent (Sigma). After culturing for 3 days, the cells were plated on a slide glass chamber (Lab-Tek) and cultured for 18 h. The cells were then incubated with 100 nM of MitoTracker Red CMXRos (Life Technologies) for 30 min at 27 °C. After DNA mounting with ProLong Gold Antifade Reagent containing DAPI (Life Technologies), images were acquired using a Nikon A1R Confocal Laser Microscope System. The fluorescence signal intensities were quantified using ImageJ software (ver. 1.48) after background subtraction with a rolling ball radius of 50 pixels.N- or C-terminal BmRNase κ cDNA was cloned into the pGEX-6P-2 plasmid, which expresses N-terminal GST-tagged and C-terminal 6× His-tagged BmRNase κ protein. A plasmid for K9A mutant BmRNase κ was produced through site-directed mutagenesis. The wild-type or K9A mutant BmRNase κ was expressed in E. coli BL21 Star (DE3) at 16 °C for 16 h in the presence of 0.5 mM IPTG. The harvested cells were suspended in the lysis buffer containing 50 mM Tris-HCl pH7.5, 500 mM NaCl, 1 mM EDTA, 0.5% Triton-X100, 10% glycerol, 1 mM PMSF, and 1 mg/ml lysozyme. After sonication, 5 mM MgCl2 and 5 mM ATP were added to the lysate. BmRNase κ protein was purified using Glutathione Sepharose 4B . The GST-tag was cleaved using PreScission Protease . The protein was further purified by Superdex 200 Increase 10/300 GL .The coding region of the 32P-labeled at their 5′-end and incubated at 37 °C in a 20-μl reaction mixture containing 100 pmol of BmRNase κ recombinant protein, 50 mM sodium phosphate (pH 6.5), 5 mM EDTA, 0.1% digitonin, and 1 mg/ml BSA. To terminate the reaction, the reaction mixture was incubated with proteinase K  for 1 min. The cleavage products were resolved on 20% denaturing PAGE. To analyze the 3′-end formation of BmRNase κ-cleavage product, a 45-nt substrate DNA/RNA hybrid  were injected into the B. mori N4 strain eggs (within 3 h after oviposition) using a microinjector . The injected embryos were incubated at 25 °C in a humidified Petri dish. At 72 h or 120 h post-injection, the embryos were collected for further analysis. The embryo’s sex was determined by quantifying the expression of Fem transcript through RT-qPCR as described previously39. The phenotypic change of female-to-male conversion was determined by the detection of a male-specific ImpM mRNA using the following primers: forward, 5′-ATGCGGGAAGAAGGTTTTATG-3′ and reverse, 5′-TCATCCCGCCTCAGACGATTG-3′41.siRNA-mediated embryonic KD was performed as reported previouslyAll experiments in this study were performed at least twice or more times independently to confirm that they yielded identical/similar results. Image quantitation was performed using Image J. Statistical analyses were performed by using Microsoft Excel or R. Bar charts with error bars denote mean values ± S.D.Further information on research design is available in the Supplementary InformationReporting Summary


Metastasis is the leading cause of cancer-related deaths. Understanding the pathogenesis of metastasis at the molecular levels is of great significance for cancer research. However, the molecular diagnosis or treatment of cancer metastasis is limited. Accumulating and growing evidence shows that epigenetic changes are present in all human cancers, and epigenetic regulation is an indispensable factor to promote tumor metastasis. With the deepening of research and the advancement of technology, the function and mechanism of epigenetic regulation, including DNA methylation, histone/RNA modification, and precursor messenger RNA alternative splicing and noncoding RNAs, has become more increasingly clear. At present, the application of epigenetic therapies in tumor treatment is becoming a feasible therapeutic route. In this review, we looked for the key molecules in epigenetic regulation and discuss their relative regulating mechanisms in cancer metastasis. Furthermore, we highlight promising therapeutic strategies, including monitoring serum DNA for diagnostic purposes and early phase clinical trial therapies that target DNA and histone methylation. This may also be beneficial in finding new targets for further prognosis and diagnosis of cancer metastasis. Cancer is one of the major public health problems which has become a dominant cause of death in the global world, while the stage of metastasis is responsible for approximately 90% of deaths . In the Epigenetic regulation refers to the heritable alterations in gene expression that are not due to the changes from the DNA sequences themselves. Since its discovery in 1983 . And in Methylation is one of the well-known modifications in DNA, RNA, and histone, which are catalyzed through methyltransferases (writers) with thorough mechanisms about how to write, read, and erase the covalent epigenetic marks . Dynamic5-Methylcytosine (5mC) is one of the most studied modifications of DNA methylation written by the family of DNA methyltransferase 1-3 (DNMT1-3) . 5mC wasIt has been more than 40 years since N6-methyladenosine (m6A) was identified . m6A, onHistone modification, as a key characteristic of epigenetic regulation, has an important role in cancer progression. A diverse set of identified histone modifications known in cancer are deregulated, such as methylation, acetylation, phosphorylation, sumoylation, adenosine diphosphate (ADP) ribosylation, deimination, and crotonylation . MisreguAlternative splicing acts on the precursor messenger RNA (pre-mRNA) of one gene to generate diverse isoforms of RNA and protein . In humaThe effects of noncoding RNAs on physiological and pathological processes are increasingly becoming clear. Now we know that noncoding RNAs interact with DNA, protein, or RNA to execute their function . For exaAsp, tRFGly, tRFGlu, and tRFTyr, competed with multiple metastasis-relative transcriptions for their binding sites in RNA-binding protein YBX1. The competitive binding to YBX1 resulted in the destabilization of metastasis-relative transcriptions that suppressed cancer cell invasion and metastasis [Leu and pre-microRNA, was significantly decreased in human colorectal cancer (CRC) tissues. And tRF/miR-1280 inhibited the Notch signaling pathway and relative metastatic features by directly downregulating JAG2 expression, which leads to the cancer stem cell-like phenotype [tRNA-derived ncRNAs (tdRNAs) have been discovered in a variety of organisms and species . tdRNAs,tastasis . In addihenotype . Furtherhenotype . Over thlncRNAs are identified by their transcription length of more than 200 nucleotides, without or with partly protein-coding potential, and many of them are uniquely expressed in tissues and cells. lncRNAs influence a number of cellular functions in pathological and physiological processes; for example, lncRNAs have a variety of functions in tumorigenesis and metastasis by regulating the expression of targeted genes . It has MicroRNAs (miRNAs) are noncoding RNAs with a length of about 18~22 nucleotides that regulate gene expression by binding to the mRNA 3′UTR of targeted genes, leading to translational repression or mRNA decay . It has Since the discovery of the first circRNA in 1976 , circRNAIn tumor progression, finding the key epigenetic molecules that control metastasis is of great clinical significance and will provide a new way to search for effective therapeutic drugs and molecular markers for diagnosis. Here, we present the available evidence from four parts on how epigenetics plays a role in cancer metastasis. First, DNA methylation or histone methylation, either alone or in combination with other epigenetic molecules (such as noncoding RNA), regulated the expression of metastasis-linked genes at the transcriptional level. Second, RNA methylation also took part in translation initiation, translation efficiency, and mRNA stability of metastasis-linked genes. Third, alternative splicing increased the diversity of mRNA and noncoding RNA that played important roles in tumor metastasis, and these processes were partly influenced by methylation modification of DNA or RNA. Fourth, noncoding RNAs were almost involved in the whole process of metastasis-linked genes from transcription initiation to protein production to biological function. It has been reported that some early clinical trials targeted epigenetics, for instance, by designing drugs that specifically target DNA methylation or histone methylation for cancer therapeutics (see the review in ). Epigen


Brain metastasis is extremely rare but predicts dismal prognosis in papillary thyroid cancer (PTC). Dynamic evaluation of stepwise metastatic lesions was barely conducted to identify the longitudinal genomic evolution of brain metastasis in PTC.Chronologically resected specimen was analyzed by whole exome sequencing, including four metastatic lymph nodes (lyn 1–4) and brain metastasis lesion (BM). Phylogenetic tree was reconstructed to infer the metastatic pattern and the potential functional mutations.TSC2 indicated different genetic originations from multifocal tumors. Lyn 3/4, particularly lyn4 exhibited high genetic similarity with BM. Besides the similar mutational compositions and signatures, shared functional mutations  were observed in lyn3/4 and BM. Frequencies of these mutations gradually increase along with the metastasis progression. Consistently, TP53 knockout and CDK4R24C introduction in PTC cells significantly decreased radioiodine uptake and increased metastatic ability.Contrasting with lyn1, ipsilateral metastatic lesions (lyn2–4 and BM) with shared biallelic mutations of CDK4 and TP53 during the tumor evolution may contribute to the lymph node and brain metastasis of PTC.Genomic mutations in  Papillary thyroid cancer (PTC) comprises approximately 85% of thyroid cancer, which is the most common endocrine malignancy . AccordiV600E and TERT promoter mutations were involved in tumorigenesis and metastasis of PTC. For instance, Xing et al. reported that PTC-specific mortality occurred in 22.7% (15/66) vs. 0.6% (4/629) in patients with genetic duet and neither respectively, suggesting independent prognostic value of these two types of somatic mutations  and TERT promoter (63.4%) were also observed (TERT promoter (~1%) rather than BRAFV600E (62%) was detected in patients with non-metastatic PTC in TCGA database  from 14 patients were sequenced to identify shared and metastatic-specific genomic mutations  on the available resected samples, aiming to illustrate the longitudinal genomic profile, address the metastatic seeding pattern, and provide the potential driver mutations in metastasis/dedifferentiation process of PTC.Totally 12,458 PTC patients were treated in West China Hospital from 2000 to 2018. Screening was conducted with the following inclusion criteria: i. Conventional PTC as initial diagnosis; ii. Experience brain metastasis; iii. Lymph node metastasis prior to BM; and Exclusion criteria: i. Medullary/follicular/anaplastic thyroid cancer or mixed with these types; ii. No lymph node and brain metastasis sample available). Finally, stepwise resected samples were only available from one PTC case with brain metastasis, including specimen from four lymph nodes (lyn 1/2/3/4) and brain metastatic lesion  and incubated at 56°C for 3 min. Genomic DNA were extracted following the GeneRead DNA FFPE Kit  manufacturer’s instruction and quantified through Qubit 2.0 fluorometer (Invitrogen). The extracted DNA was quantified through NanoDrop 2000 and agarose gel electrophoresis.via cnvkit (version 0.9.2). Tumor purities were estimated by ABSOLUTE software  was gifted from the Hu Lab. Cell, which was cultured in RPMI 1640 (Hyclone) medium containing 10% FBS (PAN Seratech) and 1% Penicillin/Streptomycin (Gibco), grew at 37°C in 5% CO2 and 95% humidified air.TP53 gene was designed from (https://zlab.bio/guide-design-resources). TPC-1 was transfected with lipo3000 (Invitrogen), and treated with puromycin for 3 days. Allelic TP53 knockout monoclones were selected and submitted to validation by Sanger sequencing (The sgRNA/Cas9 expression vector lentiCRISPRV2 was obtained from Addgene (Cambridge). The sequences for sgRNA targeting CDK4 was inserted into overexpression vector  by homologous recombination, which was subsequently introduced point mutation  by using Q5 Site-Directed Mutagenesis Kit (NEB #E0552S). Lentivirus was used to infect TPC-1 and puromycin was used to screen and construct stable transfected cell lines with CDK4/CDK4R24C overexpressing (Coding region of 4 cells/well in the upper chamber with culture medium (200 μl) alone, while the bottom of the plate was filled with culture medium (800 μl) supplemented with 10% FBS as a chemoattractant. After 24 h, cells that invaded the underside of the membrane were fixed with methanol and stained by crystal violet. The experiments were repeated for three times.Cell migration experiments were performed using 24-well cell culture chambers (BD Biosciences) containing a PET membrane with 8 μm pores according to the manufactures instructions. TPC-1 was seeded at a density of 2 × 105 cells/well were seeded in 6-well plates and incubated with 2 μCi carrier-free 131I or equal volume of HBSS for 24 h. After incubation, cells were washed twice with ice-cold HBSS and scraped from each well, and radioactivity was measured in a γ-counter. The radioactivity was normalized to the cell number for each cell type. The experiments were repeated for three times.Some 1 × 10To comprehensively analyze the lineage relationship between lymph node (lyn) metastasis and BM, we retrospectively reviewed the clinical records of all thyroid cancer patients  between 2000 and 2018. After stringent screening, 34 out of 7,135 (0.5%) cPTC patients experience brain metastasis, with no surgical indication for the majority of these patients. Only one case with stepwise resected samples was included in our study with available clinical samples  samples acquired from metastatic lyn 1/2/3/4, BM and tumor-free lymph node (as reference), but not primary tumor and lyn 0 due to unavailability or low DNA quality. Tumor purity was also estimated by using ABSOLUTE, ranging from 30 to 99%, which was used to adjust for subsequent variant allele frequencies (VAF).Taking lyn N as reference, somatic alterations were estimated by mature bioinformatics approaches. A total of 538 somatic mutations were identified , including 124 coding region or splicing sites, with a median of 39 (range from 32 to 54) in lymph node and brain metastasis . SignatuTSC2), and additional 9 among lyn3/4 and BM , while no shared mutation was found between lyn 1 and other samples (TSC2) shared by lyn2/3/4 and BM, two shared by lyn 3/4 and BM   were mostly reported as the key factor for PTC development, neither of these two common mutated genes was identified for either sample of this patient through both WES and Sanger sequencing approaches  were also estimated, agreeing on the general diploid across all tumor samples with only a few focal SCNAs except haploid for chromosome X (KIF3A)   . In anotTP53 alterations on PTC dedifferentiation has been well established and evaluated in mouse model with thyroid cancer  to represent TP53 nonsense mutation, and evaluated its impact on 131I uptake. As positive control, the ATC cell line  has a significant lower uptake of 131I compared to that in TPC-1 cells. Interestingly, knocking-out of TP53 also significantly reduced the uptake of 131I in TPC-1, which is consistent with our hypothesis   , but cannd CHD4) , and onend CHD4) . In thissplicing and TSC2R1215*) and branch mutations  may also illustrate the genomic basis of stepwise metastasis and dedifferentiation.Overall, the divergence and similarity among metastatic sites well fitted the time cross and seeding pattern of the metastasis. Since similar mutational pattern of driver genes may speculate the same origin , heterogTSC2 were identified in all differentiated and dedifferentiated samples except lyn 1, suggesting its role on tumorigenesis of PTC. Actually, TSC2 encodes a tumor suppressor, which is capable of stimulating specific GTPases  and CDK4 are well-established tumor suppressor and oncogene, respectively, involving in cell cycle checkpoint of G1/S phase. Activity of CDK4 can be inhibited by p21, which is the direct downstream target of p53  through preventing their binding  arise together with CDK4R24C, with the same trend of increased content from lyn 3 to BM  also indicated the rare TP53 mutation rate in PTC (<1%) compared to ATC (>30%) , while lowest in cPTC (the most differentiated subtype)  , and cond cancer , 47, 48,subtype) , 44, furTP53 and CDK4 mutations in the dedifferentiation process of cPTC should be validated in a large cohort. Second, due to the long duration of brain metastasis free survival of this patient (20 years from the primary surgery to brain metastasis), tumor samples were not available from the primary cPTC lesion, but several lymph nodes metastasis. The lack of primary tumor sample prevented us to determine the trunk mutations of this patient, whether bi-allelic TSC2 mutations were involved in tumorigenesis or dedifferentiation should be determined in other cohorts or functional assay. Third, because we can only conduct WES with FFPE samples, it is impossible for us to detect the fusion genes commonly reported in thyroid cancer  , mostly with no surgical indication, thus it is very difficult to obtain the clinical samples of brain metastasis. In this case, samples from only one patient were sequenced in this study, and involvement of fusions) , which cTSC2, CDK4 and TP53 may contribute to the risk of development, dedifferentiation and metastasis of PTC.In conclusion, we performed a longitudinal genomic analysis for a rare case of cPTC with brain metastasis, and revealed its molecular metastatic seeding pattern. Functionally mutations in https://bigd.big.ac.cn/gsa-human/browse/HRA000283).The original contributions presented in the study are publicly available. This data can be found here: . The ethics committee waived the requirement of written informed consent for participation.HX, JZ, and YS designed and supervised this study. XL, QH, ZX, YL, YW, and LZ conducted the experiments and data analyses. HL and HX interpreted the data. HL, YQ, YJ, LS, HC, TW and ZL collected the clinical samples and information. LD, LY, and WZ vouch for the data and the analysis. HL and HX contributed to the conception of the study and drafted the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by (1) National Natural Science Foundation of China (No. 81903735); (2) China Postdoctoral Science Foundation (2019M653416); (3) Sichuan Science and Technology Program (2019YFS0333); (4) 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University ; (5) International Cooperation Project of Chengdu Municipal Science and Technology Bureau (No. 2020-GH02-00017-HZ); (6) Post-Doctor Research Project, West China Hospital, Sichuan University (2018HXBH004).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.


Ablation of predicted upstream inhibitory neurons reduces the frequency of turn behavior, suggesting that optimization needs both calcium responses. At the molecular level, unc-130 upregulates the expression of at least three genes: nca-2, a homolog of the vertebrate cation leak channel NALCN; glr-1, an AMPA-type glutamate receptor; and eat-4, a hypothetical L-glutamate transmembrane transporter in the central neurons of optimization. unc-130 shows more limited regulation in optimizing behavior than an atonal homolog lin-32, and unc-130 and lin-32 appear to act in parallel molecular pathways. Our findings suggest that unc-130 is required for the establishment of some AIB identities to optimize avoidance behavior.The central neural network optimizes avoidance behavior depending on the nociceptive stimulation intensity and is essential for survival. How the property of hub neurons that enables the selection of behaviors is genetically defined is not well understood. We show that the transcription factor  Optimization of avoidance responses to harmful stimuli plays a critical role in defense, and failure directly leads to life-threatening situations1. Most animals choose various avoidance behaviors, such as reflexes, retreats, and U-turns, depending on the situation2. The neural circuit that evaluates risk to decide the appropriate behavior is not fully understood because of the complexity of these responses compared to simple all-or-none responses3. Thus, elucidating the neural and molecular basis is a great challenge.Behavioral optimization has a vital function shared by many species: it enables the central nervous system to take reasonable action in response to environmental informationDanio rerio) and the nematode Caenorhabditis elegans (C. elegans) have been used as model animals to elucidate the mechanism at the synaptic level. In zebrafish, two Mauthner cells in the hindbrain directly control the muscles and determine the escape direction5. Mauthner cells can alter glycine receptor expression in a stimulus-dependent manner in addition to following the genetic and developmental programs5. The excitation levels of the Mautner cells vary in response to stimulus intensity, but their association with behavioral changes remains unclear.The zebrafish  induce reversals and omega turns6. AIBs can also affect the OFF-calcium response in the AVA neurons via chemical synapses. AIBs and AVAs coordinate muscle contractions required for turns via motor/interneurons12. We have reported that a lack of AIB cells or gap junction innexin-1 (inx-1) in AIBs reduces turn frequency but increases reversal frequency. These results suggest that AIBs play a central role in optimization and that electrical synapses are components involved in turning6. Calcium imaging using a microfluidic system13 has revealed that AIBs show strong calcium induction after a hyperosmotic stimulus following a decrease during stimulation6.As part of a neural circuit, ASH sensory neurons mainly perceive nociceptive stimuli, which is the first step, and excite at least three downstream circuits17. Previously, we screened 210 neural transcription factors and found that abnormal cell LINeage 32, lin-32, a nematode homolog of the proneural gene atonal/ATOH118, is essential for the optimization of avoidance behaviors via AIB neuron development. lin-32 mutants lack a number of neural function genes in AIBs6, and lin-32 may contribute to the initiation of neurogenesis rather than the determination of AIB neuron identity. Therefore, transcription factors that establish the unique features of AIBs have been left unexplored.AIB neurons develop in two steps. First, transcription factors encoded by proneural genes determine progenitors' neuronal fates, and then the progenitors differentiate into mature neurons with unique properties through the combined activity of several transcription factorsunc-130, an ortholog of the forkhead transcription factor 3 or 4, FOXD3/410, plays an essential role in avoidance optimization, mainly by adjusting turn frequency. unc-130 null mutants lack both ON responses  and OFF responses  in AIBs. Inhibitory AIAs affect turn frequency, implying their involvement in AIB ON responses . OFF responses  in the AVAs directly downstream of the AIBs may depend on the AIBs. We report that unc-130 upregulates three genes: the putative Nematode CAlcium channel 2 (nca-2)19, a homolog of the vertebrate cation leak channel NALCN; AMPA-type glutamate receptor 1 (glr-1)21; and eat-4, a hypothetical L-glutamate transmembrane transporter22. However, it does not upregulate synaptobrevin (snb-1)25 or electrical synapse innexin 1 (inx-1)10. Our results suggest that unc-130 partially determines AIB neuron identity to aid in the optimization of avoidance behaviors.Here, we report that C. elegans tends to show reversal behavior in response to mild osmolarity changes but is biased toward omega turns in a manner correlating with stimulus strength     Fig. a–e.6. Considering that unc-130 expression is relatively restricted in cell lineages including AIB cells , during embryogenesis30, we hypothesized that the optimization defect of unc-130 mutants likely resulted from AIB neuronal dysfunction. To test this hypothesis, we attempted to analyze AIB neural responses, including calcium reductions during stimulation, using the calcium indicator inverse-pericam 2.0 (IP2.0), whose fluorescence intensity increases as the calcium concentration decreases31. As we expected, the fluorescence intensity of IP2.0 increased during osmotic stimulation and decreased after stimulation in wild-type animals, showing that AIBs received inhibitory input during stimulation and excitatory input after the stimulus was removed , implying relatively high resting calcium level in both wild-type and unc-130 animals . These data suggest that reduced responses in AIB neurons cause the optimization defect in unc-130 mutants.Previously, we showed that AIB neurons are the central neurons for turning and the biphasic response involving a calcium reduction during osmotic stimulation and an increase after stimulationved Fig. a. In conses Fig. bc and c 18, but it remains unclear whether the AIB ON response  contributes to the turning behavior. In C. elegans, ASH sensory neurons indirectly inhibit AIBs via predicted AIA inhibitory interneurons , and AIA-ablated unc-130 mutants exhibited indifferent turn frequencies , although the response was slightly dampened in mutants compared within wild-type animals  in AVAs , although it was reduced in size  on AIBs (n = 25); we used the inx-1 promoter to mark AIBs and identify AIB-specific gene expression. AIBs expressed acc-1 (Acetylcholine-gated Chloride Channel 1)38, lgc-46 , and lgc-49 39 promoter-driven GFP 38 10. We analyzed the turn behavior of deletion mutants of acc-1, lgc-46, and lgc-49, but there were no differences between the single or double mutants and wild-type animals 38 Fig. b, nor dials Fig. c and d.goa-1 , which exhibits G protein-coupled acetylcholine receptor activity40. In C. elegans, only goa-1 encodes a member of the mammalian Gi/o class of Gα subunits, and the predicted amino acid sequence of C. elegans GOA-1 is over 80% identical to that of mammalian Gαo40. Hypothetical goa-1 null mutants showed a lower turn frequency (13.3 ± 4.41%) than wild-type animals (56.3 ± 5.96)  . Thus, we concluded that the reduction in turning was independent of the goa-1 function in AIB neurons. In C. elegans, there are three types of muscarinic-type acetylcholine receptors: gar-1 (G-protein-linked Acetylcholine Receptor 1)41, gar-242, and gar-343. Although we analyzed the behavior of triple gene mutants44, turn frequency was not significantly lower in these animals than in wild-type animals . Consequently, we could not identify the AIA-AIB inhibitory receptors, but the possibility remains that an unknown G-protein coupled acetylcholine receptor participates.We next assumed a contribution of G protein-coupled acetylcholine receptors. We observed turn behavior in deletion mutants of st) Fig. a, but thins Fig. b and c   Fig. a. TherefC. elegans46. We excluded three genes from our analysis. One of them, ccb-1 , is not expressed in AIBs46. We performed a drop test for mutants of the other seven channels and found that only nca-2  mutants showed a lower turn frequency in response to a 4 M sorbitol drop than wild-type animals . The mutants for the other five channels, tag-180 47 48, nca-1 (putative Nematode CAlcium channel 1)19, cca-1 49, ccb-2 19, and unc-36 (UNCoordinated 36)47, showed no reductions in turn frequency  and ccb-2 (ok862) provide no evidence for null alleles only because they are predicted to have hypothetical reduced expression and partial loss of a domain, respectively  1) promoter-driven GFP expression. glr-1 is one of the primary excitatory receptors in AIB neurons50. GLR-1 is the primary glutamate receptor between ASHs and AIBs. Among the wild-type animals, 65.0% of individuals expressed GFP in two AIB cells, while 35.0% expressed it in one , which fills vesicles with glutamate37. We detected a significant reduction in the fluorescence intensity of eat-4 promoter-driven mCherry in the AIBs of unc-130 mutants  , a typical presynaptic marker, at synaptic sites. However, both the maximum size and the total fluorescence intensity of SNB-1::mCherry fusion puncta indicated no significant differences between the unc-130 mutants and wild-type animals  , and there was no association between the presence of ectopic neurites and turn/reversal frequency . They suggest that this morphological is not related to the optimization of avoidance behavior. Next, to determine whether unc-130 might affect the amount of inx-1 expression, we quantified the intensity of GFP driven by the inx-1 promoter in the mutants. The unc-130 mutants expressed the same amount of inx-1 as wild-type animals , indicating that unc-130 regulates genes, inx-1 different from those regulated by lin-32. Finally, we performed a drop test on the double mutants to confirm that unc-130 and lin-32 act in different molecular pathways. The double mutants exhibited more remarkable optimization defects than the single mutants  Fig. a and b. 28) Fig. a and b. ncy Fig. c  in ASHs can induce glutamate release. GLR-1, a receptor for glutamate on AIBs, induces a change in conductance to open NCA-2, a predicted voltage-gated cation channel or a cation leak channel, in order to promote the intracellular influx of cations. This contributes to the increase in the OFF-calcium concentration response in AIBs. We showed that overexpression of NCA-2a in AIBs of -1 Figs. b. These 6. Thus, the AIBs appear to integrate the two excitatory and inhibitory (E/I) pieces of information from the AIAs and ASHs to evaluate the intensity and exposure time of the stimulus and to output avoidance behavior with appropriate intensity and timing.The vesicular glutamate transporter EAT-4 transports glutamate into synaptic vesicles. Calcium induction promotes the secretion of glutamate-releasing synaptic vesicles that transmit information to downstream inter-/motor neurons, including neck motor neurons. We have shown that the contraction strength of the neck positively correlates with turn behaviorunc-130 mutants show reduced expression of glr-1 and nca-2 aapap lineage from which AIB is derived30. unc-130 specifies two glial types that arise from the neighboring lineage (ABp(l/r)aapa)54. As for the AIBs that we are focusing on in this study, they express at least inx-1, which is selective for AIBs, and show a consistent number, location, and interneuron-like morphology. We consider that they are incomplete differentiated AIBs rather than sister cells of sensory neurons or glial cells.On the other hand, since AIB-specific driven es Figs.  and S4, unc-130 is involved in both the function and development of AIBs, and comprehensive discussion summarizing the importance of both sides is more appropriate for the role of unc-130. unc-130 is required for the establishment of some AIB identities , and unc-130 contributes to behavioral optimization through its role in regulating the expression of a group of genes that are necessary for AIB identities. unc-130 has the defect in the lineage determination, so the possibility remains that it reflects an unidentified reorganization of neurons in addition to AIB defects. It might explain the synthetic defect in the behavior of unc-130;lin-32 double mutants.Now we conclude that C. elegans during stimulation. Calcium imaging results showed that ASHs and AIAs were excited and that AIBs were inhibited during the stimulus 57, it is likely due to neural circuits distinct from those involved in response to noxious osmolality. Anatomical analysis showed a direct synaptic connection between the ASHs and AIBs  response  engages the disinhibition of AIB after stimulation6.Next, we considered the neural mechanisms of calcium increases in wild-type 59. One of the next questions will be to determine whether the duration of ON-hyperpolarization of AIBs is correlated with the probability of OFF-calcium responses in C. elegans, so that similarity with a property of suprachiasmatic nucleus neurons can be clarified. Rebound responses are also observed in striatal neurons and imply the existence of an essential mechanism for fear processing and decision-making60. C. elegans AIBs share similarities in that they are involved in escape behavior optimization and behavioral choice in response to harmful stimuli. The biphasic neural response with excitatory-inhibitory association seems likely to have been evolutionarily acquired for behavioral diversity. We consider that the biphasic neural response in C. elegans may be one of the neural mechanisms of primitive excitation-inhibition association. In addition, rebound firing is also associated with diseases; for example, drug addiction pathogenesis in rodents and thalamic neurons generate unusual rebound firing at the end of inhibition in Parkinson's disease in humans61. In the future, C. elegans AIB neurons may be used as a neuron model for drug addiction pathogenesis and neurodegenerative diseases.Physiologically, such a biphasic interneuron response has also been observed in mammals. In rodents, over 60% of suprachiasmatic nucleus neurons may use such “rebound responses” or “postinhibitory rebounds,” and the probability of the response is positively correlated with the duration of hyperpolarization6, suggesting that AIA-AIB inhibition suppresses time-consuming turn behavior under conditions of toxic osmolality to expedite escape.Furthermore, AIB biphasic responses may biologically contribute to temporal control of the initiation of turn behavior. Direct AIB excitation by optogenetics facilitates turning without reversalFOXD3/4 ortholog, unc-130, specifies avoidance behavior patterns using C. elegans. FOXD3 is widely conserved from invertebrates to vertebrates, including humans10. FOXD3 defines the early pluripotency of neural crest stem cells in vertebrates to differentiate into diverse cells, such as neurons and muscles64. Even in C. elegans, unc-130 has a similarity to FOXD3/4 with regard to its expression in both neural progenitors and adult muscle cells66. Future unc-130 studies may lead to universal molecular insights into primary neurogenesis. In addition, whole-human genome analysis has implied that FOXD4 is a risk factor for suicide and obsessive–compulsive disorder67. Such outcomes may be attributable to vulnerability to stress. unc-130 might be useful for illustrating a prototypical circuit for improved coping behavior under exposure to harmful stimuli.In this study, we demonstrated, for the first time, that a C. elegans68. They speculate that combinations of homeobox (Hox) transcription factors can code for almost all neural identities69; these findings will accelerate the understanding of individual neural characters. In addition, basic helix-loop-helix (bHLH) transcription factors, including proneural genes, act in the initial phase and have a crucial role in neurogenesis61. However, forkhead box (Fox) transcription factors were grouped relatively recently in 200061, and their functions in the nervous system are still less known than those of Hox and bHLH molecules.During development, combinations of transcription factors determine neuron identities. Hobert et al. have comprehensively mapped the combination of transcription factors expressed in all neurons of FOXD3/4 ortholog, unc-130 contributes to behavioral optimization mediated by pre- and postsynaptic function to mediate biphasic neural responses. In summary, reductions in the ON and OFF calcium responses required for integrating this information and producing behavioral outputs result in incorrect behavioral choices in response to stimuli of different intensities in unc-130 mutants.Our findings suggest that a C. elegans strains using modified standard techniques70. NGM agar plates containing 67 mg/ml antibiotic streptomycin and 10 µg/ml nystatin were used. Escherichia coli OP50-1 was seeded as food. unc-130(tm320), acc-1(tm3268), nca-2(tm1305), unc-2(e55), and lin-32(tm2044) mutants were backcrossed twice with N2. lgc-46(ok2949), lgc-49(tm6556), and goa-1(sa734) mutants were backcrossed three, four, and five times with N2, respectively. cca-1(gk30), nca-1(gk9), unc-36(e251), unc-36(ok862), and ccb-2(ok862) were not backcrossed because of the pilot screening  or both the 2409 bp sra-6 promoter and GCaMP6s sequences from N2 genomic DNA and pGP-CMV-GCaMP6s into the pPD95.75_Pinx-16 or pPD95.75 plasmids instead of gfp, respectively. For expression analysis of inhibitory acetylcholine receptors . pPD95.75 was a gift from Dr. Andrew Fire. lin-44p::gfp was a gift from Dr. Yuichi Iino. pPD_gcy-28dp::ced-3(p15), pPD_gcy-28dp::ced-3(p17), pPD_Pins-1(short)::mCherry .For expression analysis of -2 Figs. c and d, a-2 Fig. , the 561t-4 Fig. , the 221rry Fig. b and pFXP6s Fig. c and d wunc-130 rescue experiments, to generate tm320;tmEx5292 , pPD_Pinx-1::UNC-130::3' UTR (2 ng/μl or 0.2 ng/μl) or pPD_Pnpr-9::UNC-130::3' UTR (2 ng/μl), pPD95.75_Pinx-1 (20 ng/μl), and pBluescript KS( +)T1 (140 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) as an injection marker into tm320 mutants, respectively. For calcium imaging, to generate tmEx5274 transgenic animals, pPD_Pinx-1::IP2.0 (20 ng/μl) and pBluescript KS( +)T1 (160 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. For AIA ablation experiments, to generate tmEx3494 transgenic animals, pPD_odr-2p::ced-3(p15) (80 ng/μl), pPD_ser-2p::ced-3(p17) (80 ng/μl), and pFX_DsRedxT-gpa-2(aa1 + int.) (20 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. To generate tmEx5137 transgenic animals for ASH calcium imaging, pPD_Psra-6::GCaMP6s (180 ng/μl) was coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. To generate the tmEx5293 transgenic animals, pFX_Pgcy-28.d::GCaMP6s (50 ng/μl), pPD_Pins-1(short)::mCherry (30 ng/μl), and pBluescript KS( +)T1 (100 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. For expression analysis of inhibitory acetylcholine receptors, acc-1, acc-2, acc-3, and acc-4, to generate tmEx5408, tmEx5409, tmEx5411, and tmEx5412 transgenic animals, pPD_Pacc-1::gfp (100 ng/μl) or pPD_Pacc-2::gfp (100 ng/μl), pPD_Pacc-3::gfp (100 ng/μl) or pPD_Pacc-4::gfp (100 ng/μl), and pPD_Pinx-1::mCherry (80 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. For lgc-46 and lgc-49, to generate tmEx5454 and tmEx5449 transgenic animals, pPD_Plgc-46::gfp (100 ng/μl) or pPD_Plgc-49::gfp (100 ng/μl), pPD_Pinx-1::mCherry (40 ng/μl), and pBluescript KS( +)T1 (40 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. To generate tmEx5614 transgenic animals, pPD_Pgoa-1::gfp (100 ng/μl), pPD_Pinx-1::mCherry (20 ng/μl) and pBluescript KS( +)T1 (60 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) into N2 animals. For expression analysis of the hypothetical voltage-dependent calcium channels and nca-2, to generate tmEx5615 and transgenic animals, pPD_Pnca-2::gfp (100 ng/μl) and pPD_Pinx-1::mCherry (40 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) and pBluescript KS( +)T1 (40 ng/μl) into N2 animals. For AIB-specific rescue, to generate tm1305; tmEx5629, tm1305;jskEx0017 and tm1305;jskEx0018 transgenic animals, pPD_Pinx-1::NCA-2a (20 ng/μl) and pPD_Pinx-1::gfp (20 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) and pBluescript KS( +)T1 (140 ng/μl) into nca-2(tm1305) animals. To generate tm1305;jskEx0019, tm1305;jskEx0020 and tm1305;jskEx0021 transgenic animals, pPD_Pinx-1::EAT-4 (20 ng/μl) and pPD_Pinx-1::gfp (20 ng/μl) were coinjected with lin-44p::gfp (20 ng/μl) and pBluescript KS( +)T1 (140 ng/μl) into unc-130(tm320) animals. For precise synaptic localization, to generate tmEx5377 transgenic animals, pPD_Pinx-1::SNB-1::mCherry (80 ng/μl) and pPD_Pinx-1::gfp (20 ng/μl) were coinjected with unc-122p::mCherry (100 ng/μl) into N2 animals. The strains tmIs825[Psra-6::ChR2(H134R)::mCherry + Pges-1::EGFP]), tmEx4532[Pnpr-4::G-CaMP6s + Pnmr-1::mCherry + Pin-44::gfp], tmEx4456[Pinx-1::ChR2(H134R)::mCherry + Pinx-1::gfp + Plin-44::gfp], rhIs4[glr-1::GFP + dpy( +)]; tmEx3532[DsRedxT-odr-2(aa1) + pBluescript KS + T1]  on NGM plates, respectively. The platinum wire has a smooth polished surface to avoid damaging the nematode. “n = x” in the figure legends indicates the number of plates analyzed. Responses were classified as omega turns and others, as previously described71. The animals were individually irradiated with 100% blue light (approximately 2.47 μW/cm2) using a CFP filter (365 nm) at their heads for 2 s. Each score was calculated for 10 ± 3 animals. We performed the experiments on at least three different days and calculated the average percentage. “n = x” in the figure legends indicates the number of plates analyzed. The experimenter was blinded to the nematode strains and to whether all-trans retinoic acid (ATR) was added or not.We performed a ChR2(H134R)-induced avoidance assay as previously described13. We used 2 M sorbitol dissolved in S basal as a stimulus. All optical recordings of neurons were performed on an IX71 microscope with a 40X immersion objective  and an ORCA-Flash2.8 CMOS camera (Hamamatsu Photonics) and analyzed with MetaMorph software (Molecular Devices). We captured time stacks of the fluorescence images at one frame per second. The images were analyzed as previously described18. We calculated the percent change in the fluorescence intensity relative to the average intensity during the 5 s before stimulation. In the IP2.0 analysis, the same baseline fluorescence was seen in both wild-type animals and unc-130 mutants, we shifted them to 0 for subtraction and normalization as corrected baselines. In the statistical analysis of AIA, we compared the maximum ΔF/F values from the 5 s before stimulation to 100 s after stimulation. We performed image tracking using a custom ImageJ  plugin. We drew a rectangular region of interest (ROI) surrounding the cell body, and the ROI was shifted according to the new position of the center for every frame.As previously described, we performed calcium imaging using an olfactory chiphttps://www.zeiss.co.jp/microscopy/downloads/zen.html). We drew an ROI surrounding the cell body  plugin. We performed ratiometric analysis for an accurate discussion of expression levels in extrachromosomal arrays, comparing marker fluorescence to nca-2 (GFP)   Fig. d, SNB-1(FP) Fig. e, and goFP) Fig. c fluoresFP) Fig. f using at-test, Mann–Whitney test, or Fisher’s exact test according to the results of the Shapiro–Wilk normality test. We used one-way ANOVA followed by Tukey’s post hoc test or the Kruskal–Wallis test with Dunn's multiple comparisons test as a post hoc test according to the results of the Shapiro–Wilk normality test for multiple groups. We produced bar graphs with the mean ± SEM values from three or more independent experiments. In both cases, “n” is the number of plates (cohorts) of 10 ± 3 animals each. In the bar graphs without error bars . Pairwise comparisons of omega turn frequencies within two groups were carried out via Student's ars Fig. b, “n” isSupplementary Information 1.Supplementary Information 2.


Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed  The propagation of life requires the rapid and accurate assembly of the microtubule-based mitotic spindle , 2. DuriThe majority of spindle assembly factors are inhibited by the canonical and abundant karyopherin complex, the importin-α/β heterodimer , 9, 10. via branching microtubule nucleation (in vitro overlaps with a known importin-α/β-binding site (in vitro (Xenopus egg cytosol (The spindle assembly factor and microtubule-binding protein targeting protein for XKlp2 (TPX2)  promotescleation , which ccleation , 20, 21.cleation , 20. TPXcleation , 23. Theing site . Howeverin vitro  and does cytosol , 25. Fur cytosol . Finallyvia liquid–liquid phase separation, to achieve compartmentalization or reaction enhancement . Previously, we used this method to reveal that the importin-α/β heterodimer inhibits TPX2-mediated branching microtubule nucleation . We added TPX2 mixed with either importin-αΔIBB or importin-β to Xenopus egg cytosol, keeping TPX2 constant at 150 nM and including importins at a range of fold excesses—2.5× (375 nM), 5× (750 nM), 10× (1.5 μM), or 20× (3 μM). When only importin-α-ΔIBB was included at 20-fold molar excess of TPX2 the total number of microtubules nucleated relative to the no importin condition was drastically reduced (A and C). Furthermore, TPX2 addition with only importin-β at 10-fold molar excess led to an even greater reduction in microtubule number than importin-αΔIBB at 20-fold molar excess, with branching microtubule nucleation being reduced to background levels (B and C). These data show that, surprisingly, importin-α and importin-β can each independently inhibit TPX2-mediated branching microtubule nucleation, with importin-β being particularly effective. Next, we investigated the nature of the interactions between TPX2 and importin-α or importin-β to gain mechanistic insight into this inhibition.In the importin-α/β heterodimer, importin-β binds to importin-α’s autoinhibitory IBB domain to expose the NLS-binding pocket on importin-α. To mimic this state, we used a truncated form of importin-α, which does not contain the IBB domain (importin-αΔIBB) , 24 FigD. We add reduced , A and Cd levels , B and CTPX2 binding to microtubules serves as the first essential step to build a branch site, from which TPX2 recruits additional branching factors , 23. Morvia fluorescence microscopy with the inclusion of either importin-αΔIBB or importin-β alone or the importin-α/β heterodimer. TPX2 concentrations were held constant at 1 μM, and importins were included at a range of molar excess concentrations. Mixtures of importin-αΔIBB and TPX2 resulted in an enhancement of TPX2 condensation at lower concentrations followed by an inhibition of TPX2 condensation at approximately 20-fold excess (A). In contrast, importin-β inhibited TPX2 condensation between 4- and 8-fold excess and importin-α/β did so at 2-fold excess, making them both more effective than importin-α (B and C).We monitored TPX2 condensation d excess A. In conportin-α , B and CD). Importin-β, in turn, suppressed TPX2 condensation more efficiently than importin-αΔIBB. The efficiency of inhibiting TPX2 condensation is correlated with importin-β’s ability to inhibit branching microtubule nucleation more strongly than importin-αΔIBB (A–C). Yet, this was surprising because, although TPX2 has an NLS that is known to interact with importin-α, it does not harbor any known IBB sites. Therefore, we next interrogated how TPX2 interacts with the importin-α/β heterodimer, as well as importin-αΔIBB and importin-β alone (D).To ensure that importin-inhibited TPX2 solutions are indeed monodisperse and do not contain any subresolution condensates, we tested each sample with dynamic light scattering, which measures an intensity–intensity autocorrelation function of scattered light from the solution. Because the scattered intensity is proportional to the sixth power of particle sizes, any residual amount of condensate can be detected. Our light scattering data further validated the results obtained by light microscopy: importin-α/β abrogates TPX2 condensation more efficiently than importin-β D. Importin-αΔIBB , A–C. Ye-β alone D.D). We first addressed this question by determining the molecular weight and stoichiometry of the TPX2–importin-α/β complex via size exclusion chromatography in line with multiangle light scattering (SEC-MALS) (A). GFP-TPX2 eluted off the SEC column with an average molecular weight of 159 ± 52 kDa, comparable with its predicted molecular weight of 112.1 kDa, indicating that it exists mainly as a monomer with no detectable aggregate/oligomers .Although small fragments of TXP2 have been shown to bind to importin-αΔIBB , 24, it Fig. 1EC-MALS) A. GFP-TPvia SEC-MALS. The eluted complex exhibited a molecular weight of 130.0 ± 29 kDa determined via MALS, consistent with the predicted molecular weight of the importin-α/β heterodimer in a 1:1 stoichiometry (165.0 kDa) .We then allowed GST-importin-α to bind to importin-β and assessed the formation of the importin-α/β heterodimer via MALS  similar to the predicted molecular weight of 286.8 kDa. This suggests that TPX2–importin-α/β exists as a trimer with 1:1:1 stoichiometry.When TPX2 and importins-α/β were allowed to bind, they eluted as a complex faster than TPX2 and importins-α/β alone. This rapid elution reflects a larger molecular weight of 310 ± 70 kDa via biolayer interferometry, which detects refractive index fluctuations upon binding and dissociation events of target proteins on the sensor surface (d between TPX2 and importin-α/β of 75 ± 15 nM (B), indicating that TPX2 binds strongly to importin-α/β.In order to determine how strongly TPX2 interacts with importin-α/β, we determined their equilibrium dissociation constant  surface , 34. We  ± 15 nM B, indicaWe next investigated how each individual importin binds to TPX2, both to address how importin-α and importin-β each contribute to forming the TPX2-importin-α/β trimer and to interrogate how their respective binding mode relates to their ability to inhibit TPX2.d = 61 ± 10 nM between importin-αΔIBB and TPX2 (B), indicating it alone has a strong affinity for TPX2, comparable with importin-α/β. We next asked whether this interaction is mediated by the two nuclear localization sequences (NLSs), NLS1 and NLS2, which had previously been shown to form a complex with importin-αΔIBB (A) in a crystal structure (Xenopus TPX2 (a.a. 270–350). It was demonstrated that NLS1 solely mediates the interaction with importin-αΔIBB, whereas NLS2 is dispensable and not conserved to humans (d = 45 ± 6 nM (D). This suggests that another strong binding site on TPX2 must exist to allow importin-αΔIBB to bind.By applying biolayer interferometry, we measured a dissociation constant of Kand TPX2 B, indicain-αΔIBB A in a crtructure . This cro humans . We crea5 ± 6 nM D. This sTo investigate where this potential new binding site was located, we first assessed whether it lies within the C-terminal half of TPX2 (amino acids 319–716). However, this construct exhibited an extremely weak association with importin-αΔIBB , suggestSince NLS1 and NLS2 were previously identified by scanning TPX2 amino acids 270 to 350 , we focud = 410 ± 70 nM) in the strength of its interaction with importin-αΔIBB, compared with 2M-TPX2 or WT-TPX2 (B and D). This suggests that, within the entire sequence of TPX2, NLS1 and NLS3 represent the only canonical NLS sites that promote high-affinity interactions with importin-αΔIBB.To assess whether NLS1 and NLS3 are indeed the major and only bindings sites on TPX2 for importin-αΔIBB, we created a triple-mutant TPX2 (3M-TPX2), for which all the NLS sequences were modified . Strikingly, 3M-TPX2 exhibited a 10-fold decrease . Comparison of the TPX2–importin-αΔIBB MALS profile to importin-αΔIBB alone, which displays a molecular weight of 159 kDa (implying it dimerizes), suggests TPX2–importin-αΔIBB is relatively dispersed and exists in a complex with stoichiometries above 1:1 (C). Taken together, these data show that importin-αΔIBB strongly interacts with TPX2 via two NLS sequences.Because TPX2 contains two NLS sequences to which importin-αΔIBB can bind, we investigated the stoichiometry of the TPX2-importin-αΔIBB complex  ±88 kDa C. Comparbove 1:1 C. Taken d = 530 ± 75 nM. Of interest, this association is driven by TPX2’s N terminus (a.a. 1–480), which exhibits a similar dissociation constant of 512 ± 70 nM (B) as full-length TPX2. Meanwhile, TPX2’s C terminus (a.a. 480–716) displays almost no binding with importin-β, exhibiting a Kd > 4 μM (D).Importin-β is known to associate with some nuclear proteins  and affe ± 70 nM B as fulld > 4 μM D.Figure C), which could be due to reversible assembly or irreversible aggregation. To delineate between these possibilities, we conducted a series of SEC experiments keeping TPX2 at 2 μM and varying importin-β concentration ranging from 2 to 30 μM, and integrated the area under the peak corresponding to the eluted complexes from SEC-MALS . If these complexes were irreversible aggregates, a linear dependence on importin-β concentration would be expected. Instead, we observed a sigmoidal binding curve  suggesting that there exists a dynamic equilibrium that saturates upon the addition of importin-β at 3-fold excess. To confirm that the saturation is not due to consumption of TPX2, we calculated the unbound fraction of TPX2 using the dissociation constant of Kd = 0.5 μM measured by biolayer interferometry. At the maximum concentration of importin-β, c = 30 μM, more than 5% of TPX2 is still in the unbound state. Based on these measurements, the concentration of importin-β would need to exceed 100 μM for ≥99% of the 2 μM of TPX2 to be bound. Therefore, the saturation of the SEC curve was not likely due to the depletion of TPX2 and rather due to the equilibrium nature of the TPX2:importin-β complex. Thus, our data demonstrate that TPX2 weakly and reversibly associates with importin-β and this appears to be sufficient to prevent TPX2 phase separation , which could be a mechanism for importins to prevent TPX2 phase separation when they bind. To address this possibility, we quantified the osmotic compressibility of importin solutions via the second virial coefficient (B2) obtained from static light scattering, wherein repulsion would result in a positive B2 (2 (slope in C), indicative of attractive intermolecular van der Waals forces originating from the exposed hydrophobic residues on the surface of importins (B)  and the high negative surface charge suggest that the proteins should be self-repulsive A, which itive B2 . Strikinslope in C, indicamportins B 37, 38, 38A, wh2, and therefore the strength of intermolecular van der Waals forces, follows the competency of importins to impede the condensates—Of most interest, the magnitude of Bensation , 31. Futin vitro and does not appear to reduce binding in cytosol   , and assd (≥70%) , 39. Nonith NLSs .via NLS1 (located at a.a. 284–287) , termed NLS3. The high dynamic range of biolayer interferometry allowed us to measure both strong interactions (<100 nM) mediated by NLS1 and NLS3 and relatively weak interactions (>400 nM) that persisted in their absence. Finally, NLS3 was previously demonstrated to play a role in nuclear import  was previously shown to bind to TPX2 284–287) , 24. Alt284–287) . By measr import , and ourr import . Collectvia weak interactions, which is reminiscent of other disordered proteins with a different karyopherin–Transportin-1/Karyoperhin-2β . Specifically, the hierarchy of inhibition competency is importin-α/β > importin-β >> importin-αΔIBB, whereas the hierarchy of affinity is importin-αΔIBB ≈ importin-α/β >> importin-β  in proximity to importin-α and importin-β. In so doing both importins appear to collectively suppress TPX2 intermolecular forces and abolish condensation (Collectively our data indicate that importin-α acts as a high-affinity bridge that holds TPX2 (ensation . We suspXenopus laevis versions and were purified to >95% purity (A). Size exclusion chromatography (SEC) was carried out on an AKTA Pure-25L. SEC in line with multi-angle light scattering (SEC-MALS) was done with a 3.2/300 column packed with Superdex-200-increase-in line with a Wyatt light scattering machine. Biolayer interferometry was carried out by varying importin concentration and measuring binding to TPX2 that was attached to sensors (B), according to vendor specifications of ForteBio. Condensation (phase separation) was achieved by diluting protein mixtures at 0.5 M into 0.1 M salt (KCl). Static and dynamic light scattering measurements were taken at a fixed right angle. Normalized intensity–intensity correlation functions of 20 s duration were collected. To visualize TPX2-mediated microtubule nucleation at indicated excess molar ratios of importins, naturally meiotically arrested X. laevis egg cytosol was immunodepleted of endogenous TPX2, indicated proteins were added, and the reaction was imaged over time. Images in each panel are representative crops from a single experimental set with brightness and contrast optimized to allow visualization of the relevant structures. The studies involving X. laevis egg cytosol have been approved by the American Association for Laboratory Animal Science (IACUC).Please see the % purity A. Size e sensors B, accordMatthewking@wustl.edu).All data are either provided in this article or will be provided upon request to Dr Matthew King, Washington University in St Louis (This article contains The authors declare that they have no conflicts of interest with the contents of this article.