TRIM59 knockdown (KD) promoted apoptosis and inhibited tumor growth, while TRIM59 overexpression led to the opposite effects. Importantly, we uncovered TRIM59 as a key regulator of cell contractility and adhesion to control the plasticity of metastatic tumor cells. At the molecular level, we identified programmed cell death protein 10 (PDCD10) as a target of TRIM59. TRIM59 stabilized PDCD10 by suppressing RING finger and transmembrane domain-containing protein 1 (RNFT1)-induced lysine 63 (K63) ubiquitination and subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa (p62)-selective autophagic degradation. TRIM59 promoted PDCD10-mediated suppression of Ras homolog family member A (RhoA)-Rho-associated coiled-coil kinase (ROCK) 1 signaling to control the transition between amoeboid and mesenchymal invasiveness. PDCD10 overexpression or administration of a ROCK inhibitor reversed TRIM59 loss-induced contractile phenotypes, thereby accelerating cell migration, invasion, and tumor formation. These findings establish the rationale for targeting deregulated TRIM59/PDCD10 to treat breast cancer.Cancer cells adopt various modes of migration during metastasis. How the ubiquitination machinery contributes to cancer cell motility remains underexplored. Here, we report that tripartite motif (TRIM) 59 is frequently up-regulated in metastatic breast cancer, which is correlated with advanced clinical stages and reduced survival among breast cancer patients.  Bioinformatic analysis and experiments on clinical samples and mouse models of breast cancer unveil a new signaling protein degradation pathway involving TRIM59 and PDCD10 that modulates breast cancer cell growth, survival, and metastasis. Cancer cell metastasis is the primary cause of mortality in cancer patients. Changes in cell morphology are critical for cancer cell motility and metastasis, and this process requires spatiotemporal control of the turnover or stabilization of signaling components that regulate cell polarity. Autophagy—a highly regulated intracellular degradation process—provides cancer cells with access to energy sources while preventing mutagenic oxidative stress to suppress tumorigenesis. But the mechanisms underlying how autophagy impacts cell metastasis remain mostly unknown. Here, we identify TRIM59, a protein in the E3 ligase family known to target ubiquitinated proteins for proteasomal or autophagic degradation, as a key component of a novel molecular mechanism in human breast cancer. We show that TRIM59 regulates the degradation of PDCD10, which is involved in programmed cell death and is the main factor for driving the pathogenesis of the devastating familial cerebral cavernous malformation (CCM) disease. We find that TRIM59 has an effect in cell shape and contractility by controlling PDCD10 levels. Our results suggest that targeting the TRIM59-PDCD10 interplay could lead to new therapeutic strategies to treat breast cancer and CCM. TRIM genes based on datasets from the Cancer Genome Atlas (TCGA) database . Among all the TRIM genes, TRIM59 was found to be the only member displaying marked up-regulation across all the 12 cancer types, a discovery that concurs with the recent findings that TRIM59 promotes the progression of prostate cancer ) and CCM2 (osmosensing scaffold for mitogen-activated protein kinase kinase kinase-3 [OSM]) to control the onset of a detrimental disease named CCM. CCM is characterized by vascular lesions in the central nervous system . Loss ofIn this study, we identified TRIM59 as a cancer-associated E3 Ub ligase, the expression of which was strongly associated with poor clinical outcomes in breast cancer patients. TRIM59 promoted breast cancer cell growth and migration/invasion both in vitro and in vivo. Importantly, we showed that TRIM59 was essential for the transition of breast cancer cells between mesenchymal and amoeboid movements. By means of yeast two-hybrid screening, we further uncovered PDCD10 and the E3 ligase RING finger and transmembrane domain-containing protein 1 (RNFT1)  as direcFigs S1A). Particularly, in breast invasive carcinoma (BRCA), TRIM59 mRNA level was significantly increased in tumor sites (red plot) compared with its expression in normal adjacent tissues . In parallel, we assessed the expression of TRIM59 mRNA in human and mouse tissues, as well as in a panel of human breast cancer cell lines, by using the mammary epithelial cell line MCF10A as a nonmalignant control. TRIM59 expression was relatively low in normal human and mouse mammary gland tissues when compared with cells/tissues of the immune system . We further detected expression of TRIM59 in breast cancer cell lines, with the highest expression in cell lines such as MCF7 but relatively low expression in cell lines such as MDA-MB-231 . To further verify the protein levels of TRIM59 in breast cancer patients, we performed immunohistochemistry (IHC) staining for TRIM59 on primary human breast tumors obtained from a large cohort of breast cancer patients . Among the 154 patients, 87 biopsy specimens contained both tumors and matched normal adjacent tissues, whereas the other 67 had only tumor tissues. Among the 87 matched samples, we detected markedly higher intensities of TRIM59 immunostaining in breast tumors than in tumor-adjacent normal tissues . Notably, TRIM59 protein levels were positively correlated with the pathologic grades of breast cancer  and were slightly higher in estrogen receptor (ER)-negative breast cancer . Most clinically relevant, elevated TRIM59 expression was significantly associated with shortened breast cancer patient survival (P = 0.0244) . To confirm that TRIM59 is an independent factor linked to clinical outcomes, we performed multivariate overall survival analysis by using a Cox proportional hazard model based on available clinical information, including patient cancer stages and pathological grades. Our statistical analysis confirmed TRIM59 expression as an independent prognostic factor . In addition, even among high-grade (Grade II and higher) patients, higher TRIM59 expression was significantly correlated with a shortened survival rate . Together, our results establish a positive correlation of augmented TRIM59 expression with breast cancer progression and survival.Among the approximately 70 members in the TRIM family, TRIM59 stood out as one of the most differentially expressed genes, with its expression ubiquitously up-regulated by 1.6- to 6.3-fold in 12 analyzed cancer types compared with the adjacent normal tissues  or clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (CRISPR/Cas9)-based knockout  strategy in TRIM59-high breast cancer cell line MCF7 . In parallel, we stably overexpressed TRIM59 by about 20-fold  in MDA-MB-231, a breast cancer cell line with relatively low TRIM59 expression . Using a cell proliferation colorimetric assay, we found that TRIM59 KD or KO in MCF7 pronouncedly suppressed cell proliferation (with both nontransfected wild-type [WT]) and control shRNA (shControl)-transfected cells as controls; Fig 2D). By contrast, cell proliferation was significantly enhanced by overexpressing (OE) TRIM59 in MDA-MB-231 cells . We next examined the anchorage-independent growth of TRIM59 KD/KO MCF7 cells and TRIM59 OE MDA-MB-231 cells by using a Matrigel-based 3D culture system. TRIM59 depletion dramatically reduced the frequency of colony formation of MCF7 cells from 14.7 ± 0.5% (control) to 5.1 ± 0.3% (shTRIM59) and 3.7 ± 0.9% (TRIM59 KO) . Conversely, TRIM59 overexpression promoted the colony formation of MDA-MB-231 cells from 10.8 ± 0.1% to 26.2 ± 0.4% . Furthermore, TRIM59 inactivation dramatically impaired MCF7 cell migration by 50%–60% , whilst TRIM59 overexpression increased cell migration of MDA-MB-231 cells by 60% . We further assessed the impact of TRIM59 inactivation or overexpression on cell migration and invasion using an independent assay based on Matrigel-coated transwell chambers. Again, down-regulation or loss of TRIM59 impeded MCF7 cell invasion , whereas TRIM59 overexpression increased the numbers of invaded MDA-MB-231 cells . Our findings establish that TRIM59 promotes the proliferation, migration, and invasion of breast cancer cells.To examine the biological function of TRIM59 in breast cancer, we employed a short hairpin RNA (shRNA)-based knockdown , respectively, with Matrigel (1:1) into the NOD scid gamma (NSG) mouse mammary fat-pad and monitored tumor growth up to 10 weeks. The tumor weight and volume were substantially reduced in mice bearing TRIM59 KD MCF7 cells when compared with control mice . By comparison, mammary xenograft tumor growth was greatly accelerated in mice bearing TRIM59 OE MDA-MB-231 cells, with a notable increase in tumor sizes . IHC staining of the marker of proliferation (Ki-67), revealed fewer proliferative cells in TRIM59 KD xenograft tumors  but more prominent proliferative activity in TRIM59 OE MDA-MB-231 xenografts, when compared with the control samples . Collectively, results from our in vivo studies further reinforced the conclusion that TRIM59 plays a pro-oncogenic role in breast cancer by promoting tumor cell growth and migration.In order to investigate the functional consequence of TRIM59 inactivation or overexpression in vivo, we orthotopically injected MCF7 cells and MDA-MB-231 cells stably expressing shTRIM59 KD or KO versus control) by using a high-throughput reverse phase protein array (RPPA) ) and inositol polyphosphate-4-phosphatase type II b (INPP4b), a recently discovered tumor suppressor [S2C Fig).To understand the molecular mechanisms by which TRIM59 promotes breast tumor growth, we performed quantitative functional proteomics studies on MCF7 cells (y (RPPA) . Our unbppressor –27 (S2C TRIM59-depleted MCF7 cells exhibited higher frequencies of early-stage (indicated by the Annexin V+ and 7’AAD− population) and late-stage apoptosis (Annexin V+7’AAD+ staining) . Consistently, xenograft tumors isolated from mice injected with TRIM59 KD MCF7 cells showed substantially higher staining of cleaved-caspase3. By contrast, TRIM59-OE MDA-MB-231 cells displayed reduced staining of cleaved-caspase3, an indicator of active apoptosis . Because the rat sarcoma (Ras)-mitogen-activated protein kinase (MAPK) signaling [TRIM59-depleted MCF7 cells. We set out to test this by determining the protein levels of p53 and cleaved caspase 3, as well as the phosphorylation status of p38, ERK1/2, and c-Jun N-terminal kinase (JNK) in TRIM59 KD MCF7 cells, with immunoblotting. We did not observe significant changes in expression or phosphorylation between TRIM59 KO and WT MCF7 cells . In aggregate, our findings suggest that TRIM59-mediated regulation of breast cancer cell growth is very likely independent of the MAPK and p53 signaling pathways.We next assessed the apoptotic properties of breast cancer cells by using 7’AAD and Annexin V double staining. ignaling  and p53 ignaling  are mostTRIM59 depletion or overexpression, we noticed prominent morphological changes in breast cancer cells. TRIM59 depletion in MCF7 cells caused a pronounced reduction of cell area with increased cell–cell interactions. By contrast, overexpression of TRIM59 changed the overall shape of MDA-MB-231 cells into a more elongated, mesenchymal phenotype, with longer membrane protrusions and reduced cell–cell adhesion . Cell shape is mainly controlled by the organization of cytoskeleton and actin/myosin (actomyosin)-mediated cell contractility [TRIM59 depletion. In addition, depletion of TRIM59 in MCF7 cells changed the organization of p-MLC to generate an accumulated arc of p-MLC in the cell cortex, accompanied by increased staining of E-cadherin, which mediates cell–cell adhesion . In line with the immunostaining results at single cell levels, immunoblot (IB) analysis revealed an increase in the total amounts of phosphorylated MLC and E-cadherin . Conversely, overexpression of TRIM59 (with IRES-GFP) in MDA-MB-231 cells led to a marked loss of p-MLC staining compared to non-overexpressing (GFP-negative) cells in the same imaging field . We next examined the effect of altered TRIM59 expression on another indicator of cell contractility by assessing the phosphorylation status of ERM with immunostaining and western blotting. Upon TRIM59 deletion, we observed enhanced blebbing , as well as increased p-ERM levels, but no significant changes in the total ERM levels in both MCF7 cells  and xenograft tumors . In contrast, TRIM59 overexpression in MDA-MB-231 cells suppressed ERM phosphorylation . In human samples, Spearman correlation analysis based on Clinical Proteomic Tumor Analysis Consortium (CPTAC) [Fig 4H, p-S148 and p-S366, R = −0.23). These data suggest that TRIM59 modulates phosphorylation of MLC and ERM in breast cancer cells. In the xenograft models, overexpression of TRIM59 in MCF7 or MDA-MB-231 cells promoted pulmonary metastasis of breast cancer cells, whereas TRIM59 deficiency suppressed cancer cell metastasis, as reflected by cytokeratin 8 (CK8) staining of metastasized cancer cells in the lung . We next performed in vivo imaging analysis to monitor the effect of TRIM59 on tumor metastasis in NSG mice injected with MCF7-luc or MDA-MB-231-luc cells (luciferase-expressing stable cells). TRIM59 deficiency suppressed primary tumor growth at early stages (around 8 weeks postinjection). This phenotype could be reversed through the reconstitution of TRIM59 OE . Notably, TRIM59-deficient MCF7-luc cells acquired a growth advantage later , but the metastatic ability was greatly reduced . Re-expression of TRIM59 restored the metastatic capacity of MCF7-luc cells to a level comparable to the WT group . Similarly, overexpression of TRIM59 in MDA-MB-231-luc cells promoted cancer cell metastasis at approximately 6 weeks postinjection and greatly shortened survival, because the mice died at 10 weeks . We further analyzed the expression of TRIM59 in human breast cancer patient lymph node metastases versus primary tumor with both RNA sequencing dataset analysis (GSE30480) and IHC staining on patient tissue array samples (paired primary versus metastatic tissues from the same patient). We found a significantly higher expression of TRIM59 in metastatic breast cancer at both mRNA and protein levels . Thus, our data demonstrate a positive correlation between TRIM59 expression and breast cancer metastasis.Upon actility , which cTRIM59 deletion . These findings indicate that TRIM59 might also promote β-catenin expression and the activation of the Wnt pathway.Because β-catenin is also a component for the cancer-promoting Wnt signaling that is intimately involved in tumor metastasis –35, we fTogether, our data indicate that TRIM59 promotes breast cancer cell metastasis by (1) inhibiting the phosphorylation of MLC and ERM required for actomyosin contractility and amoeboid migration, (2) reducing the expression of cell adhesion molecules such as E-cadherin, and (3) increasing the cellular level of β-catenin and promoting metastasis-associated Wnt signaling.S3 Table). PDCD10 has been recently shown to suppress Rho/ROCK-mediated actomyosin contractility [To gain insight into the direct target(s) of TRIM59 in the regulation of breast cancer cell survival and metastasis, we set out to perform an unbiased yeast two-hybrid screening assay (p53 and Large T-antigen as positive control) by using TRIM59 as prey and normalized universal human cDNA library as bait. PDCD10 stood out as a strong candidate that was repeatedly detected in the 40+ positive clones after stringent selections . To further narrow down the key domains within TRIM59 that mediate the interaction with PDCD10, we generated a set of truncated or deleted variants of TRIM59 , including the full-length TRIM59 (FL), TRIM59 without RING (R) domain (ΔR), TRIM59 without the predicted transmembrane domain (ΔTM), coiled-coil domain (CC), and B-box only domain (B). We found that PDCD10 interacted with FL and ΔTM, and that deletions of the RING domain  compromised the PDCD10-TRIM59 interaction. These findings suggest that the RING domain within TRIM59 is essential for its physical interaction with PDCD10 .To further biochemically validate the endogenous TRIM59-PDCD10 interaction, we performed a co-immunoprecipitation (co-IP) assay in MCF7 cells. After pulling down with an anti-TRIM59 antibody, we detected the co-enrichment of PDCD10 by blotting with an anti-PDCD10 antibody . The down-regulation of PDCD10 expression at protein levels was further validated by immunofluorescent staining in TRIM59 KO MCF7 xenograft tumors . Conversely, accumulation of PDCD10 was observed upon overexpression of TRIM59 in MDA-MB-231 cells . Under all these conditions, the mRNA levels of PDCD10 remained largely unaltered , thus ruling out a transcriptional regulatory mechanism. Furthermore, to rule out the possibility that TRIM59 might promote the nuclear translocation of PDCD10 to prevent its degradation in the cytoplasm, we separated the nucleus and cytoplasm fractions of WT or TRIM59 KO MCF7 cells. We found that PDCD10 is a cytoplasmic protein and its protein level is greatly reduced upon TRIM59 deletion . Thus, TRIM59 is essential for the maintenance of steady-state levels of PDCD10 protein.Because the RING domains of E3 ligases are often involved in mediating protein ubiquitination and degradation ,38, we rTRIM59 depletion or overexpression, we first examined the protein stability of PDCD10 in TRIM59 KD  or TRIM59 KO  MCF7 cells in the presence of a proteasome inhibitor, MG132, and autophagy inhibitor bafilomycin A1 (BafA1) or 3-methyladenine (3-MA), as well as DMSO as control. We found that inhibition of autophagy with BafA1 or 3-MA restored PDCD10 protein levels, even upon TRIM59 depletion, but not with the proteasome inhibitor MG132 . In parallel, we overexpressed TRIM59 in TRIM59-low MB-MDA-231 cells, with and without treatment of BafA1, and monitored PDCD10 protein levels by immunoblotting. We found that ectopic expression of TRIM59, similar to BafA1 treatment, caused a significant up-regulation of PDCD10 protein levels ( lanes 2 and 3 versus 1). In addition, the TRIM59 OE group in the presence of BafA1 did not show further up-regulation of PDCD10 ( lane 4 versus 3). Notably, depletion of TRIM59 did not seem to affect the basal autophagosome formation, as reflected by the similar puncta areas or numbers of autophagosomal marker GFP-microtubule-associated protein 1A/1B-light chain 3 (LC3) (GFP-LC3) [TRIM59 KO human embryonic kidney 293 cell line that expresses a mutant version of SV40 large T antigen (HEK293T) cells . Collectively, our results establish that TRIM59 could prevent PDCD10 from autophagic degradation.To identify the protein degradation pathways that might mediate PDCD10 stability upon GFP-LC3)  per cellFig 6A and 6B, KO of either TRIM59 or PDCD10 suppressed MCF7 xenograft tumor growth to a similar extent. Similar to TRIM59 , PDCD10 expression was correlated with shortened breast cancer patient survival , suggesting a potential pro-oncogenic role of both TRIM59 and PDCD10 in breast cancer.Given our observations that TRIM59 was up-regulated in human breast tumors and that TRIM59 stabilizes PDCD10 protein, we asked whether PDCD10 is essential for breast tumor growth and if the expression of PDCD10 is correlated with TRIM59. As shown in TRIM59 depletion impinged on the major downstream signaling components of PDCD10. Recent studies have identified several major affected pathways resulting from PDCD10 loss, including (i) MEKK3/ERK5 signaling and target genes KLF2, KLF4, and NOS3 and ADAMTS protease ADAMTS4 and ADAMTS5 [Fig 6D). At the protein level, we focused on analyzing the phosphorylation status of ERK5, a downstream effector of the PDCD10/MEKK3 signaling that is responsible for activating KLF2/4 and ADAMTS4/5 expression [Fig 6E). Together, our data suggest that TRIM59 modulates PDCD10 protein stability to affect PDCD10-associated downstream signaling.We next examined how  ADAMTS5 ,41; (ii) ADAMTS5 ; and , we performed functional rescue experiments by using a ROCK inhibitor (Y-27632) or through the lentivirus-mediated stable expression of PDCD10  in TRIM59 KO MCF7 cells. Both approaches reduced the appearance of ROCK-mediated membrane blebbing  and attenuated the phosphorylation of ERM induced by TRIM59 deletion . In MDA-MB-231 cells, depletion of PDCD10 by shRNA enhanced the phosphorylation of ERM. In MDA-MB-231 cells overexpressing TRIM59 with the resultant suppression of p-ERM, PDCD10 depletion restored p-ERM to a level that was comparable to WT . Furthermore, re-expression of PDCD10 in TRIM59 KO cells substantially restored the efficiency of MCF7 cell colony formation , enhanced cell invasion , and rescued the growth of xenograft tumors . Collectively, these findings suggest that TRIM59 promotes breast cancer growth and migration/invasion through PDCD10-induced suppression of ROCK signaling and subsequent MAT.To test whether PDCD10-ROCK signaling is downstream of TRIM59 but upstream of MLC/ERM to impact breast cancer cells’ contractility . After screening the interaction of PDCD10 with cargo receptors, we identified that PDCD10 specifically engaged p62 but not other receptors . These findings point to the possibility that TRIM59 might suppress the K63 ubiquitination and p62-selective autophagic degradation of PDCD10.Our above results showed that TRIM59 suppressed autophagy-mediated degradation of PDCD10, but it remained unclear what mechanisms TRIM59 might employ to stabilize PDCD10. An E3 ligase typically prevents protein from degradation through three major mechanisms: (1) the recruitment of deubiquitinase , (2) facFig 7B). TRIM59 overexpression was unable to further stabilize the protein level of PDCD10 upon p62 depletion . Next, we hypothesized that TRIM59 might stabilize PDCD10 by blocking the interaction between PDCD10 and p62. Indeed, the interaction of PDCD10 with p62 was abolished after overexpression of TRIM59 . However, we failed to detect a binary interaction between the full-length TRIM59 and p62, although a weak binding was detected between the ΔTM mutant and p62 . Ectopic expression of TRIM59 did not affect the protein level of p62 , IB: HA, lane 1 versus lane 2). These results hint that TRIM59 might block K63 ubiquitination of PDCD10 by exerting an inhibitory effect on a yet-to-be-identified E3 ligase that directly targets PDCD10.p62 has been demonstrated to interact with LC3 to facilitate the lysosomal degradation of ubiquitinated proteins by autophagy . Upon geS3 Table), we identified a previously uncharacterized E3 ligase, RNFT1 [Fig 7E and 7F). Endogenous K63 ubiquitination of PDCD10 was also diminished considerably in RNFT1 KO MCF7 cells compared with WT . To examine if RNFT1 bound to PDCD10 and TRIM59 blocked the interaction between PDCD10 and RNFT1, we performed co-IP experiments in MDA-MB-231 cells (WT versus TRIM59 OE) and MCF7 cells (WT versus TRIM59 KO). We detected the endogenous interaction between RNFT1 and PDCD10 in both breast cancer cell lines but not for IgG controls . TRIM59 OE or TRIM59 KO suppressed or stabilized the PDCD10-RNFT1 interaction, respectively . Following depletion of RNFT1 in MCF7 cells, we observed a dramatic reduction in K63 ubiquitination of PDCD10. In RNFT1 KD cells, TRIM59 overexpression was unable to further inhibit the K63 ubiquitination of PDCD10 . Collectively, our results suggest that TRIM59 blocks RNFT1-mediated K63 ubiquitination of PDCD10.So far, there is no reported E3 ligase for PDCD10. In our yeast two-hybrid screening for potential TRIM59 binders . We found that, as seen with WT PDCD10, TRIM59 overexpression in HEK293T cells led to the accumulation of the K132R and K183R mutants, but not the K179 mutant . In addition, K63 ubiquitination of PDCD10 was blocked when K179 was mutated . Therefore, our data imply that TRIM59-mediated stabilization of PDCD10 might require K179, a site that could be potentially ubiquitinated by RNFT1.Finally, to pinpoint the potential Ub site within PDCD10, we generated several lysine (K)-to-arginine (R) mutations on conserved key residues  in the C-terminal focal adhesion targeting-homology (FAT-H) domain of PDCD10, which might be involved in the linkage with Ub  TRIM59-deleted breast cancer cells favor the amoeboid mode through (i) augmented activation of actomyosin contractile proteins MLC2 and ERM, (ii) excessive formation of E-cadherin-mediated focal adhesion [The TRIM family proteins, most of which have E3 Ub ligase activity, play crucial roles in (i) cancer cell metabolism by regulating adenosine monophosphate (AMP)-activated protein kinase (AMPK) stability , (ii) caadhesion , and  [In addition, KD or KO of TRIM59 promotes cell apoptosis and cell cycle arrest at G0/G1 phase, as revealed by increased cleaved-caspase3 and Annexin V/7-AAD staining and elevated expression of G1/S transition inhibitors p21 and p27, with concomitant down-regulation of G1/S transition promoter CDK1 in the cell cycle  revealedn (GBLP) ,64. FurtTRIM59 depletion in breast cancer cells. We detected a significant reduction in VEGFR2 protein expression upon KD or KO of TRIM59 in MCF7 cells. Interestingly, one recent study has shown that PDCD10 colocalizes with VEGFR2. Loss of PDCD10 decreased the stability of VEGFR2 protein [TRIM59 depletion in breast cancer cells could further impinge on genes and pathways involved in cancer metabolism: (1) down-regulation of metabolism-related oncogenes such as c-MYC and IGF1R [Besides the observation of TRIM59 in the regulation of breast cancer cell survival and mode of cancer cell migration and metastasis, our unbiased RPPA study revealed that other critical oncogenic pathways are altered upon  protein , hinting protein . Moreove protein . TRIM59 nd IGF1R ,68; (2) nd IGF1R . TherefoTRIM59 sensitizes PDCD10 for autophagy-mediated degradation and subsequently induces robust ROCK activity to promote cell adhesion and suppress mesenchymal or lamellipodia movement . Hence, a therapeutic intervention that interrupts the functional interplays between TRIM59 and PDCD10 might provide a promising strategy to treat breast cancer and CCM.In summary, our study has unveiled TRIM59 as an essential tumor-promoting factor that facilitates breast cancer growth and metastasis through modulating PDCD10-associated signaling pathways, as well as other oncogenic pathways. Loss of The use of human cancer cell lines was in accordance with institutional guidelines on human cell research and the approved protocol (IBC2015-132) by the Institutional Review Board. Animal experiments in this study were approved and carried out in accordance with the protocol (2015-0342-IBT) provided by the Institutional Animal Care and Use Committee (IACUC) at Institute of Biosciences and Technology, College of Medicine, Texas A&M University. IACUC uses the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals, which is based on the United States Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.Breast cancer cell lines MDA-MB-436 (HTB-130), MDA-MB-231 (HTB-26), MDA-MB-468 (HTB-132), MDA-MB-175-VII (HTB-25), CAMA-1 (HTB-21), MDA-MB-453 (HTB-131), MDA-MB-361 (HTB-27), MCF7 (HTB-22), normal mammary epithelial cell line MCF10A (CRL-10317) cells, and HEK293T cells (CRL-3216) were purchased from American Type Culture Collection  and maintained in DMEM or RPMI-1640 medium with 10% fetal bovine serum (FBS) according to culture instructions provided by ATCC. Cell lysate was collected by RIPA buffer  with protease/phosphatase inhibitor cocktail . Protein concentration was determined by BCA assay using BCA Protein Assay Reagent . A total of 30–60 μg of cell lysate was subjected to western blot analysis.Antibodies used in western blot and immunoprecipitation include primary antibodies against PDCD10 , p53 (#sc-126), and total ERM antibody (#sc-22807) were purchased from Santa Cruz Biotechnology . PDCD10 antibody  was purchased from Millipore . TRIM59 antibody (#NBP-82625) was purchased from Novus Biologicals  and TRIM59 antibody (#ab69639) was purchased from Abcam . RNFT1 antibody (#PA5-48913) was purchased from Thermo Fisher Scientific . Cleaved-Caspase3 (#9661), Ki-67 (#9027), phosphorylated (p)-SAPK/JNK (#9251), p-p38 MAPK (#4511), p-p44/p42 (#9101), p-ERK5 (#3371), total ERK5 (#3552), p-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) antibody (#3726), Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (#9782), Cadherin-Catenin Antibody Sampler Kit (#9961), and p-myosin light chain 2 (Ser19) (#3671) were purchased from Cell Signaling Technology . anti-FLAG M2 affinity gel (#A2220), anti-FLAG M2-peroxidase (HRP) antibody (#A8592), and anti-HA−HRP antibody (#H6533) were purchased from Sigma-Aldrich .n = 90), as well as metastatic cancer and paired primary cancer tissues  were obtained during surgery at Sir Run Run Shaw Hospital of Zhejiang University. All samples were collected with patients’ informed consent and approved by Institutional Review Board. For assessing the correlation between TRIM59 IHC scores and clinical stages of breast cancer, a total of 170-spot, paraffin-embedded tissue array chips (HBre-Duc170Sur-01), including 90 paired breast tumor and normal tissues, 70 tumor tissues, and 10 normal tissues with 10 to 13.5 years of follow-up information, were purchased from Shanghai Outdo Biotech . Please note that we deleted nine cases  based on the 154 cases listed in S1and . Gene expression profiling (GEP) analysis of TRIM59 protein expression (in both primary and metastatic breast cancer samples) was performed on datasets from Gene Expression Omnibus  GSE30480. Specifically, we included 14 primary breast tumors and 6 metastatic lymph node samples. Statistical differences among multiple groups were analyzed with one-way ANOVA test. Significant differences were determined when the P value was less than 0.05.Fresh samples of human breast cancer and paired adjacent normal tissues  or RIPA buffer and shaken on ice for 15 minutes. Whole cell lysates were incubated for 4 hours at 4°C with the indicated antibodies and protein A/G beads  or with anti-FLAG  or anti-HA  agarose gels for FLAG- OR HA-tagged protein pull-down. After incubation, beads were washed five times with 1 mL low salt lysis buffer or RIPA buffer, boiled with 4× SDS loading buffer, and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes . Membranes were blocked in 5% dry milk/TBST for 1 hour and incubated using indicated antibodies. IB analysis was developed using the Luminata Western HRP Chemiluminescence Substrates  and ChemiDoc XRS+ System with Image Lab .The TRIM59 levels in breast tumors and normal breast tissues were evaluated by IHC using an anti-TRIM59 antibody on paraffin-embedded tissue samples or commercial tissue arrays  as previously described . BrieflyThe immunofluorescence assays and confocal microscopy were conducted as described previously . HEK293T6 clones. Positive clones were picked up and library plasmids were recovered and expanded in Escherichia coli. The inserted cDNAs were sequenced and then characterized with the BLAST program.Yeast two-hybrid screening was carried out with Matchmaker Gold Yeast Two-Hybrid System  using the AH109 yeast strain, as previously described . To consTRIM59, PDCD10, or RNFT1 were purchased from Dharmacon. Plasmid purification was conducted by using the QIAGEN Plasmid Maxi kit .Bacterial strains containing the plasmids for GIPZ lentiviral shRNA targeting TRIM59 KD efficiency was assessed by quantitative PCR.HEK293T cells were used to produce lentivirus. Co-transfection of the lentiviral plasmids, pMD2.G  and psPAX2 , was done by CalPhos Mammalian Transfection Kit . Forty-eight and seventy-two hours after transfection, virus-containing cell culture supernatant was collected and passed through a 0.45-μm filter for virus preparation. HEK293T, MCF7, or MDA-MB-231 cells were seeded at 70% confluence 1 day before infection. A total of 5 mL of virus-containing cell culture supernatant with 8 μg/mL polybrene  was added to each 10-cm dish for 3 hours before adding 5 mL of complete DMEM containing 10% FBS. Virus infection was repeated 24 hours later. Between 48 and 72 hours after the first infection, MCF7 or MDA-MB-231 cells were harvested and seeded in 96-well plates at 1 cell/well for single colony selection. 2, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na3VO4, and 10% glycerol, with freshly added protease/phosphatase inhibitor cocktail) for 20 minutes on ice, with occasional shaking every 5 minutes, and centrifuged at 14,000 rpm for 10 minutes at 4 oC to collect supernatant. Protein concentration was determined by BCA assay and adjusted to 1–1.5 mg/mL. Cell lysate was mixed with 4×SDS sample buffer without bromophenol blue . A 100-μL sample for each replicate was sent for analysis. RPPA assay was done at the RPPA Core Facility of M.D. Anderson Cancer Center . A full list of antibodies (Ab List_218) used in this study was described as follows: https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core/antibody-information-and-protocols.html.Cells were washed twice in ice-cold PBS, lysed in 500 μL lysis buffer  and frozen at −80°C. HEK293T, MCF7, or MDA-MB-231 cells were transduced with control sgRNA or TRIM59-, PDCD10-sgRNA–, or RNFT1-sgRNA–containing lentiCRISPR viruses. Transduced cells were selected in the presence of puromycin  for 48 hours and subjected to ligand stimulation or viral infection. The primers used for sgRNA are as follows:We designed human single guide RNAs (sgRNAs) as previously described , using tTRIM59 sgRNA: 5′-caccgCCAGCATGTACAGATCTTGAAA-3′PDCD10 sgRNA: 5′-caccgGGGCATAGAAACCATGGATG-3′RNFT1 sgRNA: 5′-caccgTCATGACCAGAAGCGGACGG-3′LC3 sgRNA: 5′-caccgACCTCCTTACAGCGGTCGGC-3′p62 sgRNA: 5′-caccgCACCGTGAAGGCCTACCTTC-3′6 MCF7 or MDA-MB-231 cells were plated and cultured in 6-well plates until they reached 100% confluence, followed by overnight starvation in medium containing 0.5% FBS. Starved cells were pretreated with mitomycin C  at 20 μg/mL for 4 hours. A wound was made in the confluent cell layer by scratching wells in vertical and horizontal directions. Cells were then washed with PBS twice and migration was induced by complete medium containing 10% FBS. Cell migration was checked every 12 hours until the wound healed in one of the samples. Migration was stopped by removing growth medium and fixing cells in 4% paraformaldehyde.A total of 1 × 10°C for 1 hour.Growth factor–reduced Matrigel  was mixed with serum-free MEM Eagle medium at 1:4. A total of 100 μL of the mixture was added to the bottom of cell culture inserts with 8-μm pore size. Cell culture inserts were incubated at 375 cells/mL in DMEM containing 0.5% FBS. A total of 100 μL of cell suspension was seeded into a cell culture insert with Matrigel at the bottom. A total of 500 μL of complete DMEM containing 10% FBS was added to the lower chamber. Ten percent FBS served as chemoattractant. Cell invasion was checked every 12 hours until a significant population of cells invaded through the 8-μm pore on the cell culture insert (48 hours in most experiments). Noninvasive cells were removed from the top of the filter with a cotton swab. Cells at the bottom of the insert were fixed by immersing cell culture inserts in 4% paraformaldehyde for 15 minutes at room temperature. Cell culture inserts were then washed in PBS. Invaded cells were stained with 0.05% crystal violet in distilled water for 30 minutes. After two washes in PBS, invaded cells were visualized under an Olympus IX70 microscope, and three randomly chosen fields per membrane were photographed and quantitated using the Fiji ImageJ software [Cells were serum starved overnight in DMEM containing 0.5% FBS. Starved cells were pretreated with mitomycin C at 20 μg/mL for 4 hours. Starved cells were harvested and resuspended at 1 × 10software .3 cells/well of control shRNA MCF7 or TRIM59 shRNA-MCF7 stable cells suspended in diluted Matrigel (1:4 dilution in RPMI1640 medium with 10% FBS) were added into the well and incubated for 30 minutes at 37°C. A total of 500 μL of culture medium was then added and culture was maintained for 10 days, with medium changed every 2 days. Colony formation was then photographed and quantified.Three-dimensional culture of MCF7 cells was performed as described previously . BrieflyGAPDH. The primers used for qPCR are as follows:Total RNA was isolated from cells or tissues, and first-strand cDNA was generated from total RNA using oligo-dT primers and SuperScript III Reverse Transcriptase . qPCR was performed using specific primers and the ABI Prism 7000 analyzer  with the PowerUp SYBR Green Master Mix . Target gene expression values were normalized to human GAPDH-Fwd: 5′-TCAAGAAGGTGGTGAAGCAG-3′GAPDH-Rev: 5′-GAGGGGAGATTCAGTGTGGT-3′MYC-Fwd: 5′-GGCTCCTGGCAAAAGGTCA-3′MYC-Rev: 5′-CTGCGTAGTTGTGCTGATGT-3′AXIN2-Fwd: 5′-CAACACCAGGCGGAACGAA-3′AXIN2-Rev: 5′-GCCCAATAAGGAGTGTAAGGACT-3′S100A4-Fwd: 5′-GATGAGCAACTTGGACAGCAA-3′S100A4-Rev: 5′-CTGGGCTGCTTATCTGGGAAG-3′NOS3-Fwd: 5′-TGATGGCGAAGCGAGTGAAG-3′NOS3-Rev: 5′-ACTCATCCATACACAGGACCC-3′KLF2-Fwd: 5′-TTCGGTCTCTTCGACGACG-3′KLF2-Rev: 5′-TGCGAACTCTTGGTGTAGGTC-3′KLF4-Fwd: 5′-CCCACATGAAGCGACTTCCC-3′KLF4-Rev: 5′-CAGGTCCAGGAGATCGTTGAA-3′ADAMTS4-Fwd: 5′-GAGGAGGAGATCGTGTTTCCA-3′ADAMTS4-Rev: 5′-CCAGCTCTAGTAGCAGCGTC-3′ADAMTS5-Fwd: 5′-GAACATCGACCAACTCTACTCCG-3′ADAMTS5-Rev: 5′-CAATGCCCACCGAACCATCT-3′ANGPT2-Fwd: 5′-AACTTTCGGAAGAGCATGGAC-3′ANGPT2-Rev: 5′-CGAGTCATCGTATTCGAGCGG-3′TRIM59-Fwd: 5′-AAGATCCTCGTGTACTGCCAT-3′TRIM59-Rev: 5′-CAATGCCAGTTGGAGCAATTTC-3′PDCD10-Fwd: 5′-GCCCCTCTATGCAGTCATGTA-3′PDCD10-Rev: 5′-AGCCTTGATGAAAGCGGCTC-3′RNFT1-Fwd: 5′-CCTGAAGCAAAGACATCTGGG-3′RNFT1-Rev: 5′-ACTGTGCAGTTGGCTACGATT-3′6 cells in 50 μL RPMI1640 with 10% FBS were mixed with an equal volume of pathogen-free BD Matrigel Basement Membrane Matrix and injected into the mammary fat-pad. Tumor growth was monitored and measured with a digital caliper, and the tumor volume in mm3 is calculated by the formula: volume = (width)2 × length/2. At 12 weeks, mice were euthanized and necropsied to remove tumors and organs potentially containing metastatic foci for formalin fixation, paraffin embedding, and tissue analysis.Orthotopic mammary fat-pad injection of 8-week-old female NOD.Cg-Prkdc<scid> Il2rg<tm1Wjl>/SzJ  mice  with MCF7 or MDA-MB-231 cells was performed. Briefly, mice were anesthetized and an abdominal incision was made to expose the mammary gland. A total of 1–10 × 10TRIM59 (empty vector or control sgRNA as negative control). Six-week-old NSG mice were used for the orthotopic injection of breast cancer cells  into mammary fat-pad. D-Luciferin (15 mg/mL) 150 mg/kg was injected intraperitoneally into tumor-bearing mice 10 minutes before imaging. The growth and metastasis of the tumors were monitored by weekly bioluminescence imaging using the IVIS imaging system . End point assays were conducted 10 or 14 weeks after the inoculation unless the animal euthanasia was required because of significant morbidity.MCF7 and MDA-MB-231 cells were transduced with a lentivirus containing pLenti-CMV-Luciferase-IRES-GFP, followed by lentiviral transduction for the overexpression or CRISPR/Cas9-mediated disruption of 7 MCF7 cells were harvested with trypsin-EDTA and washed with PBS. A 50-μL cell pellet was resuspended by vigorous vortexing in 500 μL ice-cold CER I solution and incubated on ice for 15 seconds. A total of 27.5 μL ice-cold CER II solution was added and mixed followed by 1 minute of incubation on ice and was then centrifuged for 5 minutes at 16,000g. The supernatant fraction containing the cytoplasmic extract was transferred to a clean prechilled tube until use. The insoluble pellet fraction containing the nuclei was suspended in 250 μL ice-cold NER, followed by continuous vortexing for 15 seconds every 10 minutes for 40 minutes. The mixture was then centrifuged at 16,000g for 10 minutes. The supernatant fraction containing the nuclear extract was transferred to a clean prechilled tube until use.Nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents . A total of 102 atmosphere. The absorbance was measured using a model ELX800 Micro Plate Reader  at 490 nm then the proliferation was calculated. Annexin V and 7-AAD staining kit  was used to detect apoptotic cells. WT, TRIM59 KD, or TRIM59 KO MCF7 cells were harvested with trypsin-EDTA and washed with PBS twice. Cells were then resuspended in 1× binding buffer at a concentration of 106 cells/mL. A total of 5 μL Annexin V and 5 μL 7-AAD was added to 100 μL of the cell solution and incubated at RT for 15 minutes in the dark. Stained cells were washed and resuspended in 400 μL of 1× binding buffer and analyzed by flow cytometry.Cell proliferation was determined by MTS assay. MCF7 or MDA-MB-231 cells were seeded into 96-well plates in 100 μL of culture medium and cultured for the indicated time. Ten microliters per well of MTS  was added into each well for a 2-hour incubation at 37°C in a humidified, 5% CO4 MCF7 or MDA-MB-231 cells suspended in 1 mL of 0.33% agarose 1640 medium containing 10% FBS were plated in 12-well plates on the top of existing 0.5% bottom agarose with the same medium. The medium was replenished every 3–5 days, and colonies that grew beyond 50 μm in diameter after 2–3 weeks were scored as countable.The colony formation assay was used to reflect anchorage-independent cell growth. About 1 × 105 WT, TRIM59 KD, or TRIM59 KO MCF7 cells per well were seeded into 6-well plates and allowed to attach overnight. The culture media were replaced with fresh complete 1640 containing 2 mM thymidine and cultured for 18 hours; after washing with PBS, the block was released by incubation in fresh complete 1640 for 10 hours. Cells were blocked again with fresh complete 1640 containing 2 mM thymidine and cultured for 18 hours; after washing with PBS, the block was released by incubation in fresh complete 1640 for 12 hours. Cells were collected and permeabilized with precooled 75% ethanol at 4°C overnight. The next day, the cells were washed with PBS and incubated with 0.5% PI  in the dark for 30 minutes. DNA content was detected by flow cytometry. The data were analyzed with ModFit software.A total of 5 × 10https://portal.gdc.cancer.gov/). The normal sample was extracted from the same adjacent non-tumorigenic tissue in the same patient. Comparison of tumor and paired adjacent normal samples is used in TCGA data analysis [t test P value is <0.05.Gene expression data were downloaded from the TCGA data portal . P values <0.05 were considered significant. Phosphorylation levels of Ezrin in BRCA were obtained from CPTAC [Descriptive statistics, including means, standard deviations, medians, and ranges, were computed for each group and analyzed with Student om CPTAC . We calcom CPTAC . MultivaS1 FigRelated to  (A) Fold change of TRIM59 expression across 12 cancer types compared with their paired controls (obtained from adjacent noncancerous tissues), based on TCGA dataset analysis. (B and C) mRNA expression profiles of TRIM59 in human (B) and mouse (C) tissues. (D) Quantitative RT-PCR of TRIM59 mRNA expression in human breast cancer cell lines. (E-G) TRIM59 IHC scores in subtypes of breast cancer based on HER and ER status. (H) Multivariate overall survival analysis of patient by using a Cox proportion hazards model. (I) Kaplan–Meier survival curves for Grade II or above breast cancer patients. Patients were divided into two groups according to TRIM59 staining scores . The underlying data can be found in (TIF)Click here for additional data file.S2 FigRelated to  (A) IB analysis of TRIM59 expression in MCF10A, MCF7, and MDA-MB-231 using two TRIM59 antibodies (Novus and Abcam). Purified TRIM59 protein was used as positive control . (B) TRIM59 mRNA expression levels in MCF7 and MDA-MB-231 cells revealed by RNA sequencing dataset analysis (GEO database: #GSE54326). n = 3. Data are presented as means ± SD. *P < 0.05. (C) RPPA analysis of alterations in protein expression levels or phosphorylation in Control, shTRIM59, and TRIM59 KO MCF7 cells. n = 3. The underlying data can be found in (TIF)Click here for additional data file.S3 FigRelated to  (A) Representative immunochemistry staining of β-catenin in tissue sections from xenograft tumors of TRIM59 KO MCF7 cells or TRIM59 OE MDA-MB-231 cells compared with control cells. Scale bars: 200 μm. (B) qPCR quantification of the Wnt signaling pathway downstream gene AXIN2 in WT and TRIM59 KO MCF7 cells. n = 4. Data are presented as means ± SD. ***P < 0.001 versus WT. The underlying data can be found in (TIF)Click here for additional data file.S4 FigRelated to  (A) IB analysis of endogenous TRIM59 expression in MCF7 cells expressing two different shRNAs targeting TRIM59 compared with scramble shRNA (shControl). (B) IB analysis of endogenous PDCD10 expression in MCF7 cells expressing shTRIM59-1 or shTRIM59-2 compared with shControl. MCF7 cells expressing shPDCD10-1 or shPDCD10-2 targeted to PDCD10 were used as control. (C) IB analysis of PDCD10 in WT MCF7 or TRIM59 KD MCF7 cells treated with DMSO, BafA1, 3-MA, or MG132 for 6 hours. (D) IB analysis of PDCD10 protein levels in WT or TRIM59 OE MDA-MB-231 cells treated with DMSO or BafA1 for 12 hours. Quantitative comparisons of PDCD10 levels were analyzed by densitometric scanning of blots and normalized to β-actin. (E) Co-IP and IB analysis of HEK293T cells transfected with FLAG-p53, along with HA-tagged UbWT, followed by MG132 treatment . (F) Representative confocal images of WT or TRIM59 KO HEK293T cells transfected with GFP-LC3. BafA1, bafilomycin A1; co-IP, co-immunoprecipitation; GFP, green fluorescent protein; HA, hemagglutinin; IB, immunoblot; KD, knockdown; KO, knockout; LC3, microtubule-associated protein 1A/1B-light chain 3; OE, overexpressed; PDCD10, programmed cell death protein 10; shRNA, short hairpin RNA; TRIM59, tripartite motif 59; UbWT, ubiquitin WT; WT, wild-type; 3-MA, 3-methyladenine.(TIF)Click here for additional data file.S5 FigA) HEK293T cells were transfected with FLAG-PDCD10 and HA-Ub (WT) or its mutants, along with Myc-TRIM59 or empty vector, in the presence of BafA1. Whole-cell lysates were immunoprecipitated with anti-FLAG beads and immunoblotted with indicated antibodies. (B) Co-IP and IB analysis of extracts of HEK293T cells transfected with FLAG-p62, along with HA-TRIM59 full-length WT or deletions . ΔR, TRIM59 without RING domain; ΔTM, TRIM59 without the predicted transmembrane domain; B, B-box-type zinc finger domain; BafA1, bafilomycin A1; CC, coiled-coil domain; co-IP, co-immunoprecipitation; HA, hemagglutinin; IB, immunoblot; K63, lysine 63; Myc, avian myelocytomatosis virus oncogene cellular homolog; PDCD10, programmed cell death protein 10; p62, phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa; Ub, ubiquitin; TRIM59, tripartite motif 59; WT, wild-type.((TIF)Click here for additional data file.S1 TableIHC, immunohistochemistry; TRIM59, tripartite motif 59.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableTRIM59, tripartite motif 59.(DOCX)Click here for additional data file.S1 DataData were listed in the indicated separate sheets.(XLSX)Click here for additional data file.


N-(2-hydroxypropyl)lindcarpine (1), together with four known aporphine alkaloids, (+)-boldine (2) (+)-norboldine (3), (+)-lindcarpine (4) and (+)-methyllindcarpine (5) were isolated from the stem bark of Actinodaphne pruinosa Nees (Lauraceae). (+)-N-(2-Hydroxypropyl)lindcarpine (1) exhibited cytotoxic activity against P-388 murine leukemia cells with an IC50 value of 3.9 μg/mL. Structural elucidation of all the compounds were performed by spectral methods such as 1D- and 2D- NMR, IR, UV, and HRESIMS.One new alkaloid; (+)- Actinodaphne pruinosa is a tree of moderate size (about 30-40 feet) found in Peninsular Malaysia and Jawa, Indonesia. Locally, Actinodapne is known as wuru (Indonesia) or medang kuning and medang kunyit  + . The IR spectrum revealed an absorption band at 3,180 cm-1 due to the OH stretching vibration. The overall physical properties and NMR spectral profile revealed its identity as a member of the aporphine group of isoquinoline, a characteristic and distinguishable chemical marker of Actinodaphne plants + ; 1H-NMR  and 13C-NMR  see 3: OCH3-10 to H-9 (2.6%).(+)-rpine 1, : A colorN-(2-hydroxypropyl)lindcarpine (1) against P-388 murine leukemia cells was tested using MTT-microculture tetrazolim assay [The cytotoxicity of (+)-im assay .


SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous  SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions. This has spurred the concept that SERINC5 interferes quantitatively and/or qualitatively with the HIV-1 Env-mediated membrane fusion process through inactivation of HIV-1 Env glycoproteins, resulting in diminished fusion pore formation  clones expressed HA in both SERINC5 alleles, whereas SERINC5(iHA/KO) clones expressed HA in only one allele, while the second allele bore a frameshift or deletion  analysis using a primer-probe assay that amplifies a region common to all three known isoforms  clones  and SERINC5(iHA/KO) clones displayed migration patterns that were identical to those of lysates obtained from Jurkat T cells and HEK293T cells transduced with SERINC5(iHA). SERINC5(iHA) presented as two protein species of 51 and 35 kDa, respectively. The 51-kDa, but not the 35-kDa, species was sensitive to peptide-N-glycosidase (PNGase) digestion (SERINC5(iHA/iHA) clones than in most SERINC5(iHA/KO) clones. Immunofluorescence microscopy demonstrated presence of endogenous SERINC5(iHA) at the plasma membrane and colocalization with cholera toxin-stainable ganglioside GM1, suggesting an association with lipid rafts  clones , suggest lysates . Parentaigestion , suggestigestion  and thatigestion . Endogenigestion  and F, wig. 2A clid rafts . Colocalid rafts .SERINC5(iHA/iHA) clones with individual interferon alpha (IFN-α) subtypes failed to consistently upregulate SERINC5 mRNA expression, while IFIT1 mRNA expression was induced by 40- to 44,000-fold by all active IFN-α subtypes, confirming the effectiveness of the IFN-α treatment (SERINC5(iHA/iHA) T cells increased up to 2-fold upon IFN-α treatment , SERINC5(iHA/KO), and SERINC5(KO/KO) clones shared similar susceptibilities to infection by the vesicular stomatitis virus G protein (VSV-G)-pseudotyped, internal ribosome entry site (IRES)-green fluorescent protein (GFP)-expressing wild-type (WT) HIV-1 and HIV-1 Δnef mutant, as assessed by analysis of the percentage of GFP-positive cells at 2 days postinfection (SERINC5(iHA/iHA) and SERINC5 (iHA/KO) clones produced wild-type HIV-1 particles of similar infectivities and shared the requirement of proviral Nef expression for the generation of fully infectious particles  protein in HIV-1 Δnef particles derived from all Jurkat HA knock-in clones (nef particles) derived from the SERINC5(iHA/iHA) clones P1E8 and P2G3, the SERINC5(iHA/KO) clones P1A2 and P1A8, and Jurkat T cells transduced with SERINC5(iHA). In contrast, levels of SERINC5(iHA) were unaffected or even enhanced by Nef in virions derived from the SERINC5(iHA/iHA) clone P2B1 and the SERINC5(iHA/KO) clones P1B6, P1B12, P1C1, and P3A1  is probably due to the saturating expression levels of SERINC5. Importantly, supernatants from uninfected or pVSV-G-transfected SERINC5(iHA/iHA) clones did not display HA positivity  protein. Parental Jurkat T cells and the nfection . Infectearticles . As expe virions . Interes virions , left th virions , right wn clones . In contn clones , in accon clones , 16. Surn clones  and E. Vand P3A1  and E. Tsitivity , arguingsitivity . These rSERINC5(iHA/iHA) and SERINC5(iHA/KO) clones demonstrated the susceptibility of the endogenous protein to downregulation by HIV-1 Nef (SERINC5(iHA/iHA) clones  clones . Infecti) clones  and E, aSERINC5 in HIV-1 restriction and the ability of Nef to antagonize this antiviral factor  compared to the reduction of HIV-1 infectivity . This observation has already been made in heterologous assays in which SERINC5-encoding plasmids were expressed at doses anticipated to approach physiological expression levels -fold com and is cnef virions, suggesting viral incorporation of SERINC5, as expected. HA positivity in virions was associated with a band of 35 kDa, and we failed to detect the high-molecular-weight SERINC5 species in virus preparations. In contrast, the exclusive and predominant incorporation of the 51-kDa species in virions generated by Jurkat T cells and HEK293T cells expressing transduced SERINC5(iHA) demonstrates that our assay displays sufficient sensitivity to detect this SERINC5 species in general and reproduces previously reported findings (SERINC5(iHA knock-in) clones represents impartially glycosylated SERINC5 species  analysis revealed a set of genes that are differentially expressed in the two phenotypic groups of clones. Regardless of the underlying reason, the apparent dispensability of Nef-mediated exclusion of endogenous SERINC5 from virions for infectivity rescue provides evidence for the presence of additional counteractive mechanisms of Nef, directed against virus-associated pools of SERINC5, as has been postulated by another group .SERINC5 as a useful technology that enabled us to investigate key aspects of SERINC5 antiviral restriction and HIV-1 Nef-mediated antagonism. A similar approach in other cell types in the future might reveal the extent to which our findings can be extrapolated in more physiologically relevant, primary T cells and macrophages. Future studies using this resource may help to advance our understanding of both SERINC5 restriction and viral counteraction and its physiological function.Together, data from this study establish CRISPR/Cas9-assisted epitope tagging of endogenous alleles of SERINC5(KO/KO) cell lines, Jurkat T cells were electroporated with 100 μg/ml of a plasmid expressing Cas9-2A-enhanced GFP (EGFP) and U6-driven chimeric guide RNA (SERINC5 target sequence GCTGAGGGACTGCCGAATCC[TGG]) using the Neon transfection system  at 1,500 V for 30 ms, with 1 pulse. EGFP-positive cells were sorted on a BD FACSAria cell sorter and clonally expanded. Individual clones were genotyped by Sanger sequencing  of the PCR-amplified genomic locus (forward primer TGCTGTGTTGACCAGGCTAA and reverse primer GGCATTGGATCCTGGAAAGC). Individual alleles were deduced using the Poly Peak Parser tool (SERINC5(iHA/iHA) and SERINC5(iHA/KO) Jurkat clones were generated by coelectroporation of 1.8 μM Cas9 RNPs , and CRISPR-RNA (crRNA) ; IDT, Leuven, Belgium) and 5 μM single-stranded DNA (ssDNA) repair oligonucleotide (ACTTTGTTTTTTCTTTTCAAGTTCTAGATGAATACCCATACGATGTTCCAGATTACGCTCATGGGAAAAATGTTACAATCTGTGTGCCTG [the HA tag is underlined]) using the Neon transfection system . Single-cell clones were screened by PCR , followed by Tris-borate-EDTA (TBE)-PAGE (7.5%), and genotyped by Sanger sequencing of the PCR-amplified genomic locus (forward primer TGGCACTGAGCTGGAATCTG and reverse primer AGTTCACGCTCTTCGCCTTT).HEK293T cells and Jurkat T cells were purchased from the ATCC and cultured as recommended. TZM-bl cells were obtained from the NIH AIDS Reagent Program. For the generation of ser tool  and, whenef IRES-GFP  was generated by subcloning SERINC5(iHA) from pBJ5-SERINC5(iHA) (kindly provided by Heinrich Göttlinger)  into pQCIRES-GFP  were kinIFN-α2a (Roferon) used in  was purcSERINC5 and IFIT1 mRNA levels was performed with the 7500 Fast real-time PCR system (Applied Biosystems) using TaqMan PCR technology with premade primer-probe kits (Applied Biosystems). Relative mRNA levels were determined using the ΔΔTC method, with human RNASEP mRNA (Applied Biosystems) as an internal reference. Each sample was analyzed in triplicates. Data analysis was performed using Applied Biosystems 7500 Fast system software.Total RNA extraction from cells and DNase treatment were performed with a Maxwell LEV simplyRNA purification kit (Promega), followed by cDNA synthesis . Quantification of relative SERINC5 cDNA with XbaI and NotI. For the loading control, a GAPDH plasmid  was digested by EcoRI, generating a 1.3-kbp GAPDH-specific probe.RNA extraction, gel electrophoresis, blotting, and detection with a radiolabeled probe were performed as described previously  but withCells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Sigma). Virus particles were concentrated by ultracentrifugation through a 20% sucrose cushion and lysed using 1% Triton. Laemmli sample buffer was added to cell and virus lysates before loading. Proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose using a semidry transfer system (Biometra). Blocked membranes were incubated with the following primary antibodies: rabbit anti-mitogen-activated protein kinase (MAPK) (Santa Cruz), mouse anti-HA , mouse anti-p24 (ExBio), rabbit anti-HIV-1 p24CA (ExBio), mouse anti-interferon-stimulated gene 15 (ISG15) , and mouse anti-VSV-G (Santa Cruz) . Secondary antibodies conjugated to Alexa Fluor 680/800 fluorescent dyes were used for detection and quantification with the Odyssey infrared imaging system (Li-Cor Biosciences).Cells were lysed with RIPA buffer containing a protease inhibitor cocktail (Sigma). Cell lysates were either left untreated (input) or pretreated with 1× Glycobuffer 2 (New England Biolabs) and 1% NP-40 buffer (New England Biolabs), in the presence or absence of 500 U of PNGase F (New England Biolabs). The mixture was incubated for 1 h at 37°C. Laemmli sample buffer was added to lysates before loading for immunoblotting.SERINC5(iHA) clones were incubated with 25 μg/ml of FITC-conjugated CTX in 0.1% fetal bovine serum (FBS)–phosphate-buffered saline (PBS) buffer for 30 min on ice. Cross-linking was then performed by incubation with anti-CTX antibody at a 1:200 dilution for 30 min at 4°C and for 10 min at 37°C. Cells were then stained with anti-HA antibody  at a 1:100 dilution, followed by an anti-rat Alexa Fluor 647-conjugated antibody, and fixed with 4% paraformaldehyde (PFA) for 5 min. To visualize the SERINC5(iHA) intracellular localization and the early endosomal compartment . For analysis, images were obtained with a confocal laser scanning microscope . All images were processed by using the Fiji-ImageJ Magic Montage plug-in. In order to visualize SERINC5 surface expression upon IFN-α treatment, cells were incubated with CellTracker red CMTPX dye (1 μM) in Dulbecco’s modified Eagle’s medium (DMEM) for 15 min at 37°C. Cells were then washed and immunostained with anti-HA  in PBS for 20 min at 4°C. After washing, cells were stained with the secondary antibody Alexa Fluor 647-conjugated goat anti-mouse IgG (Invitrogen) in PBS for 20 min at 4°C. After PFA fixation, cells were spinoculated in poly-l-lysine-coated 8-well chamber slides (Ibidi) for 5 min at 500 × g. Agarose (0.8%) in Fluorobrite DMEM was added carefully. For analysis, images were obtained with a confocal spinning-disk Ti-E microscope (Nikon/Andor). All images were processed by using the Fiji-ImageJ Magic Montage plug-in.In order to visualize rafts together with SERINC5(iHA) , parentapartment , cells wCells were immunostained with the primary mouse anti-HA antibody , followed by secondary staining with Alexa Fluor 647-conjugated goat anti-mouse IgG (Invitrogen). After surface HA staining, cells were PFA fixed and analyzed for surface HA and GFP by flow cytometry. In selected experiments, surface HA staining was followed by Triton-mediated permeabilization and intracellular MXA/B staining using rabbit anti-MXA/B (Santa Cruz Biotechnology) as a primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen). Flow cytometry analysis was performed using a FACSCalibur system with BD CellQuest Pro 4.0.2 software (BD Pharmingen) and FlowJo V10 software (FlowJo).The virus-containing supernatant was transferred to TZM-bl cells stably expressing long terminal repeat (LTR)-driven luciferase. Forty-eight hours later, cells were washed once with PBS and lysed using cell culture lysis buffer (Promega), and the Tat-dependent increase of luciferase enzyme activity in cell lysates was determined with a luciferase assay system (Promega). Luminometric activity was analyzed with a Centro LB 960 microplate luminometer and Ascent software 2.0.Δnef proviral DNA, 3 μg BlaM-Vpr DNA, and 1 μg pAdvantage (2-independent medium (Sigma-Aldrich) and loaded with CCF2-AM dye (LiveBLAzer FRET-B/G loading kit) prepared as indicated by the manufacturer. Briefly, each sample was loaded with a 100-μl solution containing 0.2 μl CCF2-AM dye (Thermo Fisher Scientific), 0.8 μl solution B (Thermo Fisher Scientific), 1 μl of probenecid , and 98 μl CO2-independent medium. Cells were incubated in the dark at room temperature overnight. The percentage of cells undergoing fusion with BlaM-Vpr-containing virus particles was monitored by analysis of the change from green (520 nm) to blue (450 nm) fluorescence, using the fluorescence-activated cell sorter (FACS) LSRIII system with DIVA software, and analyzed using FlowJo V10 software (FlowJo).Jurkat T cells were electroporated  using GenePulser Xcell (Bio-Rad) with 9 μg pNL4.3 WT or pNL4.3 dvantage . The virInfected T cells were immunostained on ice with the primary anti-HA antibody . Cells were then shifted to 37°C for the indicated durations, placed back on ice, and immunostained on ice with secondary Alexa Fluor 647-conjugated goat anti-mouse IgG (Invitrogen), followed by PFA fixation. Flow cytometry analysis was performed using the FACSCalibur or FACS Accuri system with BD CellQuest Pro 4.0.2 software (BD Pharmingen) and FlowJo V10 software (FlowJo).t test and are indicated in the figures .If not otherwise stated, bars and symbols show the arithmetic means from the indicated number of repetitions. Error bars indicate standard deviations (SD) from one representative experiment out of at least three or SEM from the indicated number of individual experiments. Significance values were calculated using the 2-tailed Student


Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species  Parkinson’s disease (PD) is the second most common neurodegenerative disease. Loss of dopaminergic (DA) neurons in substantia nigra pars compacta is a pathological marker of PD. The degeneration of DA neurons is induced by several genetic factors such as mutations of α-synuclein, leucine rich-repeat kinase 2 (LRRK2), DJ-1, parkin, and PTEN-induced putative kinase 1 (PINK1), as well as chemical toxins including rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). These PD-inducing factors are closely related with oxidative stress. DJ-1 is the molecular chaperone for reactive oxygen species (ROS)  for 24 h. Finally, cells were washed by ice-cold DPBS twice, and then whole cell lysates were obtained using 1X sample buffer. Cytotoxicity results of each chemical treatment or transfection were measured by LDH cytotoxicity detection kit  per the manufacturer’s instruction.For differentiation of human neuroblastoma SH-SY5Y cells (dSY5Y), cells were cultured in DMEM high glucose (Cellgro) with 10% FBS (Cellgro), 1% penicillin streptomycin (Gibco/BRL), and 10 μM all-trans retinoic acid for 7 days in a 5% COdSY5Y cells were transfected with 1.5 μg of LRRK2 G2019S mutant plasmid using Lipofectamine LTX reagent (Invitrogen) as per the manufacturer’s recommendation. Detailed information about LRRK G2019S mutant plasmid was previously provided  . Data is presented as the mean ± SEM. Each statistical analysis is described in detail in the figure legends. Significances are presented as following: *To validate effects of rotenone on LRRK2 kinase activity, we treated differentiated human neuroblastoma SH-SY5Y (dSY5Y) cells, which were differentiated for 7 days with all-trans retinoic acid to exhibit DA neuronal-like properties, with rotenone in a dose-dependent manner. We observed that rotenone clearly increased LRRK2 protein levels, as well as pS935 and pS1292 LRRK2 signal in a dose-dependent manner. . PhosphoWe speculated that the blockage of NCL and HSP70 induction using cycloheximide (CHX), a protein synthesis inhibitor, could promote severe neuronal toxicity via repression of NCL and HSP70 proteins upon increased total LRRK2 kinase activity. As expected, we confirmed that CHX co-treatment significantly increased cytotoxicity as well as cleaved PARP (In a previous study, the administration of LRRK2 kinase inhibitor rescued rotenone-mediated mitochondrial dysfunction, the increase in ROS levels and a fragment of the nucleus (Mendivil-Perez et al. ex vivo, isolated rat primary cortical neurons were treated with rotenone for 24 h. Again, rotenone significantly increased NCL, HSP70, LRRK2, and pS935 levels, but increases in pMoesin were not significant (To verify similar phenotypes Oxidative stress-derived apoptosis has been hypothesized to contribute to PD progression (Puspita et al. S. cerevisiae (Lewinska et al. Oxidative stress is also involved in DNA damage, and DNA damage has been reported as a marker of PD pathology (Alam et al. 2O2-induced nucleolar fragmentation and NCL degradation in mouse embryonic fibroblasts were rescued by increasing HSP70 (Wang K et al. 2O2-induced apoptosis via stabilization of NCL, and NCL knockdown did not rescue H2O2-induced apoptosis even in HSP70 overexpressing cells (Jiang et al. Induction of HSP70 could also be involved in an NCL-mediated cellular protective mechanism, because induction of HSP70 and NCL are co-regulated during early stages of liver regeneration (Konishi et al. The role of G2019S in PD pathogenesis in generating cellular or clinical symptoms is still unclear. However, previous studies have revealed that G2019S LRRK2 alters various cellular homeostasis mechanisms, such as autophagy-lysosome pathway, vesicle trafficking, mitochondria dysfunction, and accumulation of ROS in immortalized cells, primary cells, or animal models (Shin et al. Herein, we confirmed that rotenone-mediated cytotoxicity was accompanied by LRRK2 kinase activity. Moreover, neuronal survival against oxidative stress derived by increased LRRK2 kinase activity could be facilitated via induction of HSP70 and NCL. Overall, a LRRK2 kinase inhibitor may be a better therapeutic option in preventing oxidative stress-induced apoptosis of DA neurons rather than simply rescuing damaged DA neurons via excessive expression of HSP70 or NCL.


C. elegans protein ZHP-4 cooperates with its paralog ZHP-3 to enforce crossover formation at distinct steps during meiotic prophase: in the formation of early JMs and in transition of late CO intermediates into chiasmata. ZHP-3/4 localize to the synaptonemal complex (SC) co-dependently followed by their restriction to sites of designated COs. RING domain mutants revealed a critical function for ZHP-4 in localization of both proteins to the SC and for CO formation. While recombination initiates in zhp-4 mutants, they fail to appropriately acquire pro-crossover factors at abundant early JMs, indicating a function for ZHP-4 in an early step of the CO/NCO decision. At late pachytene stages, hypomorphic mutants exhibit significant levels of crossing over that are accompanied by defects in localization of pro-crossover RMH-1, MSH-5 and COSA-1 to designated crossover sites, and by the appearance of bivalents defective in chromosome remodelling required for segregation. These results reveal a ZHP-4 function at designated CO sites where it is required to stabilize pro-crossover factors at the late crossover intermediate, which in turn are required for the transition to a chiasma that is required for bivalent remodelling. Our study reveals an essential requirement for ZHP-4 in negotiating both the formation of COs and their ability to transition to structures capable of directing accurate chromosome segregation. We propose that ZHP-4 acts in concert with ZHP-3 to propel interhomolog JMs along the crossover pathway by stabilizing pro-CO factors that associate with early and late intermediates, thereby protecting designated crossovers as they transition into the chiasmata required for disjunction.Correct segregation of meiotic chromosomes depends on DNA crossovers (COs) between homologs that culminate into visible physical linkages called chiasmata. COs emerge from a larger population of joint molecules (JM), the remainder of which are repaired as noncrossovers (NCOs) to restore genomic integrity. We present evidence that the RNF212-like  The creation of a viable individual from the fusion of egg and sperm requires that they each contain the correct number of chromosomes. This is ensured through the meiotic divisions, which initially fasten identical chromosomes through DNA linkages that hold them together until the cell is ready to separate them. To make these linkages, called crossovers, the cell breaks the DNA in many places, and must repair them to create a crossover, or a noncrossover. We investigate here the role of ZHP-4, and its partner ZHP-3 which form a complex that associates along paired chromosomes and finally with crossover sites. ZHP-3/4 are conserved proteins found in many organisms that function in recruiting proteins required to decide which DNA event will become a crossover and how this DNA event is coordinated with changes in chromosome structure. Using mutations that reduce the function of ZHP-4, we show that the complex cannot localize normally to meiotic chromosome and that crossing over fails. Our results suggest that ZHP-3/4 work at early and late steps in the process to stabilize other factors required for crossover formation. The pattern of ZHP-3/4 localization is reminiscent of other pro-crossover factors , which similarly begin with abundant early localization that is then confined to the sites of the obligate crossovers at late pachytene stages, and finally disappears as chromosomes desynapse and chiasmata emerge  II; cosa-1(tm3298) III. jfsi38 [gfp::rmh-1 cb-unc-119+] II. dpy-18(e364) unc-25(e156) III. spo-11(ok79)/nT1 IV. spo-11(me44)/nT1 IV. dpy-3(e27) unc-3(e151) X. rmh-1(jf92[M01E11.3::unc-119+]) I. mus-81(tm1937) I. syp-2(ok307) V. msh-5::gfp IV. Brenner  and all Hermaphrodites were singled at L4 stage and transferred daily to fresh plates for three consecutive days. The number of eggs of each hermaphrodites was recorded immediately after each transfer; in the last plate, the number of eggs was recorded 24 hours after transfer. The number of hermaphroditic and male progeny were scored three days later. Embryonic lethality rate was calculated as the total number of surviving progeny divided by the total number of eggs. Incidence of males was calculated as the number of males divided by the total number of surviving progeny.zhp-4(vv96)/+ males with zhp-4(vv96) V; dpy-3(e27) unc-3(e151) X and zhp-4(vv96) V; dpy-18(e364) unc-25 (e156) III hermaphrodites. Similarly, zhp-4(H26A) males were crossed with zhp-4(H26A) V; dpy-3(e27) unc-3(e151) hermaphrodites. NonUnc, nonDpy F1 cross progeny were picked and allowed for self-fertilize. F1s that were homozygous for zhp-4(vv96) and zhp-4(H26A) respectively were identified by embryonic lethality (Emb) and high incidences of males (Him) in the F2 progeny. For wild-type, 1973 (1334 wild-type and 639 recombinants) F2 progeny were scored from 10 dpy-3 unc-3/+ + and 3451 (2966 wild-type and 485 recombinants) F2 progeny were scored from 15 dpy-18 unc-25/+ + heterozygotes. For zhp-4(vv96) mutants, 507 (437 wild-type and 70 recombinants) F2 progeny were scored from 37 dpy-3 unc-3/+ +; zhp-4(vv96)/zhp-4(vv96) and 815 (710 wild-type and 105 recombinants) F2 progeny were scored from 67 dpy-18 unc-25/+ +; zhp-4(vv96)/zhp-4(vv96). For zhp-4(H26A) mutants, 1165 (913 wild-type and 252 recombinants) F2 progeny were scored from 57 dpy-3 unc-3/+ +; zhp-4(H26A)/zhp-4(H26A) heterozygotes and 558 (524 wild-type and 34 recombinants) F2 progeny were scored from 35 dpy-18 unc-25/+ +; zhp-4(H26A)/zhp-4(H26A) heterozygotes. Recombination frequencies were calculated as previously described ) was generated by Shaolin Li (Gene Editing Services). All the other alleles were generated by directed mutagenesis using CRISPR-Cas9 protocol previously described ) and zhp-4(vv138[H26A]) harbour the following mutations respectively: CAC-to-GCC and CAT-to-GCC, both substituting a highly conserved His to an Ala.The  defects . Cloninged ZHP-4 . The wilescribed  with theTo raise antibodies against ZHP-4 and avoid cross-reactivity with other RING domain containing proteins, a fragment of 372 base pairs corresponding to the C-terminus of ZHP-4 was cloned into two bacterial expression vectors: pGEX-6p-2, containing the GST tag at the N-terminus  and pET28a (Qiagen), to generate an N-terminal 6xHis-fusion protein. Recombinant proteins were purified under native conditions using anti-GST beads  and Ni-NTA matrix (Qiagen) respectively following the manufacturer’s instructions. GST::ZHP-4 was used for antibody production in rat and 6xHis::ZHP-4 was used for sera purification (Medimab). ZHP-4 antibody was purified using activated supports according to the manufacturers’ protocols .For whole embryo staining, thirty-forty of 24-26h post-L4-staged adults were dissected in 1xPBS, followed by freeze crack using liquid nitrogen and fixation in methanol -20°C for 30 minutes. For whole germline staining, gonads of 24-26-h post-L4 staged adults were dissected in 1XPBS and fixed by 1% paraformaldehyde for 5 minutes, followed by freeze crack and fixation in 100% methanol for 5 minutes at -20°C. After fixation in methanol, slides were washed with PBS-T (0.1% Tween-20) for 5 minutes 3 times. Gonads were then blocked with 1% BSA in PBS-T for an hour and incubated with primary antibodies overnight at 4°C. The following day, slides were washed for 3 times 15-minutes each and then incubated with secondary antibodies for two hours. Afterwards, they were washed 3 times, 15 minutes each. 1μg/μL of DAPI in anti-fading agent (Vectashield) was added onto the slides. Images consisting of 15–20 stacks (of 0.2μm increments), were acquired and processed using a Delta Vision Deconvolution system equipped with an Olympus 1X70 microscope or a Spinning-disc confocal microscope (Leica DMI 6000B inverted microscope equipped with a Quorum WaveFX spinning Disc and EM CCD camera). The following antibodies were used in this study: guinea pig and rabbit α-HTP-3 (1:500–1:750), goat α-SYP-1 , rabbit α-HIM-8 , mouse α-GFP , rabbit α-RAD-51 (1:1125), guinea pig α-ZHP-3 (1:750), rabbit α-AIR-2 (1:200), rabbit α-HTP-1 (1:400) rat α-ZHP-4 (1:200), mouse α-HA , mouse monoclonal α-SMO-1 6F2 , tubulin-FITC conjugate , guinea pig α-SUN-1 S8Pi (1:700). For specificity of anti-HA staining see RMH-1 and COSA-1 are both fused with GFP and, following anti-GFP staining, all the foci were scored in each nucleus in the entire pachytene region of each gonad. For representation of the data, the pachytene region was divided into six equal zones and labelled as: early, early-mid, mid, mid-late, late pachytene, and pachytene exit; three gonads were scored for each genotype. For RAD-51 an anti RAD-51 antibody was used for the staining, and foci were scored along the whole gonad of each genotype untill the end of pachytene and then divided into six equal zones (three gonads per genotype were scored).rad-54 gene using the primers TTCAGGACGAACGGAGGAAC and TTCCACTGTCCACTGGCATC, followed by in vitro transcription with T7 RNA polymerase (Ambion). At 6–8 hours post-L4, very young hermaphrodites were injected and after 2 days processed for cytological analyses. The efficacy of the RNAi was never complete since the injected animals never showed sterility as expected by complete knockdown of rad-54 [rad-54(RNAi).RNA interference experiments were performed as described previously . In brief rad-54 . All thef rad-54 . Therefop < 0.05 was considered significant.Distributions of DAPI-stained bodies, RMH-1 foci and COSA-1 foci were statistically tested by Kruskal-Wallis test followed by Dunn’s multiple pairwise coparison tests. The significance of RAD-51 foci scoring was tested by Mann-Whitney U test while embryonic lethality and incidence of males by ANOVA followed by multiple pairwise comparison tests. All calculations were performed with Prism 5 (GraphPad) and S1 Table(DOCX)Click here for additional data file.S1 Figzhp-4 mutant animals show that nuclear morphology as assessed by DAPI staining is not affected in the mutants. Transition zone of each gonad is marked by the white boxes. Scale bars 10μm. (B) Immunostaining for HIM-8 in green (X chromosome pairing center binding protein) show the X chromosomes to be paired in all mid-pachytene nuclei of both zhp-4 mutants. (C) Representative images of mid-pachytene nuclei immunostained for SYP-1 in green (transverse element of the SC) and HTP-3 in red  on wild-type and zhp-4 germlines showing no defect in SC assembly. Scale bars 5μm.(A) Representative gonads from wild-type and (TIF)Click here for additional data file.S2 Figzhp-4::ha germlines immunostained with anti-HA (green) and HTP-3 (red) antibodies. No specific signal is detected in wild-type germlines, whereas linear tracks of ZHP-4::HA colocalize with HTP-3 in zhp-4::ha germlines. (C) Immunostaining for ZHP-4 (green) and HTP-3 (red) in wild-type and syp-2(ok307) germlines showed that the recruitment of ZHP-4 to meiotic chromosomes is dependent on the SC proteins. Scale bars 5μm.(A) Representative images of different stages of meiotic prophase I in wild-type gonads immunostained for ZHP-4 (green) and HTP-3 (red) antibodies. ZHP-4 is first seen as bright foci at transition zone. Starting from early pachytene, it can be detected along the chromosome tracks. Transitioning from mid to late pachytene, ZHP-4 is restricted to shorter stretches and eventually to six foci per nucleus on average, occasionally shown as short stretches at late pachytene. Some of these foci can be observed at diplotene but they were completely removed by early diakinesis. (B) Representative images of mid-pachytene nuclei of both wild-type and (TIF)Click here for additional data file.S3 FigMus musculus (Mm) HEI10 and RNF212, Homo sapiens (Hs) HEI10 and RNF212, Oryza sativa (Os) HEI10, Arabidopsis thaliana (At) HEI10, Caenorhabditis elegans (Ce) ZHP-3 and ZHP-4, Drosophila melanogaster (Dm) Vilya and Saccharomyces cerevisiae (Sc) Zip3. Proteins were aligned using MUSCLE and a phylogenetic maximum likelihood tree was constructed using Phylogeny Analysis (http://www.phylogeny.fr/phylogeny.cgi). Based on this maximum likelihood tree, similar to previous finds by [Sordaria macroscpora (Sm) HEI10, Mus musculus (Mm) HEI10 and RNF212 and RNF4, Homo sapiens (Hs) HEI10 and RNF212, Oryza sativa (Os) HEI10, Arabidopsis thaliana (At) HEI10, Caenorhabditis elegans (Ce) ZHP-3 and ZHP-4, Drosophila melanogaster (Dm) Vilya, Saccharomyces cerevisiae (Sc) Zip3 and Slx8, and Rattus norvegicus Brca1 using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). Conserved cysteines and histidines in the consensus sequence of the RING finger domain are in red, any residues that do not follow the consensus motif are in blue and underlined. Mutating conserved histidines (marked by black boxes) in Sc Zip3 [(A) Maximum likelihood tree constructed from a multiple whole protein sequence alignment of finds by , Zip3 ho Sc Zip3  and Sm H Sc Zip3  have bee(TIF)Click here for additional data file.S4 Figzhp-4(vv103) mutant animals were injected with rad-54 dsRNA, dissected two days post injection (approximately three days post L4), and stained with α-RAD-51 and α-HTP-3 antibodies. RAD-51 foci were scored only in zones 1–4 (corresponding to the mitotic region until early/mid-pachytene stages) because the effect of rad-54(RNAi) was not complete. In fact, the number of RAD-51 foci in our experiments does not accumulate (shown in part B) as reported previously [rad-54(RNAi) since RAD-51 numbers are significantly higher in zone 3 and 4 of injected animals versus wild types (p<0.001) and comparable to previously reported results [rad-54(RNAi) germlines (average of 13.5 foci/nucleus in zhp-4(vv103);rad-54(RNAi) versus 9.6 in rad-54(RNAi), p<0.00001), supporting the conclusion that the elevated number of RAD-51 foci in our mutants is consistent with ZHP-4 being required to negatively regulate DSB formation rather than a manifesting a defect in their processing. Three gonads were analyzed for zhp-4(vv103) and two for wild types . (B) Immunostaining of RAD-51 (green) and the axis component HTP-3 (red) of injected animals dissected 3 days post injection (4 days post L4) show that the increased number of RAD-51 foci resulting from rad-54(RNAi) affects the germlines only until mid-pachytene since the foci start disappearing and are completely removed by the end of late pachytene. Scale bars, 10 μm.(A) The numbers of RAD-51 foci were scored in each nucleus of the gonads of the indicated genotypes as reported for eviously . The sco results . Removin(TIF)Click here for additional data file.S5 Figzhp-4::ha tagged gene immunostained with anti-HA antibody (green) recapitulate the endogenous localization of ZHP-4. By mid-pachytene the HA tag is continuously associated with synapsed chromosomes and by late pachytene it is restricted to 6 foci/nucleus. (B) Representative images of early, mid and late pachytene nuclei of zhp-4(vv96::ha) germlines immunostained with anti-HA (green) and HTP-3 (red) antibodies showing punctate localization of ZHP-4::HAvv96 throughout pachytene stages. Scale bars, 5 μm.(A) Nuclei from early, mid and late pachytene of transgenic worms expressing (TIF)Click here for additional data file.S6 FigC. elegans SUMO homolog) and HTP-3 in red. SMO-1 is associated with chromatin in all genotypes with no detectable differences. Scale bars, 5 μm.Representative images of late pachytene nuclei of indicated genotypes immunostained for SMO-1 in green ((TIF)Click here for additional data file.S7 Figzhp-4(vv96), both congression and spindle formation are disrupted. (B) Partial projections of the oocyte anaphase I spindle in the indicated genotypes. In wild-type oocytes, chromosomes segregate as two distinct chromatin masses towards opposite poles with the microtubule channels evident between them. An example of zhp-4(vv96) mutant nucleus at the same stage shows chromosomes masses that are still connected and appear tangled. Also the tubulin localization is disrupted and partially overlaps with the chromatin masses. (C) Additional examples of metaphase oocytes from zhp-4(vv96) mutants displayed a wide range of phenotypes: (left) chromosomes failed to align to the metaphase plate; (middle) chromosomes managed to align to the metaphase plate; (right) chromatin was not condensed despite proper spindle formation. Anaphase oocytes from zhp-4(vv96) animals show a defect in clean and neat chromosome segregation. Scale bars, 5 μm.(A) Partial projections of the oocyte metaphase I spindle in the indicated genotypes stained with DAPI (blue) and anti-tubulin (green) antibody. In wild-type oocytes, tubulin organizes about the congressed bivalents to form a bipolar spindle in which the tubulin channels are evident while in (TIF)Click here for additional data file.


Scientific Reports 10.1038/s41598-018-37017-4, published online 10 January 2019Correction to: This Article contains an error in Figure 4, where the key was omitted. The correct Figure 4 appears below as Fig.