Literature context: 8Human: U2OS cellsATCCCat#HTB-96Human: A549 cellsATCCCat#CCL-185Cercopithecus aethiops: Vero Cel
Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization to exert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. Here we report that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. We show that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (γ-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. We mapped two separate domains in CnnT: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit γ-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit γ-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.
Literature context: B-126Human: MDA-MB-231ATCCHTB-26Human: A549ATCCCCL-185Human: HEK293TATCCCRL-3216Mouse:
Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.
Literature context: (RRID:CVCL_0023) were prov
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Literature context: CCL-185; RRID:CVCL_0023 Bax+/+Bak+
Cytotoxic compounds are important drugs and research tools. Here, we introduce a method, scalable time-lapse analysis of cell death kinetics (STACK), to quantify the kinetics of compound-induced cell death in mammalian cells at the population level. STACK uses live and dead cell markers, high-throughput time-lapse imaging, and mathematical modeling to determine the kinetics of population cell death over time. We used STACK to profile the effects of over 1,800 bioactive compounds on cell death in two human cancer cell lines, resulting in a large and freely available dataset. 79 potent lethal compounds common to both cell lines caused cell death with widely divergent kinetics. 13 compounds triggered cell death within hours, including the metallophore zinc pyrithione. Mechanistic studies demonstrated that this rapid onset lethal phenotype was caused in human cancer cells by metabolic disruption and ATP depletion. These results provide the first comprehensive survey of cell death kinetics and analysis of rapid-onset lethal compounds.
Literature context: 7Experimental Models: Cell LinesA549ATCCCCL-185293TATCCCRL-3216MDCKA
The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.
Literature context: CCL-185; RRID:CVCL_0023 HeLa ATCC
The mammalian sex chromosomes have undergone profound changes during their evolution from an ancestral pair of autosomes [1-4]. Specifically, the X chromosome has acquired a paradoxical sex-biased function by redistributing gene contents [5, 6] and has generated a disproportionately high number of retrogenes that are located on autosomes and exhibit male-biased expression patterns . Several selection-based models have been proposed to explain this phenomenon, including a model of sexual antagonism driving X inactivation (SAXI) [6-8] and a compensatory mechanism based on meiotic sex chromosome inactivation (MSCI) [6, 8-11]. However, experimental evidence correlating the function of X-chromosome-derived autosomal retrogenes with evolutionary forces remains limited [12-17]. Here, we show that the deficiency of Rpl10l, a murine autosomal retrogene of Rpl10 with testis-specific expression, disturbs ribosome biogenesis in late-prophase spermatocytes and prohibits the transition from prophase into metaphase of the first meiotic division, resulting in male infertility. Rpl10l expression compensates for the lack of Rpl10, which exhibits a broad expression pattern but is subject to MSCI during spermatogenesis. Importantly, ectopic expression of RPL10L prevents the death of cultured RPL10-deficient somatic cells, and Rpl10l-promoter-driven transgenic expression of Rpl10 in spermatocytes restores spermatogenesis and fertility in Rpl10l-deficient mice. Our results demonstrate that Rpl10l plays an essential role during the meiotic stage of spermatogenesis by compensating for MSCI-mediated transcriptional silencing of Rpl10. These data provide direct evidence for the compensatory hypothesis and add novel insight into the evolution of X-chromosome-derived autosomal retrogenes and their role in male fertility.
Literature context: ATCCÂ® CCL-2human: A549ATCCATCCÂ® CCL-185human: SK-MEL-28ATCCATCCÂ® HTB-72
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
Literature context: and A549 (RRID:CVCL_0023) cells wer
p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27's effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway.
Literature context: A549 (RRID:CVCL_0023), H1650 (R
Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24- cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness.
Literature context: CCL-185, RRID:CVCL_0023), H1299 (A
Radiotherapy is commonly used to treat a variety of solid tumors but improvements in the therapeutic ratio are sorely needed. The aim of this study was to assess the Chk1 kinase inhibitor, MK-8776, for its ability to radiosensitize human tumor cells. Cells derived from NSCLC and HNSCC cancers were tested for radiosensitization by MK-8776. The ability of MK-8776 to abrogate the radiation-induced G2 block was determined using flow cytometry. Effects on repair of radiation-induced DNA double strand breaks (DSBs) were determined on the basis of rad51, γ-H2AX and 53BP1 foci. Clonogenic survival analyses indicated that MK-8776 radiosensitized p53-defective tumor cells but not lines with wild-type p53. Abrogation of the G2 block was evident in both p53-defective cells and p53 wild-type lines indicating no correlation with radiosensitization. However, only p53-defective cells entered mitosis harboring unrepaired DSBs. MK-8776 appeared to inhibit repair of radiation-induced DSBs at early times after irradiation. A comparison of MK-8776 to the wee1 inhibitor, MK-1775, suggested both similarities and differences in their activities. In conclusion, MK-8776 radiosensitizes tumor cells by mechanisms that include abrogation of the G2 block and inhibition of DSB repair. Our findings support the clinical evaluation of MK-8776 in combination with radiation.
Literature context: 977 A549; RRID:CVCL_0023 Human hepa
The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.
Literature context: Mycoplasma contamination. A549 (RRID: CVCL_0023), AU565 (RRID: CVCL_1074), H129
TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations.
Literature context: and A549 (RRID:CVCL_0023, purchased
Blockade of lysosomal calcium release due to lysosomal lipid accumulation has been shown to inhibit mTORC1 signaling. However, the mechanism by which lysosomal calcium regulates mTORC1 has remained undefined. Herein we report that proper lysosomal calcium release through the calcium channel TRPML1 is required for mTORC1 activation. TRPML1 depletion inhibits mTORC1 activity, while overexpression or pharmacologic activation of TRPML1 has the opposite effect. Lysosomal calcium activates mTORC1 by inducing association of calmodulin (CaM) with mTOR. Blocking the interaction between mTOR and CaM by antagonists of CaM significantly inhibits mTORC1 activity. Moreover, CaM is capable of stimulating the kinase activity of mTORC1 in a calcium-dependent manner in vitro. These results reveal that mTOR is a new type of CaM-dependent kinase, and TRPML1, lysosomal calcium and CaM play essential regulatory roles in the mTORC1 signaling pathway.
Literature context: es. A549 (RRID:CVCL_0023), HepG2 (R
Increasing evidence highlights the important roles of microRNAs in mediating p53's tumor suppression functions. Here, we report miR-139-5p as another new p53 microRNA target. p53 induced the transcription of miR-139-5p, which in turn suppressed the protein levels of phosphodiesterase 4D (PDE4D), an oncogenic protein involved in multiple tumor promoting processes. Knockdown of p53 reversed these effects. Also, overexpression of miR-139-5p decreased PDE4D levels and increased cellular cAMP levels, leading to BIM-mediated cell growth arrest. Furthermore, our analysis of human colorectal tumor specimens revealed significant inverse correlation between the expression of miR-139-5p and that of PDE4D. Finally, overexpression of miR-139-5p suppressed the growth of xenograft tumors, accompanied by decrease in PDE4D and increase in BIM. These results demonstrate that p53 inactivates oncogenic PDE4D by inducing the expression of miR-139-5p.
Literature context: -CCL-185, RRID:CVCL_0023) were grow
Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS). Previous studies have identified interferon-induced transmembrane proteins (IFITMs) as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms (SNP) rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response (NRIR). Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS.