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HeLa

RRID:CVCL_0030

Organism

Homo sapiens

Comments

Part of: Cancer Cell Line Encyclopedia (CCLE) project. Part of: COSMIC cell lines project. Part of: MD Anderson Cell Lines Project. Part of: Naval Biosciences Laboratory (NBL) collection (transferred to ATCC in 1982). Characteristics: HeLa has 5 five HPV18 integration sites: three on normal chromosomes 8 at 8q24 and two on derivative chromosomes, der(5)t(5;22;8)(q11;q11q13;q24) and der(22)t(8;22)(q24;q13). Doubling time: 1.3 days (PubMed=29156801); ~48 hours (DSMZ). HLA typing: A*68:02,03:19; B*15:03,15:03; C*12:03,12:03; DRB1*01:02,01:02 (PubMed=25960936). Microsatellite instability: Stable (MSS) (PubMed=12661003; Sanger). Transformant: NCBI_TaxID; 333761; Human papillomavirus type 18 (HPV18). Omics: Cell surface proteome. Omics: CNV analysis. Omics: Deep antibody staining analysis. Omics: Deep exome analysis. Omics: Deep membrane proteome analysis. Omics: Deep phosphoproteome analysis. Omics: Deep proteome analysis. Omics: Deep quantitative proteome analysis. Omics: Deep RNAseq analysis. Omics: DNA methylation analysis. Omics: Genome sequenced. Omics: Glycoproteome analysis by proteomics. Omics: Myristoylated proteins analysis by proteomics. Omics: Protein expression by reverse-phase protein arrays. Omics: SNP array analysis. Omics: Transcriptome analysis. Omics: Virome analysis using proteomics. Anecdotal: The HeLa cell line which was established in February 1951 is the oldest human immortal cell line. Anecdotal: The fascinating story of the HeLa cell line and of Henrietta Lacks from whom these cells originate are described in the book of Rebecca Skloot (CelloPub=CLPUB00377). Anecdotal: The HeLa cell line and its story inspired Australian artist Cynthia Verspaget to embark in 2003 on a artistic project 'The Anarchy Cell Line' (TAnCL) where she mixed her blood with HeLa cells. This work later spawned a PhD thesis (CelloPub=CLPUB00376) where among other things she makes the observation that two main taxonomical distinctions present in the zombie, living/dead and human/nonhuman, are also present in the HeLa cell line. Miscellaneous: HeLa is the most frequent contributor to cell lines contamination. Discontinued: ATCC; CRL-7923.

Proper Citation

ATCC Cat# CRM-CCL-2, RRID:CVCL_0030

Category

Cancer cell line

Sex

Female

Synonyms

HELA, Hela, He La, He-La, Henrietta Lacks cells, Helacyton gartleri

Vendor

ATCC

Cat Num

CRM-CCL-2

Cross References

BTO; BTO:0000567 CLO; CLO_0003684 CLO; CLO_0050910 EFO; EFO_0001185 MCCL; MCC:0000219 CLDB; cl1594 CLDB; cl1595 CLDB; cl1596 CLDB; cl1597 CLDB; cl1599 CLDB; cl1600 CLDB; cl1601 CLDB; cl1603 AddexBio; C0008001/44 ArrayExpress; E-MTAB-2706 ArrayExpress; E-MTAB-3610 ATCC; CCL-2 ATCC; CRM-CCL-2 ATCC; CRL-7923 BCRC; 60005 BCRJ; 0100 BioSample; SAMN03471148 BioSample; SAMN03472333 CCLE; HELA_CERVIX CCLV; CCLV-RIE 0082 CCRID; 3111C0001CCC000011 CCRID; 3111C0001CCC000332 CCRID; 3111C0002000000057 CCRID; 3131C0001000700019 CCRID; 3142C0001000000009 CCRID; 3153C0001000000007 Cell_Model_Passport; SIDM00846 ChEMBL-Cells; CHEMBL3308376 ChEMBL-Targets; CHEMBL399 CLS; 300194/p772_HeLa Cosmic; 760219 Cosmic; 760490 Cosmic; 801357 Cosmic; 809231 Cosmic; 877477 Cosmic; 911999 Cosmic; 925354 Cosmic; 928907 Cosmic; 944113 Cosmic; 1019309 Cosmic; 1057766 Cosmic; 1071911 Cosmic; 1131525 Cosmic; 1193029 Cosmic; 1201779 Cosmic; 1324208 Cosmic; 1336880 Cosmic; 1571796 Cosmic; 2301539 Cosmic; 2660190 Cosmic-CLP; 1298134 DSMZ; ACC-57 ECACC; 08011102 ECACC; 93021013 GDSC; 1298134 GEO; GSM113863 GEO; GSM226739 GEO; GSM226875 GEO; GSM253298 GEO; GSM436128 GEO; GSM436129 GEO; GSM723055 GEO; GSM723056 GEO; GSM1088663 GEO; GSM1088664 GEO; GSM1088665 GEO; GSM1088666 GEO; GSM1374528 GEO; GSM1669875 ICLC; HTL95023 IGRhCellID; HeLA IZSLER; BS TCL 20 JCRB; JCRB9004 KCB; KCB 86019YJ KCB; KCB 90024YJ KCLB; 10002 LINCS_HMS; 50061 LINCS_LDP; LCL-1512 Lonza; 51 MeSH; D006367 NCBI_Iran; C115 NIH-ARP; 153-364 PRIDE; PRD000007 PRIDE; PRD000032 PRIDE; PRD000123 PRIDE; PRD000124 PRIDE; PRD000345 PRIDE; PRD000376 PRIDE; PRD000525 PRIDE; PRD000526 PRIDE; PRD000527 PRIDE; PXD000013 PRIDE; PXD000243 PRIDE; PXD000252 PRIDE; PXD000279 PRIDE; PXD000396 PRIDE; PXD000474 PRIDE; PXD000589 PRIDE; PXD000661 PRIDE; PXD000680 PRIDE; PXD000759 PRIDE; PXD000883 PRIDE; PXD000895 PRIDE; PXD000900 PRIDE; PXD000953 PRIDE; PXD000954 PRIDE; PXD000999 PRIDE; PXD001047 PRIDE; PXD001118 PRIDE; PXD001154 PRIDE; PXD001175 PRIDE; PXD001249 PRIDE; PXD001258 PRIDE; PXD001259 PRIDE; PXD001278 PRIDE; PXD001305 PRIDE; PXD001374 PRIDE; PXD001381 PRIDE; PXD001396 PRIDE; PXD001441 PRIDE; PXD001541 PRIDE; PXD001548 PRIDE; PXD001574 PRIDE; PXD001660 PRIDE; PXD001781 PRIDE; PXD001798 PRIDE; PXD001805 PRIDE; PXD001806 PRIDE; PXD001810 PRIDE; PXD001863 PRIDE; PXD001907 PRIDE; PXD002001 PRIDE; PXD002039 PRIDE; PXD002066 PRIDE; PXD002252 PRIDE; PXD002277 PRIDE; PXD002378 PRIDE; PXD002383 PRIDE; PXD002395 PRIDE; PXD002572 PRIDE; PXD002591 PRIDE; PXD002704 PRIDE; PXD002844 PRIDE; PXD002880 PRIDE; PXD003186 PRIDE; PXD003209 PRIDE; PXD003258 PRIDE; PXD003370 PRIDE; PXD003503 PRIDE; PXD003530 PRIDE; PXD003560 PRIDE; PXD003792 PRIDE; PXD003813 PRIDE; PXD003909 PRIDE; PXD003913 PRIDE; PXD003917 PRIDE; PXD003924 PRIDE; PXD004182 PRIDE; PXD004271 PRIDE; PXD004273 PRIDE; PXD004613 PRIDE; PXD004736 PRIDE; PXD004900 PRIDE; PXD004914 PRIDE; PXD004933 PRIDE; PXD004934 PRIDE; PXD004940 PRIDE; PXD004995 PRIDE; PXD005018 PRIDE; PXD005107 PRIDE; PXD005181 PRIDE; PXD005346 PRIDE; PXD005349 PRIDE; PXD005366 PRIDE; PXD005509 PRIDE; PXD005572 PRIDE; PXD005712 PRIDE; PXD005955 PRIDE; PXD006112 PRIDE; PXD006293 RCB; RCB0007 RCB; RCB3680 SKY/M-FISH/CGH; 5351 TKG; TKG 0331 TOKU-E; 1434 Wikidata; Q847482

Publications that use this research resource

Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2.

  • Higgs MR
  • Mol. Cell
  • 2018 Jul 5

Literature context: ato et al., 2012N/AHeLaATCCCat# CCL-2U-2-OSATCCCat# HTB-96U-2-OS Flp-


Abstract:

Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.

Funding information:
  • NCRR NIH HHS - RR024574(United States)

Mapping Active Gene-Regulatory Regions in Human Repopulating Long-Term HSCs.

  • Wünsche P
  • Cell Stem Cell
  • 2018 Jul 5

Literature context: RRID:CVCL_0030 Experimental Models: Organisms/


Abstract:

Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.

Funding information:
  • NCI NIH HHS - K08 CA120060-04(United States)

Single-color, ratiometric biosensors for detecting signaling activities in live cells.

  • Ross BL
  • Elife
  • 2018 Jul 3

Literature context:


Abstract:

Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.

Funding information:
  • National Institutes of Health - R01 DK073368()
  • National Institutes of Health - R01 DK077140()
  • National Institutes of Health - R01 HL122827()
  • National Institutes of Health - R01 MH111516()
  • National Institutes of Health - R01 MH111527()
  • National Institutes of Health - R35 CA197622()
  • NHLBI NIH HHS - R01 HL122827()
  • NIA NIH HHS - P50 AG16573(United States)
  • NIDDK NIH HHS - R01 DK077140()
  • NIMH NIH HHS - R01 MH111527()

Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency.

  • Stein CS
  • Cell Rep
  • 2018 Jun 26

Literature context: C12 myoblastATCCCRL-1772HeLaATCCCCL-2Neuro-2a (N2a)ATCCCCL-131Experim


Abstract:

Mitochondria are composed of many small proteins that control protein synthesis, complex assembly, metabolism, and ion and reactive oxygen species (ROS) handling. We show that a skeletal muscle- and heart-enriched long non-coding RNA, LINC00116, encodes a highly conserved 56-amino-acid microprotein that we named mitoregulin (Mtln). Mtln localizes to the inner mitochondrial membrane, where it binds cardiolipin and influences protein complex assembly. In cultured cells, Mtln overexpression increases mitochondrial membrane potential, respiration rates, and Ca2+ retention capacity while decreasing mitochondrial ROS and matrix-free Ca2+. Mtln-knockout mice display perturbations in mitochondrial respiratory (super)complex formation and activity, fatty acid oxidation, tricarboxylic acid (TCA) cycle enzymes, and Ca2+ retention capacity. Blue-native gel electrophoresis revealed that Mtln co-migrates alongside several complexes, including the complex I assembly module, complex V, and supercomplexes. Under denaturing conditions, Mtln remains in high-molecular-weight complexes, supporting its role as a sticky molecular tether that enhances respiratory efficiency by bolstering protein complex assembly and/or stability.

Funding information:
  • NIGMS NIH HHS - GM034869(United States)

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE.

  • Oikonomidi I
  • Elife
  • 2018 Jun 13

Literature context:


Abstract:

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.

Funding information:
  • Deutsche Forschungsgemeinschaft - Emmy Noether scholarship DFG STE2310/1-1()
  • European Cooperation in Science and Technology - BM1406()
  • European Molecular Biology Organization - Installation Grant no. 2329()
  • European Regional Development Fund - CZ.2.16/3.1.00/24016()
  • Fundação Calouste Gulbenkian - 91/BD/14()
  • Fundação para a Ciência e a Tecnologia - 91/BD/14()
  • Fundação para a Ciência e a Tecnologia - BEX-BCM/3015/2014()
  • Fundação para a Ciência e a Tecnologia - LISBOA-01-0145-FEDER-031330()
  • Fundação para a Ciência e a Tecnologia - PTDC/BEX-BCM/3015/2014()
  • Fundação para a Ciência e a Tecnologia - SFRH/ BPD/117216/2016()
  • Fundação para a Ciência e a Tecnologia - SFRH/BCC/52507/2014()
  • Ministerstvo Školství, Mládeže a T?lovýchovy - LO1302()
  • Ministry of Education, Youth and Sports of the Czech Republic - LO1302()
  • NIGMS NIH HHS - R01 GM070743(United States)
  • Science Foundation Ireland - 14/IA/2622()
  • Seventh Framework Programme - Marie Curie Career Integration Grant (project no. 618769()
  • Worldwide Cancer Research - 14-1289()

Lipid Peroxidation Drives Gasdermin D-Mediated Pyroptosis in Lethal Polymicrobial Sepsis.

  • Kang R
  • Cell Host Microbe
  • 2018 Jun 5

Literature context: CTIB-202HL-60ATCCCCL-240HeLaATCC


Abstract:

Sepsis is a life-threatening condition caused by pathogen infection and associated with pyroptosis. Pyroptosis occurs upon activation of proinflammatory caspases and their subsequent cleavage of gasdermin D (GSDMD), resulting in GSDMD N-terminal fragments that form membrane pores to induce cell lysis. Here, we show that antioxidant defense enzyme glutathione peroxidase 4 (GPX4) and its ability to decrease lipid peroxidation, negatively regulate macrophage pyroptosis, and septic lethality in mice. Conditional Gpx4 knockout in myeloid lineage cells increases lipid peroxidation-dependent caspase-11 activation and GSDMD cleavage. The resultant N-terminal GSDMD fragments then trigger macrophage pyroptotic cell death in a phospholipase C gamma 1 (PLCG1)-dependent fashion. Administration of the antioxidant vitamin E that reduces lipid peroxidation, chemical inhibition of PLCG1, or genetic Caspase-11 deletion or Gsdmd inactivation prevents polymicrobial sepsis in Gpx4-/- mice. Collectively, this study suggests that lipid peroxidation drives GSDMD-mediated pyroptosis and hence constitutes a potential therapeutic target for lethal infection.

Funding information:
  • NCI NIH HHS - N01 CA015083(United States)
  • NCI NIH HHS - R01 CA160417()
  • NIGMS NIH HHS - R01 GM115366()

Immunomimetic Designer Cells Protect Mice from MRSA Infection.

  • Liu Y
  • Cell
  • 2018 Jun 12

Literature context: BHK-21ATCCCCL-10Human: HeLaATCCCCL-2Human: HT-1080ATCCCCL-121Human:


Abstract:

Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/J015652/1(United Kingdom)

The Cellosaurus, a Cell-Line Knowledge Resource.

  • Bairoch A
  • J Biomol Tech
  • 2018 May 10

Literature context: es, such as “we have used HeLa (RRID:CVCL_0030) obtained from ATCC (catalog nu


Abstract:

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences.

Funding information:
  • NINDS NIH HHS - 5R01NS063974(United States)

SRC-3 Coactivator Governs Dynamic Estrogen-Induced Chromatin Looping Interactions during Transcription.

  • Panigrahi AK
  • Mol. Cell
  • 2018 May 17

Literature context: College of Medicine ATCC CCL-2; RRID:CVCL_0030 HeLa S3 (for making bulk NE) Ce


Abstract:

Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription.

Funding information:
  • NCI NIH HHS - P30 CA125123()
  • NICHD NIH HHS - F32 HD007875()
  • NICHD NIH HHS - R01 HD007857()
  • NIGMS NIH HHS - R01 GM062591(United States)

The homeodomain transcription factor Prox1 is a direct target of SoxC proteins during developmental vertebrate neurogenesis.

  • Jacob A
  • J. Neurochem.
  • 2018 May 11

Literature context: -2, RRID:CVCL_0030) were used. Cell lines were not


Abstract:

The high-mobility-group-domain containing SoxC transcription factors Sox4 and Sox11 are expressed and required in the vertebrate central nervous system in neuronal precursors and neuroblasts. To identify genes that are widely regulated by SoxC proteins during vertebrate neurogenesis we generated expression profiles from developing mouse brain and chicken neural tube with reduced SoxC expression and found the transcription factor Prox1 strongly downregulated under both conditions. This led us to hypothesize that Prox1 expression depends on SoxC proteins in the developing central nervous system of mouse and chicken. By combining luciferase reporter assays and overexpression in the chicken neural tube with in vivo and in vitro binding studies, we identify the Prox1 gene promoter and two upstream enhancers at -44 kb and -40 kb relative to the transcription start as regulatory regions that are bound and activated by SoxC proteins. This argues that Prox1 is a direct target gene of SoxC proteins during neurogenesis. Electroporations in the chicken neural tube furthermore show that Prox1 activates a subset of SoxC target genes, whereas it has no effects on others. We propose that the transcriptional control of Prox1 by SoxC proteins may ensure coupling of two types of transcription factors that are both required during early neurogenesis, but have at least in part distinct functions. This article is protected by copyright. All rights reserved.

Funding information:
  • NCRR NIH HHS - S10 RR024615(United States)

Combined Human Genome-wide RNAi and Metabolite Analyses Identify IMPDH as a Host-Directed Target against Chlamydia Infection.

  • Rother M
  • Cell Host Microbe
  • 2018 May 9

Literature context: HeLa cells (ATCC CCL2; RRID:CVCL_0030; authenticated via short tandem


Abstract:

Chlamydia trachomatis (Ctr) accounts for >130 million human infections annually. Since chronic Ctr infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As an obligate intracellular bacterium, Ctr strictly depends on the functional contribution of the host cell. Here, we combined a human genome-wide RNA interference screen with metabolic profiling to obtain detailed understanding of changes in the infected cell and identify druggable pathways essential for Ctr growth. We demonstrate that Ctr shifts the host metabolism toward aerobic glycolysis, consistent with increased biomass requirement. We identify key regulator complexes of glucose and nucleotide metabolism that govern Ctr infection processes. Pharmacological targeting of inosine-5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits Ctr growth both in vitro and in vivo. These results highlight the potency of genome-scale functional screening for the discovery of drug targets against bacterial infections.

Funding information:
  • Wellcome Trust - 094879(United Kingdom)

The HIV-1 Tat protein recruits a ubiquitin ligase to reorganize the 7SK snRNP for transcriptional activation.

  • Faust TB
  • Elife
  • 2018 May 30

Literature context: HeLaprovirusΔtat, HeLa (RRID:CVCL_0030), and HEK 293T (RRID:CVCL_0063)


Abstract:

The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.

Funding information:
  • National Institute of Allergy and Infectious Diseases - R01AI114362()
  • National Institute of Allergy and Infectious Diseases - RO1AI114362()
  • National Institute of General Medical Sciences - P50GM082250()
  • NHLBI NIH HHS - R15 HL071526(United States)
  • NIAID NIH HHS - P30 AI027763()
  • Welch Foundation - I-1782()

Angiogenin/Ribonuclease 5 Is an EGFR Ligand and a Serum Biomarker for Erlotinib Sensitivity in Pancreatic Cancer.

  • Wang YN
  • Cancer Cell
  • 2018 Apr 9

Literature context: g Logsdon)NAHuman: HeLaATCCATCC#CCL-2Mouse: NIH-3T3ATCCATCC#CRL-1658H


Abstract:

Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment. These results identify a role of ANG as a serum biomarker that may be used to stratify patients for EGFR-targeted therapies, and offer insights into the ligand-receptor relationship between RNase and RTK families.

Funding information:
  • NCI NIH HHS - P30 CA016672()
  • NCI NIH HHS - R01 CA211615()
  • NCI NIH HHS - T32 CA186892()
  • NCI NIH HHS - U01 CA201777()
  • NIGMS NIH HHS - R01 GM098294(United States)

SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform.

  • Hatcher JM
  • Cell Chem Biol
  • 2018 Apr 19

Literature context: Models: Cell LinesHeLaATCCCat# CCL-2Ba/F3Pasi Jänne lab in Dana-Farb


Abstract:

The SRPK family of kinases regulates pre-mRNA splicing by phosphorylating serine/arginine (SR)-rich splicing factors, signals splicing control in response to extracellular stimuli, and contributes to tumorigenesis, suggesting that these splicing kinases are potential therapeutic targets. Here, we report the development of the first irreversible SRPK inhibitor, SRPKIN-1, which is also the first kinase inhibitor that forms a covalent bond with a tyrosine phenol group in the ATP-binding pocket. Kinome-wide profiling demonstrates its selectivity for SRPK1/2, and SRPKIN-1 attenuates SR protein phosphorylation at submicromolar concentrations. Vascular endothelial growth factor (VEGF) is a known target for SRPK-regulated splicing and, relative to the first-generation SRPK inhibitor SRPIN340 or small interfering RNA-mediated SRPK knockdown, SRPKIN-1 is more potent in converting the pro-angiogenic VEGF-A165a to the anti-angiogenic VEGF-A165b isoform and in blocking laser-induced neovascularization in a murine retinal model. These findings encourage further development of SRPK inhibitors for treatment of age-related macular degeneration.

Funding information:
  • NIGMS NIH HHS - R01-GM043778(United States)

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates.

  • Sepulveda G
  • Elife
  • 2018 Apr 30

Literature context: Nashville, TN ATCC Cat# CCL-2, RRID:CVCL_0030


Abstract:

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Funding information:
  • NIA NIH HHS - R01 AG039756(United States)
  • University of California, Davis - New Faculty Startup Funds()

Cell-Autonomous Regulation of Dendrite Self-Avoidance by the Wnt Secretory Factor MIG-14/Wntless.

  • Liao CP
  • Neuron
  • 2018 Apr 18

Literature context:


Abstract:

Self-avoidance allows sister dendrites from the same neuron to form non-redundant coverage of the sensory territory and is important for neural circuitry functions. Here, we report an unexpected, cell-autonomous role of the Wnt-secretory factor MIG-14/Wntless in mediating dendrite self-avoidance in the C. elegans multidendritic PVD neurons. Similar findings in Drosophila suggest that this novel function of Wntless is conserved. The mig-14 mutant shows defects in dendrite self-avoidance, and ectopic MIG-14 expression triggers dendrite repulsion. Functions of dendrite self-avoidance and Wnt secretion could be mapped to distinct MIG-14 domains, indicating that these two functions of MIG-14 are genetically separable, consistent with lack of self-avoidance defects in the Wnt mutants. We further demonstrate that MIG-14 engages Wiskott-Aldrich syndrome protein (WASP)-dependent actin assembly to regulate dendrite self-avoidance. Our work expands the repertoire of self-avoidance molecules and uncovers a previously unknown, Wnt-independent function of MIG-14/Wntless.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/D020190/1(United Kingdom)

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

  • Zhang X
  • Dev. Cell
  • 2018 Apr 23

Literature context: l Models: Cell LinesHeLaATCCCat#CCL-2U2OSGift from Dr. Xiaochun Yu, C


Abstract:

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation.

Funding information:
  • NINDS NIH HHS - NS43987(United States)

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

  • Guo L
  • Cell
  • 2018 Apr 19

Literature context: Human: HeLa ATCC Cat# CCL-2; RRID:CVCL_0030 ReNcell VM Human Neural Progeni


Abstract:

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-β2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-β1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-β2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-β2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

Funding information:
  • NCI NIH HHS - P01CA684841(United States)
  • NIGMS NIH HHS - R01 GM069909()
  • NIGMS NIH HHS - R01 GM099836()
  • NIGMS NIH HHS - T32 GM008275()
  • NIGMS NIH HHS - T32 GM071339()
  • NINDS NIH HHS - F31 NS079009()
  • NINDS NIH HHS - R01 NS081303()
  • NINDS NIH HHS - R01 NS087227()
  • NINDS NIH HHS - R21 NS090205()
  • NINDS NIH HHS - R21 NS094921()
  • NINDS NIH HHS - R21 NS100055()
  • NINDS NIH HHS - R35 NS097263()
  • NINDS NIH HHS - R35 NS097974()

Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.

  • Sorkina T
  • Elife
  • 2018 Apr 9

Literature context: Cell line (human) HeLa ATCC RRID:CVCL_0030


Abstract:

Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.

Funding information:
  • National Institutes of Health - DA014204()
  • NIAID NIH HHS - U01 AI101981(United States)

Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length.

  • Liu S
  • Elife
  • 2018 Mar 29

Literature context: HeLa (ATCC, RRID:CVCL_0030) and the retinal pigmented epit


Abstract:

Animal cells within a tissue typically display a striking regularity in their size. To date, the molecular mechanisms that control this uniformity are still unknown. We have previously shown that size uniformity in animal cells is promoted, in part, by size-dependent regulation of G1 length. To identify the molecular mechanisms underlying this process, we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression. Small cells display higher p38 activity and spend more time in G1 than larger cells. Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells, resulting in faster proliferation, smaller cell size and increased size heterogeneity. We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly, to increase cell size uniformity.

Funding information:
  • Biotechnology and Biological Sciences Research Council - 095831(United Kingdom)
  • Canadian Institutes of Health Research - FRN-343437()
  • National Institute of General Medical Sciences - GM26875()

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions.

  • Dao TP
  • Mol. Cell
  • 2018 Mar 15

Literature context: Cell LinesU2OSATCCHTB-96HeLaATCCCCL-2Recombinant DNAP4455 FLAG-hPLIC-


Abstract:

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Funding information:
  • NCI NIH HHS - 1R21CA131774(United States)

Reshaping of the Dendritic Cell Chromatin Landscape and Interferon Pathways during HIV Infection.

  • Johnson JS
  • Cell Host Microbe
  • 2018 Mar 14

Literature context: HeLa cells ATCC Cat# CRL-7923, RRID:CVCL_0030 THP-1 cells ATCC Cat# TIB-202,


Abstract:

Myeloid dendritic cells (DCs) have the innate capacity to sense pathogens and orchestrate immune responses. However, DCs do not mount efficient immune responses to HIV-1, primarily due to restriction of virus reverse transcription, which prevents accumulation of viral cDNA and limits its detection through the cGAS-STING pathway. By allowing reverse transcription to proceed, we find that DCs detect HIV-1 in distinct phases, before and after virus integration. Blocking integration suppresses, but does not abolish, activation of the transcription factor IRF3, downstream interferon (IFN) responses, and DC maturation. Consistent with two stages of detection, HIV-1 "primes" chromatin accessibility of innate immune genes before and after integration. Once primed, robust IFN responses can be unmasked by agonists of the innate adaptor protein, MyD88, through a process that requires cGAS, STING, IRF3, and nuclear factor κB. Thus, HIV-1 replication increases material available for sensing, and discrete inflammatory inputs tune cGAS signaling to drive DC maturation.

Funding information:
  • NIAID NIH HHS - R21 AI084633()
  • NINDS NIH HHS - R21 NS050463(United States)

A Sub-population of Group A Streptococcus Elicits a Population-wide Production of Bacteriocins to Establish Dominance in the Host.

  • Hertzog BB
  • Cell Host Microbe
  • 2018 Mar 14

Literature context: aATCC (Baruch et al., 2014)Cat# CCL-2Human: HaCaTKohen R. lab (Portug


Abstract:

Bacteria use quorum sensing (QS) to regulate gene expression. We identified a group A Streptococcus (GAS) strain possessing the QS system sil, which produces functional bacteriocins, through a sequential signaling pathway integrating host and bacterial signals. Host cells infected by GAS release asparagine (ASN), which is sensed by the bacteria to alter its gene expression and rate of proliferation. We show that upon ASN sensing, GAS upregulates expression of the QS autoinducer peptide SilCR. Initial SilCR expression activates the autoinduction cycle for further SilCR production. The autoinduction process propagates throughout the GAS population, resulting in bacteriocin production. Subcutaneous co-injection of mice with a bacteriocin-producing strain and the globally disseminated M1T1 GAS clone results in M1T1 killing within soft tissue. Thus, by sensing host signals, a fraction of a bacterial population can trigger an autoinduction mechanism mediated by QS, which acts on the entire bacterial community to outcompete other bacteria within the infection.

Funding information:
  • NIGMS NIH HHS - R01GM090077(United States)

The Histone Chaperones ASF1 and CAF-1 Promote MMS22L-TONSL-Mediated Rad51 Loading onto ssDNA during Homologous Recombination in Human Cells.

  • Huang TH
  • Mol. Cell
  • 2018 Mar 1

Literature context: HeLa cells ATCC Cat# CCL-2; RRID:CVCL_0030 K-562 cells ATCC Cat# CCL-243;


Abstract:

The access-repair-restore model for the role of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or a mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence of reduced recruitment of the Rad51 loader MMS22L-TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end resection, persistent activation of the ATR-Chk1 pathway, and cell cycle arrest. In agreement, histones occupy ssDNA during DNA repair in yeast. We also uncovered DNA-PKcs-dependent DNA damage-induced ASF1A phosphorylation, which enhances chromatin assembly, promoting MMS22L-TONSL recruitment and, hence, Rad51 loading. We propose that transient assembly of newly synthesized histones onto ssDNA serves to recruit MMS22L-TONSL to efficiently form the Rad51 nucleofilament for strand invasion, suggesting an active role of chromatin assembly in homologous recombination.

Funding information:
  • Intramural NIH HHS - (United States)
  • NCI NIH HHS - R01 CA095641()

Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

  • Lukeš T
  • Gigascience
  • 2018 Mar 1

Literature context: HeLa cells (RRID:CVCL_0030) were maintained in phenol red-


Abstract:

Background: Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Findings: Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. Conclusions: This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

Funding information:
  • NEI NIH HHS - T32 EY013933(United States)

TOM70 Sustains Cell Bioenergetics by Promoting IP3R3-Mediated ER to Mitochondria Ca2+ Transfer.

  • Filadi R
  • Curr. Biol.
  • 2018 Feb 5

Literature context: CCat# CRL-2991HeLaATCCCat# ATCC CCL-2VDAC1/3 −/− MEF[51]N/AIP3Rs-1/2/


Abstract:

The mitochondrial translocase of the outer membrane (TOM) is a protein complex that is essential for the post-translational import of nuclear-encoded mitochondrial proteins. Among its subunits, TOM70 and TOM20 are only transiently associated with the core complex, suggesting their possible additional roles within the outer mitochondrial membrane (OMM). Here, by using different mammalian cell lines, we demonstrate that TOM70, but not TOM20, clusters in distinct OMM foci, frequently overlapping with sites in which the endoplasmic reticulum (ER) contacts mitochondria. Functionally, TOM70 depletion specifically impairs inositol trisphosphates (IP3)-linked ER to mitochondria Ca2+ transfer. This phenomenon is dependent on the capacity of TOM70 to interact with IP3-receptors and favor their functional recruitment close to mitochondria. Importantly, the reduced constitutive Ca2+ transfer to mitochondria, observed in TOM70-depleted cells, dampens mitochondrial respiration, affects cell bioenergetics, induces autophagy, and inhibits proliferation. Our data reveal a hitherto unexpected role for TOM70 in pro-survival ER-mitochondria communication, reinforcing the view that the ER-mitochondria signaling platform is a key regulator of cell fate.

Funding information:
  • NIEHS NIH HHS - ES11047(United States)

Serum Interleukin-23 in Polish Patients with Systemic Lupus Erythematosus: Association with Lupus Nephritis, Obesity, and Peripheral Vascular Disease.

  • Fischer K
  • Mediators Inflamm.
  • 2018 Feb 13

Literature context: HEp-2 cell line contaminated by RRID:CVCL_0030 cervical adenocarcinoma human H


Abstract:

Objectives: To analyze the correlation between the serum concentration of interleukin- (IL-) 23 and atherosclerotic changes, traditional atherosclerotic risk factors, the autoantibody profile, and involvement of selected organs in systemic lupus erythematosus (SLE) patients. Patients and Methods: We studied 94 SLE patients and 27 controls. We analyzed the IL-23 serum concentration, autoantibodies, carotid intima-media thickness and atherosclerotic plaque, the ankle-brachial index, atherosclerotic risk factors, and organ manifestations. Results: Concentrations of IL-23 significantly differed between SLE patients and the controls (p = 0.0015). On the basis of multivariate stepwise analysis, we revealed that high levels of IL-23 were associated with atherosclerotic plaque in common femoral arteries (OR = 12.67; 95% CI: 1.41-113.84), lupus nephritis (OR = 3.69; 95% CI: 1.16-12.22), and obesity (OR = 4.21; 95% CI: 1.40-12.67). Autoantibodies related to IL-23 were anti-phosphatidylethanolamine antibodies (OR = 11.06; 95% CI: 1.24-98.65) and anti-SS-B/La antibodies (OR = 15.43; 95% CI: 1.73-137.25). Conclusions: IL-23 may be involved in lupus nephritis pathogenesis. Through its association with obesity and selected antiphospholipid antibodies, IL-23 might promote a hypercoagulable state contributing to atherothrombosis development in SLE patients.

Funding information:
  • NIAID NIH HHS - HHSN272200700038C(United States)

Decoding a Salmonella Typhi Regulatory Network that Controls Typhoid Toxin Expression within Human Cells.

  • Fowler CC
  • Cell Host Microbe
  • 2018 Jan 10

Literature context: RRID:CVCL_0030 Oligonucleotides


Abstract:

Salmonella Typhi is the cause of typhoid fever, a major global health concern. An essential virulence factor of this pathogen is typhoid toxin. In contrast to most AB-type toxins, typhoid toxin is exclusively expressed by intracellular bacteria. The regulatory networks that ensure this unique gene expression pattern are unknown. Here, we developed FAST-INSeq, a genome-wide screening approach to identify S. Typhi genes required for typhoid toxin expression within infected cells. We find that typhoid toxin expression is controlled by a silencing and counter-silencing mechanism through the opposing actions of the PhoP/PhoQ two-component regulatory system and the histone-like protein H-NS. The screen also identified bacterial mutants that alter the proportion of intracellular S. Typhi that reside within an intravacuolar environment, which was essential for toxin expression. Collectively, these data describe a regulatory mechanism that allows a bacterial pathogen to exclusively express a virulence factor when located within a specific intracellular compartment.

Funding information:
  • NIAID NIH HHS - R01 AI079022()
  • NIAID NIH HHS - R56 AI079022()
  • NIEHS NIH HHS - R01ES08744(United States)

Nek9 Phosphorylation Defines a New Role for TPX2 in Eg5-Dependent Centrosome Separation before Nuclear Envelope Breakdown.

  • Eibes S
  • Curr. Biol.
  • 2018 Jan 8

Literature context: ental Models: Cell LinesHeLaATCCCCL-2HEK293TATCCCRL-3216TPX2 lox/lox


Abstract:

Centrosomes [1, 2] play a central role during spindle assembly in most animal cells [3]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4, 5], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7, 8]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10-17]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19] and the kinase Nek9 [20], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system.

Funding information:
  • NIGMS NIH HHS - R01 GM041804(United States)

Endosomal Rab cycles regulate Parkin-mediated mitophagy.

  • Yamano K
  • Elife
  • 2018 Jan 23

Literature context: (Homo sapiens) HeLa ATCC RRID:CVCL_0030


Abstract:

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Funding information:
  • Japan Science and Technology Agency - JPMJCR13M7(International)
  • Japan Society for the Promotion of Science - 16K15095(International)
  • Japan Society for the Promotion of Science - JP15H01196(International)
  • Japan Society for the Promotion of Science - JP16K18545(International)
  • Japan Society for the Promotion of Science - JP26000014(International)
  • Japan Society for the Promotion of Science - JP26111729(International)
  • Japan Society for the Promotion of Science - JP26840033(International)
  • NIDCR NIH HHS - R03 DE018415-02(United States)
  • NINDS NIH HHS - Intramural program(United States)

NF-κB Dynamics Discriminate between TNF Doses in Single Cells.

  • Zhang Q
  • Cell Syst
  • 2017 Dec 27

Literature context: HeLa ATCC RRID:CVCL_0030 HCT116 ATCC RRID: VCL_0291


Abstract:

Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune their cytokine responses beyond the decision to activate.

Funding information:
  • NIGMS NIH HHS - P41 GM103712()
  • NIGMS NIH HHS - R35 GM119462()

The Adenovirus E1A C Terminus Suppresses a Delayed Antiviral Response and Modulates RAS Signaling.

  • Zemke NR
  • Cell Host Microbe
  • 2017 Dec 13

Literature context: ory RRID:CVCL_0030 2fTGH George Stark Lab RRID: CV


Abstract:

The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3.

Funding information:
  • NCI NIH HHS - CA136905(United States)
  • NCI NIH HHS - R01 CA025235()
  • NIAID NIH HHS - T32 AI060567()

New tools for "hot-wiring" clathrin-mediated endocytosis with temporal and spatial precision.

  • Wood LA
  • J. Cell Biol.
  • 2017 Dec 4

Literature context: rch Resource Identifier [RRID]: CVCL_0030) were maintained in DMEM plus 1


Abstract:

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment ("hook") is inducibly attached to an "anchor" protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook-anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it "hot-wiring." We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the β2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement.

An Alkynyl-Fucose Halts Hepatoma Cell Migration and Invasion by Inhibiting GDP-Fucose-Synthesizing Enzyme FX, TSTA3.

  • Kizuka Y
  • Cell Chem Biol
  • 2017 Dec 21

Literature context: CCCL-185Hep3BATCCHB-8064HeLaATCCCCL-2HepG2ATCCHB-8065FTO2B(Kitazume-K


Abstract:

Fucosylation is a glycan modification critically involved in cancer and inflammation. Although potent fucosylation inhibitors are useful for basic and clinical research, only a few inhibitors have been developed. Here, we focus on a fucose analog with an alkyne group, 6-alkynyl-fucose (6-Alk-Fuc), which is used widely as a detection probe for fucosylated glycans, but is also suggested for use as a fucosylation inhibitor. Our glycan analysis using lectin and mass spectrometry demonstrated that 6-Alk-Fuc is a potent and general inhibitor of cellular fucosylation, with much higher potency than the existing inhibitor, 2-fluoro-fucose (2-F-Fuc). The action mechanism was shown to deplete cellular GDP-Fuc, and the direct target of 6-Alk-Fuc is FX (encoded by TSTA3), the bifunctional GDP-Fuc synthase. We also show that 6-Alk-Fuc halts hepatoma invasion. These results highlight the unappreciated role of 6-Alk-Fuc as a fucosylation inhibitor and its potential use for basic and clinical science.

Funding information:
  • NIGMS NIH HHS - GM068763(United States)

Shigella entry unveils a calcium/calpain-dependent mechanism for inhibiting sumoylation.

  • Lapaquette P
  • Elife
  • 2017 Dec 12

Literature context: ine (Hela) CCL-2 ATCC ATCC CCL2/RRID:CVCL_0030 cell line (HT1080) CCL-121 ATCC


Abstract:

Disruption of the sumoylation/desumoylation equilibrium is associated with several disease states such as cancer and infections, however the mechanisms regulating the global SUMO balance remain poorly defined. Here, we show that infection by Shigella flexneri, the causative agent of human bacillary dysentery, switches off host sumoylation during epithelial cell infection in vitro and in vivo and that this effect is mainly mediated by a calcium/calpain-induced cleavage of the SUMO E1 enzyme SAE2, thus leading to sumoylation inhibition. Furthermore, we describe a mechanism by which Shigella promotes its own invasion by altering the sumoylation state of RhoGDIα, a master negative regulator of RhoGTPase activity and actin polymerization. Together, our data suggest that SUMO modification is essential to restrain pathogenic bacterial entry by limiting cytoskeletal rearrangement induced by bacterial effectors. Moreover, these findings identify calcium-activated calpains as powerful modulators of cellular sumoylation levels with potentially broad implications in several physiological and pathological situations.

Funding information:
  • NCI NIH HHS - 5 R01CA 140729-02(United States)

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

  • Clift D
  • Cell
  • 2017 Dec 14

Literature context: D:RRID:CVCL_0030 Human: NHLF Lonza Cat#CC-2512


Abstract:

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Funding information:
  • NIDCD NIH HHS - P30 DC04657(United States)

Pharmacological augmentation of nicotinamide phosphoribosyltransferase (NAMPT) protects against paclitaxel-induced peripheral neuropathy.

  • LoCoco PM
  • Elife
  • 2017 Nov 10

Literature context: HeLa cervical cancer cells (RRID:CVCL_0030), Panc-1 prostate cancer cells


Abstract:

Chemotherapy-induced peripheral neuropathy (CIPN) arises from collateral damage to peripheral afferent sensory neurons by anticancer pharmacotherapy, leading to debilitating neuropathic pain. No effective treatment for CIPN exists, short of dose-reduction which worsens cancer prognosis. Here, we report that stimulation of nicotinamide phosphoribosyltransferase (NAMPT) produced robust neuroprotection in an aggressive CIPN model utilizing the frontline anticancer drug, paclitaxel (PTX). Daily treatment of rats with the first-in-class NAMPT stimulator, P7C3-A20, prevented behavioral and histologic indicators of peripheral neuropathy, stimulated tissue NAD recovery, improved general health, and abolished attrition produced by a near maximum-tolerated dose of PTX. Inhibition of NAMPT blocked P7C3-A20-mediated neuroprotection, whereas supplementation with the NAMPT substrate, nicotinamide, potentiated a subthreshold dose of P7C3-A20 to full efficacy. Importantly, P7C3-A20 blocked PTX-induced allodynia in tumored mice without reducing antitumoral efficacy. These findings identify enhancement of NAMPT activity as a promising new therapeutic strategy to protect against anticancer drug-induced peripheral neurotoxicity.

Funding information:
  • NIMH NIH HHS - T32 MH065214(United States)

TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes.

  • Willett R
  • Nat Commun
  • 2017 Nov 17

Literature context: 100 μg/ml streptomycin (Gibco). HeLa cells (ATCC) and U2OS cells (ATCC) we


Abstract:

Lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions.

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS.

  • Ly T
  • Elife
  • 2017 Oct 20

Literature context: Human Cell Line HeLa ATCC RRID:CVCL_0030 Tested negative for mycoplasma


Abstract:

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

Tetherin Suppresses Type I Interferon Signaling by Targeting MAVS for NDP52-Mediated Selective Autophagic Degradation in Human Cells.

  • Jin S
  • Mol. Cell
  • 2017 Oct 19

Literature context: KO HEK293TThis paperN/AHeLaATCCCCL-2THP-1ATCCTIB-202A549ATCCCRM-CCL-


Abstract:

Tetherin (BST2/CD317) is an interferon-inducible antiviral factor known for its ability to block the release of enveloped viruses from infected cells. Yet its role in type I interferon (IFN) signaling remains poorly defined. Here, we demonstrate that Tetherin is a negative regulator of RIG-I like receptor (RLR)-mediated type I IFN signaling by targeting MAVS. The induction of Tetherin by type I IFN accelerates MAVS degradation via ubiquitin-dependent selective autophagy in human cells. Moreover, Tetherin recruits E3 ubiquitin ligase MARCH8 to catalyze K27-linked ubiquitin chains on MAVS at lysine 7, which serves as a recognition signal for NDP52-dependent autophagic degradation. Taken together, our findings reveal a negative feedback loop of RLR signaling generated by Tetherin-MARCH8-MAVS-NDP52 axis and provide insights into a better understanding of the crosstalk between selective autophagy and optimal deactivation of type I IFN signaling.

IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

  • Zhou H
  • Elife
  • 2017 Oct 9

Literature context: re the experiments. Hela cells (RRID:CVCL_0030) were purchased from ATCC (Mana


Abstract:

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

Funding information:
  • NCI NIH HHS - P01 CA062220()
  • NHLBI NIH HHS - P01 HL029582()
  • NIDA NIH HHS - R00DA024754(United States)

Regulation of m6A Transcripts by the 3'→5' RNA Helicase YTHDC2 Is Essential for a Successful Meiotic Program in the Mammalian Germline.

  • Wojtas MN
  • Mol. Cell
  • 2017 Oct 19

Literature context: HeLa cells Sigma 93021013; RRID:CVCL_0030 HEK293T cells GE Dharmacon #HCL


Abstract:

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'→5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'→3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

  • Kleino A
  • Immunity
  • 2017 Oct 17

Literature context: 1972N/AHuman HeLa cell lineATCCCCL-2Experimental Models: Organisms/S


Abstract:

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

Funding information:
  • NCI NIH HHS - 5P01CA027502-27(United States)
  • NIAID NIH HHS - R01 AI045937()
  • NIAID NIH HHS - R01 AI060025()
  • NIAID NIH HHS - R01 AI119030()
  • NIAID NIH HHS - R37 AI050872()
  • NIAID NIH HHS - R56 AI060025()

Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion.

  • Shin D
  • Elife
  • 2017 Oct 2

Literature context: Collection (RRID: CVCL_0063 and CVCL_0030, respectively) and maintained i


Abstract:

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

Survival Motor Neuron Protein is Released from Cells in Exosomes: A Potential Biomarker for Spinal Muscular Atrophy.

  • Nash LA
  • Sci Rep
  • 2017 Oct 24

Literature context: (ATCC CCL 18586, and HeLa (ATCC CCL 287) cells were grown in Minimum


Abstract:

Spinal muscular atrophy (SMA) is caused by homozygous mutation of the survival motor neuron 1 (SMN1) gene. Disease severity inversely correlates to the amount of SMN protein produced from the homologous SMN2 gene. We show that SMN protein is naturally released in exosomes from all cell types examined. Fibroblasts from patients or a mouse model of SMA released exosomes containing reduced levels of SMN protein relative to normal controls. Cells overexpressing SMN protein released exosomes with dramatically elevated levels of SMN protein. We observed enhanced quantities of exosomes in the medium from SMN-depleted cells, and in serum from a mouse model of SMA and a patient with Type 3 SMA, suggesting that SMN-depletion causes a deregulation of exosome release or uptake. The quantity of SMN protein contained in the serum-derived exosomes correlated with the genotype of the animal, with progressively less protein in carrier and affected animals compared to wildtype mice. SMN protein was easily detectable in exosomes isolated from human serum, with a reduction in the amount of SMN protein in exosomes from a patient with Type 3 SMA compared to a normal control. Our results suggest that exosome-derived SMN protein may serve as an effective biomarker for SMA.

A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

  • Gabrielsen M
  • Mol. Cell
  • 2017 Oct 19

Literature context: HeLa DSMZ Cat#ACC-57; RRID:CVCL_0030 Recombinant DNA


Abstract:

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

  • Gulluni F
  • Cancer Cell
  • 2017 Oct 9

Literature context: odels: Cell LinesHeLaATCCCat. # CCL-2HEK293TATCCCat. # CRL-3216; RRID


Abstract:

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.

  • Samandi S
  • Elife
  • 2017 Oct 30

Literature context: authenticated by STR profiling, RRID:CVCL_0030) cultures tested negative for m


Abstract:

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

Funding information:
  • NINDS NIH HHS - R01 NS084396(United States)

Diverse Viruses Require the Calcium Transporter SPCA1 for Maturation and Spread.

  • Hoffmann HH
  • Cell Host Microbe
  • 2017 Oct 11

Literature context: ATCC ATCC Cat# CCL-2; RRID:CVCL_0030 Monkey: VERO (kidney epithelial


Abstract:

Respiratory and arthropod-borne viral infections are a global threat due to the lack of effective antivirals and vaccines. A potential strategy is to target host proteins required for viruses but non-essential for the host. To identify such proteins, we performed a genome-wide knockout screen in human haploid cells and identified the calcium pump SPCA1. SPCA1 is required by viruses from the Paramyxoviridae, Flaviviridae, and Togaviridae families, including measles, dengue, West Nile, Zika, and chikungunya viruses. Calcium transport activity is required for SPCA1 to promote virus spread. SPCA1 regulates proteases within the trans-Golgi network that require calcium for their activity and are critical for virus glycoprotein maturation. Consistent with these findings, viral glycoproteins fail to mature in SPCA1-deficient cells preventing viral spread, which is evident even in cells with partial loss of SPCA1. Thus, SPCA1 is an attractive antiviral host target for a broad spectrum of established and emerging viral infections.

Funding information:
  • NIDDK NIH HHS - F32 DK095666()
  • NIGMS NIH HHS - GM43644(United States)

In Situ Architecture and Cellular Interactions of PolyQ Inclusions.

  • Bäuerlein FJB
  • Cell
  • 2017 Sep 21

Literature context: an: HeLa cells ATCC ATCC CCL-2; RRID:CVCL_0030 Experimental Models: Organisms/


Abstract:

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.

Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis.

  • Trabanelli S
  • Nat Commun
  • 2017 Sep 19

Literature context: B-26, ATCC), ovarian (HeLa, cn: CCL-2, ATCC), colon (HCT116, cn: CCL-


Abstract:

Group 2 innate lymphoid cells (ILC2s) are involved in human diseases, such as allergy, atopic dermatitis and nasal polyposis, but their function in human cancer remains unclear. Here we show that, in acute promyelocytic leukaemia (APL), ILC2s are increased and hyper-activated through the interaction of CRTH2 and NKp30 with elevated tumour-derived PGD2 and B7H6, respectively. ILC2s, in turn, activate monocytic myeloid-derived suppressor cells (M-MDSCs) via IL-13 secretion. Upon treating APL with all-trans retinoic acid and achieving complete remission, the levels of PGD2, NKp30, ILC2s, IL-13 and M-MDSCs are restored. Similarly, disruption of this tumour immunosuppressive axis by specifically blocking PGD2, IL-13 and NKp30 partially restores ILC2 and M-MDSC levels and results in increased survival. Thus, using APL as a model, we uncover a tolerogenic pathway that may represent a relevant immunosuppressive, therapeutic targetable, mechanism operating in various human tumour types, as supported by our observations in prostate cancer.Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth.

Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins.

  • Whiteley AM
  • Elife
  • 2017 Sep 21

Literature context: and 10% FBS (Sigma). HeLa cells RRID:CVCL_0030 (originally from ATCC, Manassus


Abstract:

Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1-/- mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1-/- B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.

Funding information:
  • NIDDK NIH HHS - K01 DK098285()
  • NINDS NIH HHS - R01 NS078279(United States)

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP.

  • Pawellek A
  • Elife
  • 2017 Sep 8

Literature context: , RNA isolation and RT-PCRHeLa (RRID:CVCL_0030), HEK293 (RRID:CVCL_0045) and N


Abstract:

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.

Funding information:
  • Medical Research Council - G0400132(United Kingdom)

A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication.

  • Miller CN
  • Cell Host Microbe
  • 2017 Sep 13

Literature context: . HeLa cells (ATCC clone CCL-2; RRID:CVCL_0030; sex of cells: female) were cul


Abstract:

Many intracellular pathogens exploit host secretory trafficking to support their intracellular cycle, but knowledge of these pathogenic processes is limited. The bacterium Brucella abortus uses a type IV secretion system (VirB T4SS) to generate a replication-permissive Brucella-containing vacuole (rBCV) derived from the host ER, a process that requires host early secretory trafficking. Here we show that the VirB T4SS effector BspB contributes to rBCV biogenesis and Brucella replication by interacting with the conserved oligomeric Golgi (COG) tethering complex, a major coordinator of Golgi vesicular trafficking, thus remodeling Golgi membrane traffic and redirecting Golgi-derived vesicles to the BCV. Altogether, these findings demonstrate that Brucella modulates COG-dependent trafficking via delivery of a T4SS effector to promote rBCV biogenesis and intracellular proliferation, providing mechanistic insight into how bacterial exploitation of host secretory functions promotes pathogenesis.

Funding information:
  • NIAID NIH HHS - R01 AI129992()
  • NIAID NIH HHS - R21 AI112649()
  • NIAID NIH HHS - T32 AI007025()

Delta-Secretase Phosphorylation by SRPK2 Enhances Its Enzymatic Activity, Provoking Pathogenesis in Alzheimer's Disease.

  • Wang ZH
  • Mol. Cell
  • 2017 Sep 7

Literature context: EK293ATCCCRL-1573Human: HeLaATCCCCL-2Human: SH-SY5YATCCCRL-2266Experi


Abstract:

Delta-secretase, a lysosomal asparagine endopeptidase (AEP), simultaneously cleaves both APP and tau, controlling the onset of pathogenesis of Alzheimer's disease (AD). However, how this protease is post-translationally regulated remains unclear. Here we report that serine-arginine protein kinase 2 (SRPK2) phosphorylates delta-secretase and enhances its enzymatic activity. SRPK2 phosphorylates serine 226 on delta-secretase and accelerates its autocatalytic cleavage, leading to its cytoplasmic translocation and escalated enzymatic activities. Delta-secretase is highly phosphorylated in human AD brains, tightly correlated with SRPK2 activity. Overexpression of a phosphorylation mimetic (S226D) in young 3xTg mice strongly promotes APP and tau fragmentation and facilitates amyloid plaque deposits and neurofibrillary tangle (NFT) formation, resulting in cognitive impairment. Conversely, viral injection of the non-phosphorylatable mutant (S226A) into 5XFAD mice decreases APP and tau proteolytic cleavage, attenuates AD pathologies, and reverses cognitive defects. Our findings support that delta-secretase phosphorylation by SRPK2 plays a critical role in aggravating AD pathogenesis.

Pimozide reduces toxic forms of tau in TauC3 mice via 5' adenosine monophosphate-activated protein kinase-mediated autophagy.

  • Kim YD
  • J. Neurochem.
  • 2017 Sep 20

Literature context: HeLa (RRID:CVCL_0030) and SH-SY


Abstract:

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease-associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non-mTOR pathways is a new option for autophagy-based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK-Unc-51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase-cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40-insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR-independent AMPK-ULK1 axis.

The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation.

  • Zhao YG
  • Mol. Cell
  • 2017 Sep 21

Literature context: cellsATCCCRL-1651HeLa cellsATCCCCL-2WT MEF cellsThis paperN/AVmp1 KO


Abstract:

During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.

Manganese-induced trafficking and turnover of GPP130 is mediated by sortilin.

  • Venkat S
  • Mol. Biol. Cell
  • 2017 Sep 15

Literature context: HeLa cells (Cat# ATCC-CCL-2, RRID:CVCL_0030) were grown in MEM (Cat#10-010-


Abstract:

Elevated, nontoxic doses of manganese (Mn) protect against Shiga toxin-1-induced cell death via down-regulation of GPP130, a cycling Golgi membrane protein that serves as an endosome-to-Golgi trafficking receptor for the toxin. Mn binds to GPP130 in the Golgi and causes GPP130 to oligomerize/aggregate, and the complexes are diverted to lysosomes. In fact, based on experiments using the self-interacting FM domain, it appears generally true that aggregation of a Golgi protein leads to its lysosomal degradation. How such oligomers are selectively sorted out of the Golgi is unknown. Here we provide evidence that Mn-induced exit of GPP130 from the trans-Golgi network (TGN) toward lysosomes is mediated by the sorting receptor sortilin interacting with the lumenal stem domain of GPP130. In contrast, FM-induced lysosomal trafficking of the Golgi protein galactosyltransferase was sortilin independent and occurred even in the absence of its native lumenal domain. Thus sortilin-dependent as well as sortilin-independent sorting mechanisms target aggregated Golgi membrane proteins for lysosomal degradation.

Funding information:
  • NIAID NIH HHS - R01 AI068112-05(United States)

A novel c-Src recruitment pathway from the cytosol to focal adhesions.

  • Machiyama H
  • FEBS Lett.
  • 2017 Aug 18

Literature context: parent vector.Cell cultureHeLa (RRID:CVCL_0030) and SYF (RRID:CVCL_6461) cells


Abstract:

The role of myristoylation in the localization and catalytic activity of Src at focal adhesions was investigated by live-cell imaging and site-directed mutagenesis. Although the majority of activated Src molecules are localized at focal adhesions, it is unclear how activated Src molecules are recruited to focal adhesions. Because Src is activated at the cell membrane, translocation of Src to cell membranes is considered to be essential for its recruitment to focal adhesions. Membrane-targeting-deficient Src mutant SrcG2A localizes at focal adhesions, indicating direct recruitment of Src from cytosol to focal adhesions. Furthermore, directly recruited Src molecules are shown to enhance paxillin dynamics at focal adhesions. These results reveal that the regulation of Src activation and translocation is more complex than previously suggested.

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

  • Hamperl S
  • Cell
  • 2017 Aug 10

Literature context: Human: HeLa ATCC Cat#CCL-2; RRID:CVCL_0030 Oligonucleotides


Abstract:

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.

Funding information:
  • NIGMS NIH HHS - R01 GM119334()

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

  • Kang Y
  • Mol. Cell
  • 2017 Aug 3

Literature context: cells N/A RRID:CVCL_0030 HEK293T N/


Abstract:

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport.

  • Shan P
  • Curr. Biol.
  • 2017 Aug 7

Literature context: HeLa ATCC RRID:CVCL_0030 HCT116 ATCC RRID: CVCL_0291


Abstract:

Most eukaryotic mRNAs are polyadenylated in the nucleus, and the poly(A)-tail is required for efficient mRNA export and translation. However, mechanisms governing mRNA transport remain unclear. Here, we report that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 acts as an energy sensor and negatively regulates poly(A)RNA transport via deacetylating a poly(A)-binding protein, PABP1. Upon energy starvation, SIRT1 interacts with and deacetylates PABP1 and deactivates its poly(A)RNA binding, leading to nuclear accumulation of PABP1 and poly(A)RNA and thus facilitating eukaryotic cells to attenuate protein synthesis and energy consumption to adapt to energy stress. Moreover, AMPK-directed SIRT1 phosphorylation is required for energy starvation-induced PABP1-SIRT1 association, PABP1 deacetylation, and poly(A)RNA nuclear retention. In addition, the SIRT1-PABP1 association is not specific to energy starvation but represents a common stress response. These observations provide insights into dynamic modulation of eukaryotic mRNA transport and translation, suggesting that the poly(A)-tail also provides a basis for eukaryotes to effectively shut down mature mRNA transport and thereby tailor protein synthesis to maintain energy homeostasis under stress conditions.

Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

  • Solis GP
  • Cell
  • 2017 Aug 24

Literature context: 31Human epithelial HeLaATCCCat# CCL-2Human neuroblastoma BE(2)CKarim


Abstract:

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions.

  • Chen K
  • Elife
  • 2017 Aug 25

Literature context: DMEM for 24 hr and fixed. HeLa (RRID:CVCL_0030) and MDCK (RRID: CVCL_0422) cel


Abstract:

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins.

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

  • Mackenzie IR
  • Neuron
  • 2017 Aug 16

Literature context: HeLa cells ATCC Cat #CCL-2; RRID:CVCL_0030 U-2 OS cells ATCC Cat #HTB-96;


Abstract:

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 10-6). Postmortem neuropathology of five TIA1 mutations carriers showed a consistent pathological signature with numerous round, hyaline, TAR DNA-binding protein 43 (TDP-43)-positive inclusions. TIA1 mutations significantly increased the propensity of TIA1 protein to undergo phase transition. In live cells, TIA1 mutations delayed stress granule (SG) disassembly and promoted the accumulation of non-dynamic SGs that harbored TDP-43. Moreover, TDP-43 in SGs became less mobile and insoluble. The identification of TIA1 mutations in ALS/FTD reinforces the importance of RNA metabolism and SG dynamics in ALS/FTD pathogenesis.

Funding information:
  • Howard Hughes Medical Institute - R35 NS097974()

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

  • Arduino DM
  • Mol. Cell
  • 2017 Aug 17

Literature context: Models: Cell LinesHeLa cellsATCCCCL-2HEK293T cellsATCCCRL-11268SH-SY5


Abstract:

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.

Funding information:
  • NIMH NIH HHS - P50 MH090963(United States)

Ccdc3: A New P63 Target Involved in Regulation Of Liver Lipid Metabolism.

  • Liao W
  • Sci Rep
  • 2017 Aug 21

Literature context: lls (RRID:CVCL_0030), human keratinocyte HaCat cell


Abstract:

TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, upregulates CCDC3 expression by directly binding to its enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity, but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but conversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance and steatosis formation in transgenic CCDC3 mice on high-fat diet (HFD) by reducing the expression of hepatic PPARγ and its target gene CIDEA as well as other genes involved in de novo lipogenesis. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on HFD. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of the TIM22 Protein Translocase in Mitochondria.

  • Vukotic M
  • Mol. Cell
  • 2017 Aug 3

Literature context: nesHuman: HEK293InvitrogenR75007Human: HeLaATCCCCL-2Human: HEK293T AGK−/−This studyN


Abstract:

Mutations in mitochondrial acylglycerol kinase (AGK) cause Sengers syndrome, which is characterized by cataracts, hypertrophic cardiomyopathy, and skeletal myopathy. AGK generates phosphatidic acid and lysophosphatidic acid, bioactive phospholipids involved in lipid signaling and the regulation of tumor progression. However, the molecular mechanisms of the mitochondrial pathology remain enigmatic. Determining its mitochondrial interactome, we have identified AGK as a constituent of the TIM22 complex in the mitochondrial inner membrane. AGK assembles with TIMM22 and TIMM29 and supports the import of a subset of multi-spanning membrane proteins. The function of AGK as a subunit of the TIM22 complex does not depend on its kinase activity. However, enzymatically active AGK is required to maintain mitochondrial cristae morphogenesis and the apoptotic resistance of cells. The dual function of AGK as lipid kinase and constituent of the TIM22 complex reveals that disturbances in both phospholipid metabolism and mitochondrial protein biogenesis contribute to the pathogenesis of Sengers syndrome.

Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5.

  • Flack JE
  • Mol. Cell
  • 2017 Jul 20

Literature context: ell LinesHEK293TATCCCat#CRL-3216HeLaATCCCat#CCL-2HCT116ATCCCat#CCL-247Experimenta


Abstract:

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription.

Mechanisms of Paradoxical Activation of AMPK by the Kinase Inhibitors SU6656 and Sorafenib.

  • Ross FA
  • Cell Chem Biol
  • 2017 Jul 20

Literature context: ermoFisher ScientificCat# R78007HeLaECACCCat# 93021013Recombinant DNAPlasmid encoding


Abstract:

SU6656, a Src kinase inhibitor, was reported to increase fat oxidation and reduce body weight in mice, with proposed mechanisms involving AMP-activated protein kinase (AMPK) activation via inhibition of phosphorylation of either LKB1 or AMPK by the Src kinase, Fyn. However, we report that AMPK activation by SU6656 is independent of Src kinases or tyrosine phosphorylation of LKB1 or AMPK and is not due to decreased cellular energy status or binding at the ADaM site on AMPK. SU6656 is a potent AMPK inhibitor, yet binding at the catalytic site paradoxically promotes phosphorylation of Thr172 by LKB1. This would enhance phosphorylation of downstream targets provided the lifetime of Thr172 phosphorylation was sufficient to allow dissociation of the inhibitor and subsequent catalysis prior to its dephosphorylation. By contrast, sorafenib, a kinase inhibitor in clinical use, activates AMPK indirectly by inhibiting mitochondrial metabolism and increasing cellular AMP:ADP and/or ADP:ATP ratios.

DGAT1-Dependent Lipid Droplet Biogenesis Protects Mitochondrial Function during Starvation-Induced Autophagy.

  • Nguyen TB
  • Dev. Cell
  • 2017 Jul 10

Literature context: Kopito (Stanford University)N/AHeLaATCCCat. # CCL-2HEK 293T/17ATCCCat. # CRL-11268H


Abstract:

Lipid droplets (LDs) provide an "on-demand" source of fatty acids (FAs) that can be mobilized in response to fluctuations in nutrient abundance. Surprisingly, the amount of LDs increases during prolonged periods of nutrient deprivation. Why cells store FAs in LDs during an energy crisis is unknown. Our data demonstrate that mTORC1-regulated autophagy is necessary and sufficient for starvation-induced LD biogenesis. The ER-resident diacylglycerol acyltransferase 1 (DGAT1) selectively channels autophagy-liberated FAs into new, clustered LDs that are in close proximity to mitochondria and are lipolytically degraded. However, LDs are not required for FA delivery to mitochondria but instead function to prevent acylcarnitine accumulation and lipotoxic dysregulation of mitochondria. Our data support a model in which LDs provide a lipid buffering system that sequesters FAs released during the autophagic degradation of membranous organelles, reducing lipotoxicity. These findings reveal an unrecognized aspect of the cellular adaptive response to starvation, mediated by LDs.

Funding information:
  • NIDDK NIH HHS - R00 DK095921()
  • NIGMS NIH HHS - R01 GM112948()

Viral Replication Complexes Are Targeted by LC3-Guided Interferon-Inducible GTPases.

  • Biering SB
  • Cell Host Microbe
  • 2017 Jul 12

Literature context: t# CCL-2; RRID:CVCL_0030 Human: HeL


Abstract:

All viruses with positive-sense RNA genomes replicate on membranous structures in the cytoplasm called replication complexes (RCs). RCs provide an advantageous microenvironment for viral replication, but it is unknown how the host immune system counteracts these structures. Here we show that interferon-gamma (IFNG) disrupts the RC of murine norovirus (MNV) via evolutionarily conserved autophagy proteins and the induction of IFN-inducible GTPases, which are known to destroy the membrane of vacuoles containing bacteria, protists, or fungi. The MNV RC was marked by the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system of autophagy and then targeted by immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs) upon their induction by IFNG. Further, the LC3 conjugation system and the IFN-inducible GTPases were necessary to inhibit MNV replication in mice and human cells. These data suggest that viral RCs can be marked and antagonized by a universal immune defense mechanism targeting diverse pathogens replicating in cytosolic membrane structures.

Funding information:
  • NIAID NIH HHS - R01 AI103197()

MLKL, the Protein that Mediates Necroptosis, Also Regulates Endosomal Trafficking and Extracellular Vesicle Generation.

  • Yoon S
  • Immunity
  • 2017 Jul 18

Literature context: CRL-7923, RRID:CVCL_0030 Murine Emb


Abstract:

Activation of the pseudokinase mixed lineage kinase domain-like (MLKL) upon its phosphorylation by the protein kinase RIPK3 triggers necroptosis, a form of programmed cell death in which rupture of cellular membranes yields release of intracellular components. We report that MLKL also associated with endosomes and controlled the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of extracellular vesicles. This role was exerted on two quantitative grades: a constitutive one independent of RIPK3, and an enhanced one, triggered by RIPK3, where the association of MLKL with the endosomes was enhanced, and it was found to bind endosomal sorting complexes required for transport (ESCRT) proteins and the flotillins and to be excluded, together with them, from cells within vesicles. We suggest that release of phosphorylated MLKL within extracellular vesicles serves as a mechanism for self-restricting the necroptotic activity of this protein.

Sensing Self and Foreign Circular RNAs by Intron Identity.

  • Chen YG
  • Mol. Cell
  • 2017 Jul 20

Literature context: 1Experimental Models: Cell LinesHeLaATCCCCL-2HEK293TATCCCRL-3216HaCaTATCCPCS-


Abstract:

Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.

How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells.

  • Shemetov AA
  • Cell Chem Biol
  • 2017 Jun 22

Literature context: t# CCL-2; RRID:CVCL_0030 HEK293 ATC


Abstract:

Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher's choice rather difficult. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells. For instance, in common HeLa cells miRFP703, mIFP, and smURFP were 2-, 9-, and 53-fold dimmer than miRFP670. Either co-expression of heme oxygenase or incubation of cells with heme precursor weakly affected NIR fluorescence, however, in the latter case elevated cellular autofluorescence. Exogenously added chromophore substantially increased smURFP brightness but only slightly enhanced brightness of other NIR FPs. mIFP showed intermediate, while monomeric miRFP670 and miRFP703 exhibited high binding efficiency of endogenous biliverdin chromophore. This feature makes them easy to use as GFP-like proteins for spectral multiplexing with FPs of visible range.

Funding information:
  • NIGMS NIH HHS - R35 GM122567()
  • NINDS NIH HHS - U01 NS099573()

Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles.

  • Gupta KH
  • Dev. Cell
  • 2017 Jun 19

Literature context: TCC CCL-2 RRID:CVCL_0030 Human: HEK


Abstract:

Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.

Funding information:
  • NIAID NIH HHS - R21 AI110685()

LTR-Retrotransposon Control by tRNA-Derived Small RNAs.

  • Schorn AJ
  • Cell
  • 2017 Jun 29

Literature context: Hospital, Toronto/Canada)#TS6.5human HeLa cellsATCCCCL-2Oligonucleotidesall oligonucleot


Abstract:

Transposon reactivation is an inherent danger in cells that lose epigenetic silencing during developmental reprogramming. In the mouse, long terminal repeat (LTR)-retrotransposons, or endogenous retroviruses (ERV), account for most novel insertions and are expressed in the absence of histone H3 lysine 9 trimethylation in preimplantation stem cells. We found abundant 18 nt tRNA-derived small RNA (tRF) in these cells and ubiquitously expressed 22 nt tRFs that include the 3' terminal CCA of mature tRNAs and target the tRNA primer binding site (PBS) essential for ERV reverse transcription. We show that the two most active ERV families, IAP and MusD/ETn, are major targets and are strongly inhibited by tRFs in retrotransposition assays. 22 nt tRFs post-transcriptionally silence coding-competent ERVs, while 18 nt tRFs specifically interfere with reverse transcription and retrotransposon mobility. The PBS offers a unique target to specifically inhibit LTR-retrotransposons, and tRF-targeting is a potentially highly conserved mechanism of small RNA-mediated transposon control.

Funding information:
  • NCI NIH HHS - P30 CA045508()
  • NIGMS NIH HHS - R01 GM076396()

The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms.

  • Hu W
  • Elife
  • 2017 Jun 28

Literature context: (RRID:CVCL_0030) cells wer


Abstract:

Adenylyl cyclases (ACs) generate cAMP, a second messenger of utmost importance that regulates a vast array of biological processes in all kingdoms of life. However, almost nothing is known about how AC activity is regulated through protein degradation mediated by ubiquitination or other mechanisms. Here, we show that transcriptional regulator interacting with the PHD-bromodomain 1 (TRIP-Br1, Sertad1), a newly identified protein with poorly characterized functions, acts as an adaptor that bridges the interaction of multiple AC isoforms with X-linked inhibitor of apoptosis protein (XIAP), a RING-domain E3 ubiquitin ligase. XIAP ubiquitinates a highly conserved Lys residue in AC isoforms and thereby accelerates the endocytosis and degradation of multiple AC isoforms in human cell lines and mice. XIAP/TRIP-Br1-mediated degradation of ACs forms part of a negative-feedback loop that controls the homeostasis of cAMP signaling in mice. Our findings reveal a previously unrecognized mechanism for degrading multiple AC isoforms and modulating the homeostasis of cAMP signaling.

Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

  • Nillegoda NB
  • Elife
  • 2017 May 15

Literature context: Culture Collection (ATCC-CCL2; RRID:CVCL_0030) were grown in DMEM (Gibco, The


Abstract:

Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

A Tunable Brake for HECT Ubiquitin Ligases.

  • Chen Z
  • Mol. Cell
  • 2017 May 4

Literature context: ls: Cell LinesHeLa cellATCCATCC CCL-2OligonucleotidesPrimers used in


Abstract:

The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.

Funding information:
  • NCI NIH HHS - P50 CA062924()
  • NCI NIH HHS - R01 CA074305()
  • NCI NIH HHS - R37 CA043460()
  • NIGMS NIH HHS - F32 GM120855()
  • NIGMS NIH HHS - R01 GM034933()
  • NIGMS NIH HHS - R01 GM109102()
  • NIGMS NIH HHS - R35 GM118177()

Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming Cells Exposed to Intrauterine Diabetes Mellitus.

  • Gohn CR
  • J. Cell. Physiol.
  • 2017 May 15

Literature context: t# CCL-2, RRID:CVCL_0030) and Lenti


Abstract:

Diabetes mellitus (DM) during pregnancy has long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin-dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. J. Cell. Physiol. 232: 1885-1892, 2017. © 2016 Wiley Periodicals, Inc.

RhoD Inhibits RhoC-ROCK-Dependent Cell Contraction via PAK6.

  • Durkin CH
  • Dev. Cell
  • 2017 May 8

Literature context: l LinesHuman: HeLa cellsATCCCat#CCL-2Human: U-2 OS cellsThe Francis C


Abstract:

RhoA-mediated regulation of myosin-II activity in the actin cortex controls the ability of cells to contract and bleb during a variety of cellular processes, including cell migration and division. Cell contraction and blebbing also frequently occur as part of the cytopathic effect seen during many different viral infections. We now demonstrate that the vaccinia virus protein F11, which localizes to the plasma membrane, is required for ROCK-mediated cell contraction from 2 hr post infection. Curiously, F11-induced cell contraction is dependent on RhoC and not RhoA signaling to ROCK. Moreover, RhoC-driven cell contraction depends on the upstream inhibition of RhoD signaling by F11. This inhibition prevents RhoD from regulating its downstream effector Pak6, alleviating the suppression of RhoC by the kinase. Our observations with vaccinia have now demonstrated that RhoD recruits Pak6 to the plasma membrane to antagonize RhoC signaling during cell contraction and blebbing.

RPL10L Is Required for Male Meiotic Division by Compensating for RPL10 during Meiotic Sex Chromosome Inactivation in Mice.

  • Jiang L
  • Curr. Biol.
  • 2017 May 22

Literature context: t# CCL-2; RRID:CVCL_0030 U-2 OS ATC


Abstract:

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 [6]. 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.

Kinetics of Endogenous CaMKII Required for Synaptic Plasticity Revealed by Optogenetic Kinase Inhibitor.

  • Murakoshi H
  • Neuron
  • 2017 Apr 5

Literature context: Models: Cell LinesHeLa cellsATCCATCC CCL-2HEK293 cellsATCCATCC CRL-11268Ex


Abstract:

Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.

Funding information:
  • NIMH NIH HHS - R01 MH080047()
  • NIMH NIH HHS - R01 MH111486()
  • NINDS NIH HHS - DP1 NS096787()

Systematic Analysis of Human Protein Phosphatase Interactions and Dynamics.

  • Yadav L
  • Cell Syst
  • 2017 Apr 26

Literature context: man: HeLa cell lineSigma-AldrichCat# 93021013Human: HCT116 cell lineATCCCat#


Abstract:

Coordinated activities of protein kinases and phosphatases ensure phosphorylation homeostasis, which, when perturbed, can instigate diseases, including cancer. Yet, in contrast to kinases, much less is known about protein phosphatase functions and their interactions and complexes. Here, we used quantitative affinity proteomics to assay protein-protein interactions for 54 phosphatases distributed across the three major protein phosphatase families, with additional analysis of their 12 co-factors. We identified 838 high-confidence interactions, of which 631, to our knowledge, have not been reported before. We show that inhibiting the activity of phosphatases PP1 and PP2A by okadaic acid disrupts their specific interactions, supporting the potential of therapeutics that target these proteins. Additional analyses revealed candidate physical and functional interaction links to phosphatase-based regulation of several signaling pathways and to human cancer. Our study provides an initial glimpse of the protein interaction landscape of phosphatases and their functions in cellular regulation.

Funding information:
  • NIMH NIH HHS - R01 MH080420-05(United States)

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.

  • Legnini I
  • Mol. Cell
  • 2017 Apr 6

Literature context: (DMD)Telethon BiobankN/AHeLaATCCCat# CCL-2N2AATCCCat# CCL-131Mouse embryon


Abstract:

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.

Funding information:
  • Telethon - GGP16213()

A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis.

  • Treiber T
  • Mol. Cell
  • 2017 Apr 20

Literature context: CRL-3216human: HeLaour labATCC® CCL-2human: A549ATCCATCC® CCL-185huma


Abstract:

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.

A Metabolic Function for Phospholipid and Histone Methylation.

  • Ye C
  • Mol. Cell
  • 2017 Apr 20

Literature context: RL-11268Human: Hep G2ATCCHB-8065Human: HeLaATCCCCL-2Experimental Models: Or


Abstract:

S-adenosylmethionine (SAM) is the methyl donor for biological methylation modifications that regulate protein and nucleic acid functions. Here, we show that methylation of a phospholipid, phosphatidylethanolamine (PE), is a major consumer of SAM. The induction of phospholipid biosynthetic genes is accompanied by induction of the enzyme that hydrolyzes S-adenosylhomocysteine (SAH), a product and inhibitor of methyltransferases. Beyond its function for the synthesis of phosphatidylcholine (PC), the methylation of PE facilitates the turnover of SAM for the synthesis of cysteine and glutathione through transsulfuration. Strikingly, cells that lack PE methylation accumulate SAM, which leads to hypermethylation of histones and the major phosphatase PP2A, dependency on cysteine, and sensitivity to oxidative stress. Without PE methylation, particular sites on histones then become methyl sinks to enable the conversion of SAM to SAH. These findings reveal an unforeseen metabolic function for phospholipid and histone methylation intrinsic to the life of a cell.

Funding information:
  • NCI NIH HHS - P30 CA142543()
  • NIGMS NIH HHS - R01 GM094314()

The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover.

  • Tawo R
  • Cell
  • 2017 Apr 20

Literature context: ells (female))Sigma-AldrichCat# 93021013Experimental Models: Organisms/S


Abstract:

Aging is attended by a progressive decline in protein homeostasis (proteostasis), aggravating the risk for protein aggregation diseases. To understand the coordination between proteome imbalance and longevity, we addressed the mechanistic role of the quality-control ubiquitin ligase CHIP, which is a key regulator of proteostasis. We observed that CHIP deficiency leads to increased levels of the insulin receptor (INSR) and reduced lifespan of worms and flies. The membrane-bound INSR regulates the insulin and IGF1 signaling (IIS) pathway and thereby defines metabolism and aging. INSR is a direct target of CHIP, which triggers receptor monoubiquitylation and endocytic-lysosomal turnover to promote longevity. However, upon proteotoxic stress conditions and during aging, CHIP is recruited toward disposal of misfolded proteins, reducing its capacity to degrade the INSR. Our study indicates a competitive relationship between proteostasis and longevity regulation through CHIP-assisted proteolysis, providing a mechanistic concept for understanding the impact of proteome imbalance on aging.

Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels.

  • Song L
  • Elife
  • 2017 Mar 31

Literature context: ., 2014). HeLa (Cat#ATCC-CCL-2, RRID:CVCL_0030), MDA-MB231 (Cat#ATCC-HTB-26, C


Abstract:

Small molecule inhibitors of site-specific O-glycosylation by the polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) family are currently unavailable but hold promise as therapeutics, especially if selective against individual ppGalNAc-T isozymes. To identify a compound targeting the ppGalNAc-T3 isozyme, we screened libraries to find compounds that act on a cell-based fluorescence sensor of ppGalNAc-T3 but not on a sensor of ppGalNAc-T2. This identified a hit that subsequent in vitro analysis showed directly binds and inhibits purified ppGalNAc-T3 with no detectable activity against either ppGalNAc-T2 or ppGalNAc-T6. Remarkably, the inhibitor was active in two medically relevant contexts. In cell culture, it opposed increased cancer cell invasiveness driven by upregulated ppGalNAc-T3 suggesting the inhibitor might be anti-metastatic. In cells and mice, it blocked ppGalNAc-T3-mediated glycan-masking of FGF23 thereby increasing its cleavage, a possible treatment of chronic kidney disease. These findings establish a pharmacological approach for the ppGalNAc-transferase family and suggest that targeting specific ppGalNAc-transferases will yield new therapeutics.

Funding information:
  • NIDCR NIH HHS - R21 DE026714(United States)
  • NIGMS NIH HHS - P20 GM103648(United States)

Imaging ERK and PKA Activation in Single Dendritic Spines during Structural Plasticity.

  • Tang S
  • Neuron
  • 2017 Mar 22

Literature context:


Abstract:

Extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) play important roles in LTP and spine structural plasticity. While fluorescence resonance energy transfer (FRET)-based sensors for these kinases had previously been developed, they did not provide sufficient sensitivity for imaging small neuronal compartments, such as single dendritic spines in brain slices. Here we improved the sensitivity of FRET-based kinase sensors for monitoring kinase activity under two-photon fluorescence lifetime imaging microscopy (2pFLIM). Using these improved sensors, we succeeded in imaging ERK and PKA activation in single dendritic spines during structural long-term potentiation (sLTP) in hippocampal CA1 pyramidal neurons, revealing that the activation of these kinases spreads widely with length constants of more than 10 μm. The strategy for improvement of sensors used here should be applicable for developing highly sensitive biosensors for various protein kinases. VIDEO ABSTRACT.

Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.

  • Boeynaems S
  • Mol. Cell
  • 2017 Mar 16

Literature context: ental Models: Cell LinesHeLaATCCCCL-2MEF eIF2a SSDr. Nancy KedershaEm


Abstract:

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Funding information:
  • European Research Council - 647458()
  • NIGMS NIH HHS - P20 GM104937()
  • NIGMS NIH HHS - R01 GM111700()
  • NIGMS NIH HHS - R01 GM118530()
  • NIMH NIH HHS - T32 MH020068()

Palmitoylated SCP1 is targeted to the plasma membrane and negatively regulates angiogenesis.

  • Liao P
  • Elife
  • 2017 Mar 31

Literature context:


Abstract:

SCP1 as a nuclear transcriptional regulator acts globally to silence neuronal genes and to affect the dephosphorylation of RNA Pol ll. However, we report the first finding and description of SCP1 as a plasma membrane-localized protein in various cancer cells using EGFP- or other epitope-fused SCP1. Membrane-located SCP1 dephosphorylates AKT at serine 473, leading to the abolishment of serine 473 phosphorylation that results in suppressed angiogenesis and a decreased risk of tumorigenesis. Consistently, we observed increased AKT phosphorylation and angiogenesis followed by enhanced tumorigenesis in Ctdsp1 (which encodes SCP1) gene - knockout mice. Importantly, we discovered that the membrane localization of SCP1 is crucial for impeding angiogenesis and tumor growth, and this localization depends on palmitoylation of a conserved cysteine motif within its NH2 terminus. Thus, our study discovers a novel mechanism underlying SCP1 shuttling between the plasma membrane and nucleus, which constitutes a unique pathway in transducing AKT signaling that is closely linked to angiogenesis and tumorigenesis.

p27Kip1 promotes invadopodia turnover and invasion through the regulation of the PAK1/Cortactin pathway.

  • Jeannot P
  • Elife
  • 2017 Mar 13

Literature context: on. HeLa (RRID:CVCL_0030), HEK 293


Abstract:

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.

Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk.

  • Galarza-Muñoz G
  • Cell
  • 2017 Mar 23

Literature context: 0Experimental Models: Cell LinesHuman: HeLa cell lineDuke University Cell Culture Cor


Abstract:

Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Funding information:
  • NIEHS NIH HHS - R01 ES017080()
  • NINDS NIH HHS - F32 NS087899()
  • NINDS NIH HHS - R01 NS060925()

YOD1/TRAF6 association balances p62-dependent IL-1 signaling to NF-κB.

  • Schimmack G
  • Elife
  • 2017 Feb 28

Literature context: HeLa (RRID:CVCL_0030), HEK-293


Abstract:

The ubiquitin ligase TRAF6 is a key regulator of canonical IκB kinase (IKK)/NF-κB signaling in response to interleukin-1 (IL-1) stimulation. Here, we identified the deubiquitinating enzyme YOD1 (OTUD2) as a novel interactor of TRAF6 in human cells. YOD1 binds to the C-terminal TRAF homology domain of TRAF6 that also serves as the interaction surface for the adaptor p62/Sequestosome-1, which is required for IL-1 signaling to NF-κB. We show that YOD1 competes with p62 for TRAF6 association and abolishes the sequestration of TRAF6 to cytosolic p62 aggregates by a non-catalytic mechanism. YOD1 associates with TRAF6 in unstimulated cells but is released upon IL-1β stimulation, thereby facilitating TRAF6 auto-ubiquitination as well as NEMO/IKKγ substrate ubiquitination. Further, IL-1 triggered IKK/NF-κB signaling and induction of target genes is decreased by YOD1 overexpression and augmented after YOD1 depletion. Hence, our data define that YOD1 antagonizes TRAF6/p62-dependent IL-1 signaling to NF-κB.

A conformational switch regulates the ubiquitin ligase HUWE1.

  • Sander B
  • Elife
  • 2017 Feb 14

Literature context: HeLa (RRID:CVCL_0030) and HEK29


Abstract:

The human ubiquitin ligase HUWE1 has key roles in tumorigenesis, yet it is unkown how its activity is regulated. We present the crystal structure of a C-terminal part of HUWE1, including the catalytic domain, and reveal an asymmetric auto-inhibited dimer. We show that HUWE1 dimerizes in solution and self-associates in cells, and that both occurs through the crystallographic dimer interface. We demonstrate that HUWE1 is inhibited in cells and that it can be activated by disruption of the dimer interface. We identify a conserved segment in HUWE1 that counteracts dimer formation by associating with the dimerization region intramolecularly. Our studies reveal, intriguingly, that the tumor suppressor p14ARF binds to this segment and may thus shift the conformational equilibrium of HUWE1 toward the inactive state. We propose a model, in which the activity of HUWE1 underlies conformational control in response to physiological cues-a mechanism that may be exploited for cancer therapy.

Mutant KRAS Enhances Tumor Cell Fitness by Upregulating Stress Granules.

  • Grabocka E
  • Cell
  • 2016 Dec 15

Literature context: Cat#CCL2; RRID:CVCL_0030 Experiment


Abstract:

There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation of cytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.

Funding information:
  • NCI NIH HHS - F32 CA139922()
  • NCI NIH HHS - P30 CA016087()
  • NCI NIH HHS - R01 CA055360()

IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact.

  • Bonneau B
  • Elife
  • 2016 Dec 20

Literature context: La cells (RRID:CVCL_0030) were obta


Abstract:

IRBIT is a molecule that interacts with the inositol 1,4,5-trisphosphate (IP3)-binding pocket of the IP3 receptor (IP3R), whereas the antiapoptotic protein, Bcl2l10, binds to another part of the IP3-binding domain. Here we show that Bcl2l10 and IRBIT interact and exert an additive inhibition of IP3R in the physiological state. Moreover, we found that these proteins associate in a complex in mitochondria-associated membranes (MAMs) and that their interplay is involved in apoptosis regulation. MAMs are a hotspot for Ca2+ transfer between endoplasmic reticulum (ER) and mitochondria, and massive Ca2+ release through IP3R in mitochondria induces cell death. We found that upon apoptotic stress, IRBIT is dephosphorylated, becoming an inhibitor of Bcl2l10. Moreover, IRBIT promotes ER mitochondria contact. Our results suggest that by inhibiting Bcl2l10 activity and promoting contact between ER and mitochondria, IRBIT facilitates massive Ca2+ transfer to mitochondria and promotes apoptosis. This work then describes IRBIT as a new regulator of cell death.

Funding information:
  • Howard Hughes Medical Institute - R01 NS036715(United States)
  • NCATS NIH HHS - UL1 TR001105(United States)

Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells.

  • Zhong G
  • Elife
  • 2016 Nov 2

Literature context: oma HeLa (RRID:CVCL_0030) cells wer


Abstract:

Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes's activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules.

Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

  • Berger S
  • Elife
  • 2016 Nov 2

Literature context: rom ATCC, RRID:CVCL_0030) were gene


Abstract:

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

Funding information:
  • NIDDK NIH HHS - R21 DK106584(United States)

Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes.

  • Pylypenko O
  • Elife
  • 2016 Sep 13

Literature context: t# CCL-2, RRID:CVCL_0030) were cult


Abstract:

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.

Funding information:
  • NIDCD NIH HHS - R01DC009236(United States)

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

  • Zhang X
  • Cell
  • 2016 Aug 25

Literature context: nesMouse: Neuro2aATCCCat#CCL-131Human: HeLaATCCCat#CCL-2Human: U2OSATCCCat#HTB-96Human:


Abstract:

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability.

  • Kang Y
  • Elife
  • 2016 Aug 24

Literature context: cells (RRID:CVCL_0030); kindly p


Abstract:

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it's C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.

NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.

  • Moutel S
  • Elife
  • 2016 Jul 19

Literature context: La cells (RRID:CVCL_0030) used in t


Abstract:

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.

Centriole distal appendages promote membrane docking, leading to cilia initiation.

  • Tanos BE
  • Genes Dev.
  • 2013 Jan 15

Literature context: penicillin–streptomycin. 293T, HeLa, and U2OS cells were cultured i


Abstract:

The distal appendages (DAPs) of centrioles have been proposed to anchor cilia to the plasma membrane, but their molecular composition, assembly, and exact function in ciliogenesis remain poorly understood. Using quantitative centrosome proteomics and superresolution microscopy, we identified five DAP components, including one previously described (CEP164), one partially characterized (CEP89 [ccdc123]), and three novel (CEP83 [ccdc41], SCLT1, and FBF1) DAP proteins. Analyses of DAP assembly revealed a hierarchy. CEP83 recruits both SCLT1 and CEP89 to centrioles. Subsequent recruitment of FBF1 and CEP164 is independent of CEP89 but mediated by SCLT1. All five DAP components are essential for ciliogenesis; loss of CEP83 specifically blocks centriole-to-membrane docking. Undocked centrioles fail to recruit TTBK2 or release CP110, the two earliest modifications found on centrioles prior to cilia assembly, revealing centriole-to-membrane docking as a temporal and spatial cue promoting cilia initiation.

Funding information:
  • NCI NIH HHS - P30 CA008748()
  • NCRR NIH HHS - S10 RR031855()
  • NCRR NIH HHS - S10RR031855()
  • NIGMS NIH HHS - F32 GM096558()
  • NIGMS NIH HHS - GM088253()
  • NIGMS NIH HHS - GM096558()
  • NIGMS NIH HHS - R01 GM088253()
  • NINDS NIH HHS - R21 NS054143(United States)

Mitofusin 1 is degraded at G2/M phase through ubiquitylation by MARCH5.

  • Park YY
  • Cell Div
  • 2012 Dec 20

Literature context: sion in detail, we synchronized HeLa cells at G1/S using the double-


Abstract:

BACKGROUND: Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated. FINDINGS: Here, we report that mitochondria were found to be fragmented in G2 phase prior to mitotic entry. Mitofusin 1 (Mfn1), a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G2/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G2/M phase. CONCLUSIONS: Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G2/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

Funding information:
  • NIDCR NIH HHS - R01 DE-015945(United States)