OpenFreezer: a reagent information management software system

Olhovsky, Marina; Williton, Kelly; Dai, Anna Yue; Pasculescu, Adrian; Lee, John Paul; Goudreault, Marilyn; Wells, Clark D.; Park, Jin Gyoon; Gingras, Anne‐Claude; Linding, Rune; Pawson, Tony; Colwill, Karen

doi:10.1038/nmeth.1658

Cited by 23 publications

(21 citation statements)

References 2 publications

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“…Entry clones for HIST1H2BG (accession # EU446968), HIST1H3A (accession # HQ448409), MED4 (accession # DQ893076), MED20 (accession # BC012618) and MED23 (accession # EU832308) were obtained from the ORFeome collection (http://horfdb.dfci.harvard.edu/) [18, 19], archived at the Lunenfeld-Tanenbaum using OpenFreezer [20]. Bait proteins of interest were stably expressed in T-REx Flp-In HEK293 cells as described [17].…”

Section: Methodsmentioning

confidence: 99%

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

Lambert

Tucholska

et al. 2015

Journal of Proteomics

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254

269

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Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes.

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Section: Methodsmentioning

confidence: 99%

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

Lambert

Tucholska

et al. 2015

Journal of Proteomics

Self Cite

254

269

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“…Further processing of the embryos and ISH were carried out as described (Wilkinson and Nieto, 1993). Riboprobes were obtained from Open Freezer at the Lunenfeld-Tanenbaum Research Institute (Toronto, Canada) (Olhovsky et al, 2011).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Yap and Taz are required for Ret-dependent urinary tract morphogenesis

et al. 2015

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Despite the high occurrence of congenital abnormalities of the lower urinary tract in humans, the molecular, cellular and morphological aspects of their development are still poorly understood. Here, we use a conditional knockout approach to inactivate within the nephric duct (ND) lineage the two effectors of the Hippo pathway, Yap and Taz. Deletion of Yap leads to hydronephrotic kidneys with blind-ending megaureters at birth. In Yap mutants, the ND successfully migrates towards, and contacts, the cloaca. However, close analysis reveals that the tip of the Yap −/− ND forms an aberrant connection with the cloaca and does not properly insert into the cloaca, leading to later detachment of the ND from the cloaca. Taz deletion from the ND does not cause any defect, but analysis of Yap −/− ;Taz −/− NDs indicates that both genes play partially redundant roles in ureterovesical junction formation. Aspects of the Yap −/− phenotype resemble hypersensitivity to RET signaling, including excess budding of the ND, increased phospho-ERK and increased expression of Crlf1, Sprouty1, Etv4 and Etv5. Importantly, the Yap ND−/− ND phenotype can be largely rescued by reducing Ret gene dosage. Taken together, these results suggest that disrupting Yap/Taz activities enhances Ret pathway activity and contributes to pathogenesis of lower urinary tract defects in human infants.

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“…Some Flag-tagged constructs were previously created and obtained directly as outlined in Table S5. For other cDNA, vectors were obtained and used as a template to clone genes into the gateway entry vector pDONR223 for subsequent epitope tagging; cDNA templates or entry clones were obtained from OpenFreezer (Olhovsky et al, 2011) or Addgene and primers used for this cloning are listed in Table S3. Llgl2 template cDNA ( pEGFP2-LGL2) was obtained from William Nelson (Stanford, CA).…”

Section: Constructs and Sirnasmentioning

confidence: 99%

The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin

Campbell

Samavarchi-Tehrani

Barrios‐Rodiles

et al. 2016

Journal of Cell Science

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The Crumbs complex is an important determinant of epithelial apicalbasal polarity that functions in regulation of tight junctions, resistance to epithelial-to-mesenchymal transitions and as a tumour suppressor. Although the functional role of the Crumbs complex is being elucidated, its regulation is poorly understood. Here, we show that suppression of RNF146, an E3 ubiquitin ligase that recognizes ADP-ribosylated substrates, and tankyrase, a poly(ADP-ribose) polymerase, disrupts the junctional Crumbs complex and disturbs the function of tight junctions. We show that RNF146 binds a number of polarity-associated proteins, in particular members of the angiomotin (AMOT) family. Accordingly, AMOT proteins are ADPribosylated by TNKS2, which drives ubiquitylation by RNF146 and subsequent degradation. Ablation of RNF146 or tankyrase, as well as overexpression of AMOT, led to the relocation of PALS1 (a Crumbs complex component) from the apical membrane to internal puncta, a phenotype that is rescued by AMOTL2 knockdown. We thus reveal a new function of RNF146 and tankyrase in stabilizing the Crumbs complex through downregulation of AMOT proteins at the apical membrane.

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OpenFreezer: a reagent information management software system

Cited by 23 publications

References 2 publications

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

Yap and Taz are required for Ret-dependent urinary tract morphogenesis

The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin

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