lifeact  (Addgene inc)

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: ( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing miniTurbo-tagged Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .

Article Snippet: The cDNA encoding miniTurbo-tagged Itga5 was generated by fusing the miniTurbo tag (a gift from Alice Ting, Addgene plasmid # 107168) in frame to the 3′ end of the human Itag5 cDNA (a gift from Rick Horwitz, Addgene plasmid # 15238) and subsequently cloned into pRetroQ-N1 vector.

Techniques: CRISPR, Expressing, Transduction, Flow Cytometry, Software, Clone Assay, Comparison, Control, Stable Transfection

Journal: Computational and Structural Biotechnology Journal

Article Title: Family-wide analysis of integrin structures predicted by AlphaFold2

doi: 10.1016/j.csbj.2023.09.022

Figure Lengend Snippet: AlphaFold2 structures of α 10 and β 1 integrins from different species . ( A ) Structures of α 10 integrins from human, mouse, rat, and zebrafish. The structures were aligned based on the calf-2 domain and oriented perpendicularly to the cell membrane. The N-glycan site at the thigh/calf-1 interface is shown as a red stick. ( B ) Relative positions of α 10 thigh and calf-1 domains in the fully bent conformation, illustrating the interfacial location of the N-glycan site shown as red sticks. The thigh and calf-1 domains of α 10 were superimposed on those of bent α IIb integrin structure. ( C ) Structures of β 1 integrins from human, mouse, cat, and chicken. The structures were aligned based on the βI domain and oriented perpendicularly to the cell membrane. ( D ) Conformation of β 1 integrin co-expressed with selected integrin α subunit. Human integrin α subunits with a C-terminal EGFP tag were co-expressed with human β 1 in 293T cells. The binding of mAb 9EG7 or MAR4 was measured by flow cytometry in a buffer containing 1 mM Ca 2+ /Mg 2+ or 0.1 mM Ca 2+ plus 2 mM Mn 2+ . The data are presented as the MFI of 9EG7 binding as a percentage of the MFI of MAR4 binding.

Article Snippet: The α 5 with C-terminal EGFP tag (α 5 -EGFP) was a gift from Rick Horwitz (Addgene plasmid #15238; http://n2t.net/addgene:15238; RRID:Addgene_15238) .

Techniques: Membrane, Glycoproteomics, Binding Assay, Flow Cytometry