INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling
- Boston Children’s Hospital, United States
- Harvard Medical School, United States
- Yale University, United States
- Novartis Institutes for Biomedical Research, United States
- Massachusetts General Hospital, United States
Figures
Figure 1 with 1 supplement
INAVA localizes to cell-cell junctions.
(A) Domain architecture of the long and short INAVA isoforms.(B) Brightfield images of Caco2BBe WT, INAVA-GFP over-expressing, and INAVA CRISPR knockout cells grown 2 days on coverslips. Scalebar = 2…
Figure 1—figure supplement 1
INAVA is important for barrier function in epithelial Caco2BBe cells.
(A) Verification of Caco2BBe INAVA knockdown by qPCR. (B) Genomic DNA PCR of INAVA locus in Caco2BBe WT and INAVA exon deleted CRISPR knockout lines. (C) Top, brightfield images of control or INAVA …
Figure 2 with 1 supplement
INAVA recruits ARNO to cell-cell junctions to promote GEF-independent actin assembly.
(A) GST pulldowns with recombinant GST-INAVA-CUPID domain and His-MBP-CC (coiled-coil) of cytohesin members (see also Figure 2—figure supplement 1C). (B) Immunoblot of Caco2BBe stably transduced …
Figure 2—figure supplement 1
Cytohesins interact with INAVA to Promote GEF Independent Actin Assembly.
(A) Protein interaction Platform (PIP) Assay in Yeast. ARNO full length, CC-SEC7 and CC domain constructs were fused to YFP, and INAVA was fused to μNS, and transformed in yeast. Yeast were mated …
Figure 3 with 1 supplement
INAVA is an activator of IL-1β signaling and is inhibited by ARNO.
(A and B) Confocal images of (A) Caco2BBe and (B) HCT8 or HCT8 TRAF6 KO cells stably expressing INAVA-GFP long isoform treated with or without 10 ng/ml IL-1β for 30 min. Graph display quantification …
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Figure 3—source data 1
Source Data for Figure 3C.
- https://doi.org/10.7554/eLife.38539.007
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Figure 3—source data 2
Source Data for Figure 3D.
- https://doi.org/10.7554/eLife.38539.008
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Figure 3—source data 3
Source Data for Figure 3E.
- https://doi.org/10.7554/eLife.38539.009
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Figure 3—source data 4
Source Data for Figure 3F.
- https://doi.org/10.7554/eLife.38539.010
Figure 3—figure supplement 1
INAVA-GFP puncta formation is relatively less in HCT8 cells stimulated with TNFα.
(A) Verification of HCT8 TRAF6 knockout cells. Immunoblot of p65 activation in HCT8 wildtype and TRAF6 knockout cells treated with IL-1β (10 ng/ml) for 15 min. (B) Quantification of %positive GFP …
Figure 4 with 1 supplement
IL-1β-initiated NFκB and MAPK signaling and cytokine secretion is decreased in MDMs from INAVA rs7554511 CC risk carriers.
(A–C) MDMs from rs7554511 AA (INAVA high-expressing) and CC (INAVA low-expressing) (n = 10/genotype) carriers were treated with 10 ng/ml IL-1β. (A) Summary graph of INAVA expression as assessed by …
Figure 4—figure supplement 1
ARNO inhibits INAVA-dependent NOD2-induced signaling and cytokine secretion.
(A–D) MDMs from rs7554511 CC homozygote (low-expressing) INAVA carriers were transfected with empty vector (EV) or the indicated HA-INAVA constructs. (A) Representative flow cytometry of INAVA …
Figure 5
INAVA enhances TRAF6 dependent polyubiquitination in cells and in vitro.
(A) HEK293T cells were transfected with HA-Ubiquitin and with or without myc-TRAF6. Whole cell lysates were prepared in RIPA buffer with protease inhibitors and analyzed by immunoblot for HA …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rat anti- Ecadherin | Sigma | U3254 | (1:1000) |
| Antibody | Rat anti-HA | Sigma | 11867423001 | (1:1000) |
| Antibody | Rabbit anti-HA | Cell Signaling | C29F4 | (1:1000) |
| Antibody | Rabbit anti-GFP | Sigma | G1544 | (1:1000) |
| Antibody | Mouse anti-b-actin | Sigma | A5441 | (1:5000) |
| Antibody | Mouse anti-ARNO | Sigma | SAB1404698 | (1:1000) |
| Antibody | anti-mouse HRP | Sigma | A4416 | (1:500) |
| Antibody | anti-rabbit HRP | Sigma | A6154 | (1:500) |
| Antibody | anti-rat HRP | Sigma | A9037 | (1:500) |
| Antibody | Rabbit anti-p65 | Cell Signaling | 3033 | (1:1000) |
| Antibody | Rabbit anti-phospho- p65 | Cell Signaling | 4764 | (1:1000) |
| Antibody | Mouse anti-myc | Cell Signaling | 9B11 | (1:1000) |
| Antibody | Mouse phospho-ERK | Cell Signaling | E10 | (1:1000) |
| Antibody | Mouse phospho-p38 | Cell Signaling | 3D7 | (1:1000) |
| Antibody | Mouse phospho-JNK | Cell Signaling | G9 | (1:1000) |
| Antibody | Rabbit phospho-IkBa | Cell Signaling | 14D4 | (1:1000) |
| Antibody | Mouse anti-Traf6 | Santa Cruz | sc-8409 | (1:500) |
| Antibody | DRAQ5 | ThermoFisher | 62251 | (1:1000) |
| Antibody | anti-mouse Alexa-647 | ThermoFisher | A21237 | (1:500) |
| Antibody | Mouse anti-ZO-1 | Thermo Fisher | 339100 | (1:1000) |
| Antibody | Mouse AntiT-TNF | BD Bioscience | MAb1; MAb11 | (1:1000) |
| Antibody | Rat anti- IL6 | BD Bioscience | MQ2-13A5; MQ2-39C3 | (1:1000) |
| Antibody | Mouse anti-IL8 | BD Bioscience | G265-5; G265-8 | (1:1000) |
| Antibody | Rat anti- IL10 | BD Bioscience | JES3-9D7; JES3-12G8 | (1:1000) |
| Antibody | Mouse anti-IL-1b | Thermo Fisher | CRM56; CRM57 | (1:1000) |
| Antibody | Mouse anti-IL12 | Thermo Fisher | C8.3; C8.6 | (1:1000) |
| Antibody | Rabbit anti-INAVA | Abcam | ab121945 | (1:1000) |
| Cell Line (Homo sapiens) | HEK293T | ATCC | ||
| Cell Line (Homo sapiens) | MCF7 | ATCC | ||
| Cell Line (Homo sapiens) | Caco2BBe (C2BBe1) | ATCC | ||
| Cell Line (Homo sapiens) | HCT8 | ATCC | ||
| Software, algorithm | ImageJ | ImageJ (https://i magej.nih.gov/ij/) | ||
| Software, algorithm | GraphPad Prism | Graphpad Prism (https://www.graphpad .com/scientific- software/prism/) | ||
| Software, algorithm | Slidebook | Intelligent Imaging Innovations (https://www.inte lligent-imaging.com/) | ||
| Recombinant DNA reagent | INAVA | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | Cytohesin1 | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | ARNO | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | GRP1 | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | Cytohesin 4 | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | TRAF6 | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | pHAGE-CMV-MCS- IRES-ZsGreen | Harvard Plasmid Repository | ||
| Recombinant DNA reagent | pGEXTEV | Kim Orth, UT Southwestern | ||
| Recombinant DNA reagent | pLVX-Puro | Clontech | ||
| Recombinant DNA reagent | pLVX-EF1α- AcGFP-N1 | Clontech | ||
| Recombinant DNA reagent | pLVX-EF1α -AcGFP-C1 | Clontech | ||
| Recombinant DNA reagent | pET24a | Clontech | ||
| Recombinant DNA reagent | pET28a | Clontech | ||
| Recombinant DNA reagent | pAG413 Gal-YFP | Cammie Lesser, Mass Gen. Hospital | ||
| Recombinant DNA reagent | pAG413Gal- mcherry | Cammie Lesser, Mass Gen. Hospital | ||
| Recombinant DNA reagent | pag413Gal-μNS | Cammie Lesser, Mass Gen. Hospital | ||
| Recombinant DNA reagent | pTY-shRNA- EF1a-Puro2a-GFP | Yi Zhang, Harvard Medical School | ||
| Recombinant DNA reagent | SV40- Renilla | Promega | ||
| Recombinant DNA reagent | NF-κB luciferase | Jonathan Kagan, Boston Children's Hospital | ||
| Recombinant DNA reagent | pCW57.1 | Addgene, gift from David Root | ||
| Recombinant DNA reagent | psPAX2 | Addgene, gift from Didier Trono | ||
| Recombinant DNA reagent | pCMV-VSVG | Addgene, gift from Bob Weinberg | ||
| Recombinant DNA reagent | UBE1 | Addgene, gift from Cynthia Wolberger | ||
| Recombinant DNA reagent | UBC13 | Addgene, gift from Cynthia Wolberger | ||
| Recombinant DNA reagent | UEV1a | Addgene, gift from Cheryl Arrowsmith | ||
| Recombinant DNA reagent | plentiC RISPRv2 | Addgene, gift from Feng Zhang | ||
| Recombinant DNA reagent | pcDNA3.0 | ThermoFisher | ||
| Biological sample (Homo sapien) | Human Peripheral Blood Mononuclear Cells | this study | Donors recruited at Yale University | |
| Peptide, recombinant protein | UbiquitinΔ 2G-HA-His6 | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | His-ΔN17ARF1 | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | GST-INAVA-CUPID | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | His-MBP- INAVA-CUPID | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | UBC13 | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | UEV1a | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | His-Cytohesin- CC-SEC7 | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | Traf6-R ING-His6 | this study | Recombinantly expressed in house | |
| Peptide, recombinant protein | ubiquitin | Boston Biochem | ||
| Chemical compound, drug | MANT-GTP | Jena Bioscience | ||
| Chemical compound, drug | TRITC-phalloidin | American Peptide | 92014A | |
| Sequence- based reagent | gRNAs | this study | Recombinantly expressed in house | |
| Sequence- based reagent | shRNAs | this study | Recombinantly expressed in house | |
| Sequence- based reagent | qPCR-primers | this study | Recombinantly expressed in house | |
| Sequence- based reagent | genotyping primers | this study | Recombinantly expressed in house | |
| Strain, strain background (Escherichia coli) | Rosetta2D E3pLyS | EMD Millipore | ||
| Strain, strain background (E.coli) | Shuffle T7 Express | New England Biolabs | ||
| Other | HiTrap QHP | GE Healthcare | ||
| Other | Superdex 200 10/300 GL | GE Healthcare |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.38539.015
