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. 2009 Mar 6;284(10):6486-94.
doi: 10.1074/jbc.M807547200. Epub 2009 Jan 3.

Retinoic Acid-Induced gene-1 (RIG-I) Associates With the Actin Cytoskeleton via Caspase Activation and Recruitment Domain-Dependent Interactions

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Free PMC article

Retinoic Acid-Induced gene-1 (RIG-I) Associates With the Actin Cytoskeleton via Caspase Activation and Recruitment Domain-Dependent Interactions

Amitava Mukherjee et al. J Biol Chem. .
Free PMC article

Abstract

The actin cytoskeleton serves as a barrier that protects mammalian cells from environmental pathogens such as bacteria, fungi, and viruses. Several components of antimicrobial signaling pathways have been shown to associate directly with the actin cytoskeleton, indicating that the cytoskeleton may also serve as a platform for immune-associated molecules. Here we report that retinoic acid-induced gene-I (RIG-I), an important viral RNA recognition molecule, is associated with the actin cytoskeleton and localizes predominantly to actin-enriched membrane ruffles in non-polarized epithelial cells. Subcellular localization and fractionation experiments revealed that the association between RIG-I and the actin cytoskeleton was mediated by its N-terminal caspase activation and recruitment domains (CARDs), which were necessary and sufficient to induce cytoskeletal association. We also show that RIG-I plays a role in cellular migration, as ectopic expression of RIG-I enhanced cellular migration in a wound healing assay and depletion of endogenous RIG-I significantly reduced wound healing. We further show that in both cultured intestinal epithelial cells (IEC) and human colon and small intestine biopsies, RIG-I is enriched at apico-lateral cell junctions and colocalizes with markers of the tight junction. Depolymerization of the actin cytoskeleton in polarized IEC led to the rapid relocalization of RIG-I and to the induction of type I interferon signaling. These data provide evidence that RIG-I is associated with the actin cytoskeleton in non-polarized epithelial cells and with the junctional complex in polarized IECs and human intestine and colon biopsies and may point to a physiological role for RIG-I in the regulation of cellular migration.

Figures

FIGURE 1.
FIGURE 1.
RIG-I localizes to actin-enriched membrane ruffles in HEK293 and U2OS cells. A and B, HEK293 (A) or U2OS (B) cells were fixed and stained with phalloidin to detect actin (red) and with anti-RIG-I goat polyclonal antibody (green). C, HEK293 cells were subjected to cellular fractionation as described under “Experimental Procedures” to obtain cytosolic, membrane/organelle, nuclear, and cytoskeletal fractions. Isolated fractions were subjected to immunoblot analysis for RIG-I (top). Membranes were stripped and reprobed for calpain-2 (middle) and vimentin (bottom) to confirm the purity of cytosolic and cytoskeletal fractions, respectively. D and E, HEK293 cells were transfected with EGFP-RIG-I and fixed and stained for actin (red). F, HEK293 cells were transfected with EGFP-RIG-I and cellular fractionation was performed. Fractions were immunoblotted for GFP (top), calpain-2 (middle), and vimentin (bottom). Data are representative of experiments performed at least three times.
FIGURE 2.
FIGURE 2.
RIG-I is relocalized by Rac inhibition and phorbol 12-myristate 13-acetate (PMA) treatment. HEK293 cells were exposed to the specific (A) Rac inhibitor (200 μm, NSC23766) or (B) phorbol 12-myristate 13-acetate (2 μm) for 2 h and then stained for endogenous RIG-I (green) and actin (red).
FIGURE 3.
FIGURE 3.
The CARDs of RIG-I are necessary and sufficient for lamellipodia localization. A, schematic of EGFP-tagged wild-type and mutant RIG-I proteins. Numbers represent amino acid residues in RIG-I. B, confocal micrographs of U2OS cells transfected with EGFP control (EGFP-C2), EGFP-RIG-I, EGFP-ΔCARDs, or EGFP-CARDs and costained for actin (red) 48 h following transfection. C, HEK293 cells were transfected with EGFP-RIG-I, EGFP-ΔCARDs, or EGFP-CARDs and cellular fractionation was performed. Fractions were subjected to immunoblot analysis for GFP (top) and vimentin (below). Data are representative of experiments performed at least three times.
FIGURE 4.
FIGURE 4.
RIG-I regulates cell migration. Wound healing assays were performed in HEK293 cells transfected with EGFP-C2, EGFP-RIG-I, EGFP-Δ-CARDs (A) or with scrambled (control) or RIG-I siRNAs (B). Shown is the percent of wound closure at the indicated times (left) and corresponding representative brightfield images (right). The efficacy of RIG-I siRNA is shown in B. Data are representative of experiments performed at least three times in triplicate. *, p ≤ 0.05 (A and B), significant from control cells. Statistical analysis was performed as described under “Experimental Procedures.”
FIGURE 5.
FIGURE 5.
RIG-I associates with apical tight junctions in Caco-2 cells. A, immunofluorescence microscopy for endogenous RIG-I (green) and actin (red) in subconfluent Caco-2 cells grown on collagen-coated glass chamber slides for 24 h. Inset shows ×3 magnification of colocalized (yellow) membrane ruffles. B, confocal microscopy of endogenous RIG-I (green) and ZO-1 (red) in polarized Caco-2 cells cultured for 72 h on collagen-coated glass chamber slides. C, immunofluorescence microscopy for endogenous MDA5 in confluent Caco-2 cells (4′,6-diamidino-2-phenylindole (DAPI) stained nuclei in blue). D, Caco-2 cells grown for 72 h were subjected to cellular fractionation as described under “Experimental Procedures” to obtain cytosolic, membrane/organelle, nuclear, and cytoskeletal fractions. Isolated fractions were subjected to immunoblot analysis for RIG-I (top). Membranes were stripped and reprobed for calpain-2 (middle) and vimentin (bottom) to confirm the purity of cytosolic and cytoskeletal fractions, respectively. E, Caco-2 cells were transfected with EGFP-RIG-I and 48 h later, EGFP-RIG-I localization was assessed. Data are representative of experiments performed at least three times.
FIGURE 6.
FIGURE 6.
RIG-I localizes to the junctional complex of human colon and small intestine biopsies. RIG-I immunohistochemistry was performed on formalin-fixed paraffin-embedded normal human colon and small intestine biopsies. Black arrows indicate examples of RIG-I association with apical junctional complexes.
FIGURE 7.
FIGURE 7.
Disruption of actin cytoskeleton leads to RIG-I relocalization and the induction of type I interferon signaling. A, immunofluorescence microscopy of RIG-I (green), and actin (red, top) or ZO-1 (red, bottom) in Caco-2 cells exposed to cytoD (8 μm) for 2 h. 4′,6-Diamidino-2-phenylindole (DAPI)-stained nuclei are shown in blue. B, luciferase assays (expressed in relative luminescence activity) from control (No Inh) and cytoD-treated Caco-2 cells transfected with IFNβ and NFκB promoted luciferase constructs. C, Caco-2 cells were treated with vehicle (dimethyl sulfoxide, No Inh) or with cytoD for 2 h and fixed and stained for IRF3 (green) and actin (red). D and E, subconfluent Caco-2 cells cultured for 24 h were exposed to cytoD for 2 h and fixed and stained for RIG-I (green, D) or IRF3 (green, E) and actin (red). F, time-lapse live cell microscopy from EGFP-RIG-I transfected non-confluent Caco-2 cells. Images were taken before and after the addition of cytoD at 10-s intervals. Shown are still images captured at the indicated times following the addition of cytoD. Data are representative of experiments performed at least three times (*, p ≤ 0.05) (B).

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