Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic invadosome clusters

Figures

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  FIGURE 1:. Dynamic clusters of actin structures form in Sf21 insect cells during early AcMNPV infection. (A) Confocal and TIRF images of Sf21 insect cells transiently transfected with GFP-actin and mock infected or infected with AcMNPV at an MOI of 10. Images were taken from 4 to 7 hpi. Scale bars = 5 μm. (B) Clusters of actin structures in AcMNPV-infected Sf21 cells were quantified from 2 to 32 hpi. Data are mean ± SD of n = 3 biological replicates imaging ∼5000 cells each. P values were calculated with a one-way analysis of variance (ANOVA) with Tukey’s posthoc test relative to the 2 h time point and are indicated as follows: ** = p < 0.005, **** = p < 0.0001. (C) Confocal time-lapse images of Sf21 insect cells transiently transfected with GFP-actin and infected with AcMNPV. Inset images in the bottom row show red, yellow, and green circles enclosing stationary actin puncta that disappear, are maintained, or appear, respectively. Scale bars = 5 μm. Time postinfection is indicated.

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  FIGURE 2:. AcMNPV-induced invadosomes degrade underlying extracellular matrix. (A) Images of Sf21 cells plated on rhodamine-fibronectin matrix, transiently transfected with GFP-actin, infected with an AcMNPV MOI of 10, and imaged at 3 and 5 hpi. Insets at 5 hpi show areas where invadosome clusters correspond with areas of cleared matrix. The white dotted line was used to make a kymograph showing GFP-actin accumulation and matrix clearing over time. Scale bars = 5 μm. (B) Quantification of Pearson’s correlation coefficients of GFP-actin and matrix signal intensities measured over 2 h in cells infected with AcMNPV or mock infected (control). Intensities of actin and matrix signals were measured at the site of invadosome clusters in AcMNPV-infected cells or at random sites in mock infected cells. Negative values indicate an inverse correlation between GFP-actin signal and matrix signal intensity at a single site. Data represent individual measured sites and were taken from n = 3 biological replicates of 11 cells for each treatment. The p value was calculated by an unpaired t test; **** = p < 0.0001.

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  FIGURE 3:. ARIF-1 is necessary and sufficient for the formation of invadosome clusters. (A) Confocal images of Sf21 cells transiently transfected with GFP-actin and infected with an MOI of 10 of the indicated virus. Images were taken at 4 hpi and are representative of three biological replicates. Scale bars = 5 μm. (B) Clusters of invadosomes in infected cells were quantified at 4 hpi. Data are mean ± SD of n = 3 biological replicates of ∼5000 cells each. P values were calculated by one-way ANOVA with multiple comparisons and are indicated as follows: ns = not significant, **** = p < 0.0001. (C) Images of Sf21 cells transiently expressing GFP-actin and ARIF-1. Images were taken 2 d posttransfection and are representative of three biological replicates. Scale bars = 5 μm. (D) Quantification of invadosome clusters in cells transfected with GFP-actin and ARIF-1. Structures were quantified by eye 2 d posttransfection. Data are mean ± SD of n = 3 biological replicates of 60 cells each treatment. The p value was calculated by an unpaired t test; **** = p = 0.0002.

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  FIGURE 4:. ARIF-1 colocalizes with invadosome clusters. (A) Confocal immunofluorescence microscopy images of Sf21 cells infected with the indicated virus. Cells were fixed at 5 hpi and stained with anti–ARIF-1 antibody and fluorescent phalloidin. Insets show selected invadosome clusters. Scale bars = 5 μm. (B) Confocal images of live Sf21 cells transiently expressing F-tractin-mCherry and GFP-ARIF-1. Images were taken 2 d postransfection. Insets show selected invadosome clusters. Scale bars = 5 μm.

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  FIGURE 5:. ARIF-1 residues 303–371 are necessary for the formation of invadosome clusters. (A) Left: predicted ARIF-1 structure with three transmembrane domains and a cytoplasmic C-terminal region (aa 219–417). Right: AcMNPV transmembrane protein GP64 (red) fusion to ARIF-1 C-terminal region. (B) Left: visual representation of ARIF-1 C-terminal truncations. Right: quantification of invadosome clusters in Sf21 cells transiently expressing GFP-actin and truncated ARIF-1 2 d posttransfection. Data are mean ± SD of n = 3 biological replicates of 60 cells each. P values were calculated with a one-way ANOVA with multiple comparisons, comparing each treatment to ARIF-1 (1–417), and are indicated as follows: * = p = 0.0167, **** = p < 0.0001. (C) Left: visual representation of AcMNPV transmembrane protein GP64 (red) fused to ARIF-1(219–417) and N-terminal truncations. Right: quantification of invadosome clusters in Sf21 cells transiently expressing GFP-actin and the indicated construct 2 d posttransfection. Data are mean ± SD of n = 3 biological replicates of 60 cells each. P values were calculated with a one-way ANOVA with multiple comparisons, comparing each treatment to ARIF-1 (1–417), and are…

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  FIGURE 6:. ARIF-1 residues Y332F and P335A are important for the formation of invadosome clusters. (A) Tyrosine (Y, orange) and proline (P, purple) residues are indicated on the C-terminal region of ARIF-1. (B) Invadosome clusters in cells transiently transfected with GFP-actin and the indicated tyrosine-to-phenylalanine point mutations were quantified 2 d posttransfection. Data are mean ± SD of n = 3 biological replicates of 60 cells each. P values were calculated with a one-way ANOVA with multiple comparisons, comparing each treatment to ARIF-1 (1–417), and are indicated as follows **** = p < 0.0001. (C) Invadosome clusters in cells transiently transfected with GFP-actin and the indicated proline-to-alanine point mutations were quantified 2 d posttransfection. Data are mean ± SD of n = 3 biological replicates of 60 cells each. P values were calculated with a one-way ANOVA with multiple comparisons, comparing each treatment to ARIF-1 (1–417), and are indicated as follows: **** = p < 0.0001. (D) Confocal images of Sf21 cells transiently expressing GFP-actin and the indicated ARIF-1 mutation. Images are representative of three biological replicates. Scale bars = 5 μm.

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  FIGURE 7:. Cortactin and the Arp2/3 complex are present at invadosome clusters, and Arp2/3 complex is required for their formation and maintenance. (A) Confocal images of Sf21 cells transiently expressing Lifeact mCherry and GFP-tagged S. frugiperda cortactin. Images were taken 2 d posttransfection and are representative of three biological replicates. Scale bars = 5 μm. (B) Confocal images of Sf21 cells transiently expressing Lifeact mCherry and GFP-tagged Arp2/3 complex subunit ARPC3. Images were taken 2 d posttransfection and are representative of three biological replicates. Scale bars = 5 μm. (C) Invadosome clusters were quantified 1 h after the indicated drugs were added to Sf21 cells infected with an AcMNPV MOI of 10 at 4 hpi. Data are mean ± SD of n = 3 biological replicates of 40 invadosome clusters. P values were calculated with a one-way ANOVA with multiple comparisons, comparing each treatment to ARIF-1 (1–417), and are indicated as follows: ns = not significant, **** = p < 0.0001.