Fig 2: CLIC4 and CLIC1 regulate membrane blebbing during cytokinesis.(A) Live-cell imaging of stable Lifeact-RFP expressing control (top) and CLIC1/4 DKO c2 (bottom) cells. Representative frames of time-lapse movies at the indicated time points are presented. CLIC1/4 DKO cells show abnormal bleb formation at the polar cortex during cell division. Arrowheads denote the furrow regression in CLIC1/4 DKO cells and so the multinucleated cell formation. Scale bar, 10 µm. (B) Quantification of maximal bleb extension during cytokinesis in control and CLIC1/4 DKO cells. n = 129 cells for control and n = 95 cells for CLIC1/4 DKO c2 were quantified and individual blebs were plotted based on their maximal length. Red lines indicate the median of bleb maximal length for each condition. Unpaired two-tailed t test was used for significance analysis (***P < 0.001). (C) Spatiotemporal regulation of CLIC4-GFP (top) and CLIC1-GFP (bottom) during bleb retraction. Representative frames of time-lapse movies at the indicated time points are presented. Arrowheads denote membrane blebs at different phases, from expansion to retraction during cytokinesis. Both CLIC4-GFP and CLIC1-GFP localize to the bleb rim during retraction phase of the membrane blebs. Scale bar, 10 µm.

Fig 3: CLIC4 interacts with anillin, ALIX, and ezrin in cytokinesis.(A) Co-localization of endogenous CLIC4 with anillin, ALIX, and ezrin in cytokinesis. Maximum intensity projections of Z-stacks show endogenous anillin, ALIX, and ezrin (red), endogenous CLIC4 (green), and DNA staining (DAPI, blue). Scale bars, 5 µm. (B) Co-immunoprecipitation analysis of interaction partners of CLIC4 in cytokinesis arrested cells using the sensitive GFP-trap approach. Western blot analyses of whole cell lysates (input) and elute fractions obtained from stable GFP (as control) and CLIC4-GFP expressing cells were performed against anti-GFP (top), anti-CLIC4 (middle), anti-anillin, anti-ALIX, and anti-ezrin antibodies (bottom). Co-immunoprecipitation of ALIX and ezrin with only CLIC4-GFP indicates that CLIC4 resides in the same complex with ezrin and ALIX during cytokinesis. (C) Spatial analysis of the interactions of CLIC4 with anillin, ALIX, and ezrin in intact cytokinesis cells by in situ proximity ligation assay (PLA). The representative images show the interactions between the examined antibody pairs as red fluorescent PLA puncta. Cells were also stained with DAPI for DNA (blue). Control was treated with only anti-CLIC4 antibody. Each image is the maximum intensity projection of a Z-stack and represent a typical cell staining observed in 20 fields chosen randomly. Scale bars, 5 µm. (D) Quantification of the interactions of CLIC4 in cytokinesis by in situ PLA. In addition to only anti-CLIC4 antibody–treated cells (n = 533), cells subjected to only anti-anillin (n = 639), anti-ALIX (n = 538), and anti-ezrin (n = 499) antibodies (Fig S2) were also quantified as controls. The cells treated with antibody pairs of CLIC4-anillin (n = 757), CLIC4-ALIX (n = 456), and CLIC4-ezrin (n = 499) were used for the interaction analysis. Red lines indicate the mean number of PLA dots per daughter cell nucleus. Comparison of multiple groups was performed by one-way ANOVA with Bonferroni post hoc test (***P < 0.001). Mean ± SEM are shown.

Fig 4: Single and double knockouts of CLIC4 and CLIC1 lead to multinucleated cells.(A) CLIC4 knockout (KO) validation was achieved by Western blot analysis of the whole cell lysates using anti-CLIC4 antibody. a-Tubulin was used as a loading control. (B) Western blot analysis of CLIC4-GFP expression in the CLIC4 KO rescue cell lines. Whole cell lysates were immunoblotted with anti-GFP antibody to detect the expression of CLIC4-GFP. a-Tubulin was used as a loading control. (C) Quantification of multinucleation percentage of CLIC4 KO and CLIC4-GFP expressing CLIC4 KO rescue cell lines. Cells were stained with anti-a-tubulin antibody (red) and DAPI for DNA (blue). Asterisks in the representative images denote multinucleated cells. Scale bars, 20 µm. Multinucleation percentage was statistically higher in CLIC4 KO cells, and CLIC4-GFP expression in CLIC4 KO cells significantly decreased multinucleation percentage. n = 300 cells per experiment were quantified and mean ± SEM of three independent experiments are shown. One-way ANOVA with Bonferroni post hoc test was used for statistical analysis (*P < 0.05; **P < 0.01; ***P < 0.001). (D) CLIC1 KO validation was achieved by Western blot analysis of the whole cell lysates using anti-CLIC1 antibody. CLIC4 expression was not altered in CLIC1 KO cells as revealed by immunoblotting with anti-CLIC4 antibody. a-Tubulin was used as a loading control. (E) Western blot analysis of CLIC1-GFP expression in the CLIC1 KO cell lines. Whole cell lysates were immunoblotted with anti-GFP antibody to detect expression of CLIC1-GFP. a-Tubulin was used as a loading control. (F) Quantification of multinucleation percentage of CLIC1 KO and CLIC1-GFP expressing CLIC1 KO rescue cell lines. Cells were stained with anti-a-tubulin antibody (red) and DAPI for DNA (blue). Asterisks in the representative images denote multinucleated cells. Scale bars, 20 µm. Multinucleation percentage was statistically higher in CLIC1 KO cells and CLIC1-GFP re-expression in CLIC1 KO cells significantly decreased multinucleation percentage. n = 300 cells per experiment were quantified and mean ± SEM of three independent experiments are shown. One-way ANOVA with Bonferroni post hoc test was used for statistical analysis (*P < 0.05; **P < 0.01; ***P < 0.001). (G) CLIC1/4 double KO (DKO) validation was achieved by Western blot analysis of the whole cell lysates using anti-CLIC4 and anti-CLIC1 antibodies. a-Tubulin was used as a loading control. (H) Quantification of multinucleation percentage of CLIC1/4 DKO cell lines. Cells were stained with anti-a-tubulin antibody (red) and DAPI for DNA (blue). Asterisks in the representative images denote multinucleated cells. Scale bars, 20 µm. Multinucleation percentage was statistically higher in CLIC1/4 DKO cells. n = 200 cells per experiment were quantified and mean ± SEM of three independent experiments are shown. One-way ANOVA with Dunnett’s post hoc test was used for statistical analysis (***P < 0.001). (I) Quantification of successful and defective cell divisions of control and CLIC1/4 DKO cells by live-cell imaging. Cells were imaged for 24 h. n = 371 cell divisions from six experiments for control cells and n = 526 cell divisions from eight experiments for CLIC1/4 DKO cells were quantified and mean ± SEM are shown. Unpaired two-tailed t test was used for significance analysis (**P < 0.01). (J) Representative still images of time-lapse movies showing a successful cell division in control cell line and cytokinesis defect in CLIC1/4 DKO c9 cell line. Dividing cells were marked with yellow dashed lines before and after cell division. Red arrows indicate the presence of midbody. Scale bars, 10 µm.

Fig 5: Chloride intracellular channels (CLICs) facilitate CHIKV replication.A) (i) CLIC1 and CLIC4, respectively, as well as (ii) CLIC1 and CLIC4 in combination, were knocked down by transfecting siRNA into Huh7 cells. B) At 48 hrs after initial siRNA transfection, cells were infected with CHIKV (MOI 10) and released CHIKV and titered at 24 hpi (n = 3). Viral titer was normalised to the scrambled siRNA control sample. One-way ANOVA was performed to compare samples to scrambled siRNA transfected cells. Error bars represent standard deviation. *p=0.05; ** p=0.01, *** p=0.001. C) TST-tagged nsP3 from CHIKV infected (MOI 10) Huh7 cells (24 hpi) was pulled-down using Strep-Tactin Sepharose. Western blots were probed for nsP3 binding proteins.