Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 1. Localization of CLIC4 throughout the cell cycle. (A) Schematic representation of abscission that generates an extracellular post-mitotic midbody. (B,C) HeLa cells expressing exogenous GFP–CLIC4 were fixed and stained with phalloidin–Alexa 568 (B) or anti-acetylated tubulin antibodies (C). Arrow in B points to an ingressing cleavage furrow. Arrow in C points to an abscission site. Asterisk marks the midbody. (D) HeLa cells expressing exogenous GFP–CLIC4 were analysed by time-lapse microscopy. Images shown are stills representing different sequential time points during anaphase. Arrow marks cleavage furrow. (E) HeLa cells expressing endogenously tagged GFP–CLIC4 were fixed and stained with anti-anillin antibody. Arrow points to the cleavage furrow. (F) 3D volume rendition of a GFP–CLIC4 expressing HeLa cell in anaphase. Red marks central spindle microtubules labeled with anti-acetylated tubulin antibodies. The 3D image is shown at an angle to better demonstrate the GFP–CLIC4 ring at the furrow. For different angles of the 3D rendering see Movie 1. Scale bars in B–F: 10 μm

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Expressing, Staining, Time-lapse Microscopy, Labeling

Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 2. RhoA is necessary and sufficient for CLIC4 localization. (A) HeLa cells expressing endogenous GFP–CLIC4 were TCA-fixed and stained with RhoA antibodies. Image was deconvolved. (B) HeLa cells expressing endogenously labeled GFP–CLIC4 were imaged in anaphase in the presence or absence of RhoA inhibitor. Arrows point to cleavage furrows. (C) shCLIC4 HeLa cells were transfected with either wild-type GFP–CLIC4 or GFP–CLIC4 C35A. Cells were then fixed and stained with phalloidin–Alexa 568. Arrows point to cleavage furrow. (D,E) Quantification of GFP–CLIC4 (D,E) or GFP–CLIC4 C35A (E) fluorescence in cleavage furrow and poles of dividing cell. Data shown are the means and standard deviations of 15–30 anaphase cells randomly picked during three independent experiments. Scale bars in A–C: 10 μm

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Expressing, Staining, Labeling, Transfection, Fluorescence

Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 3. Depletion of CLIC4 leads to mitotic defects. (A) HeLa cells were fixed and co-stained with phalloidin–Alexa 568 and anti-acetylated tubulin antibodies. The number of multi-nucleated cells were then counted. Graph on the right shows quantification and statistical analysis of multi-nucleation induced by shRNA- dependent CLIC4 depletion. Data shown are the means and standard deviations of at least 75 cells counted in randomly chosen fields from three different experiments. *P<0.05; **P<0.025; ***P<0.01; ns, not significant. (B–D) Control (B) or shCLIC4 (C,D) HeLa cells were analysed by time-lapse microscopy. Images shown are the stills from different time points of cells undergoing mitotic cell division. Arrows point to blebs during anaphase. (E) Quantification of time required for cells to go from metaphase to abscission. Data shown are means and standard deviations derived from randomly picked cells in two different experiments. WT, wild type. (F) Quantification of blebbing frequency in control or shCLIC4 HeLa cells. Data shown are means and standard deviations derived from randomly picked cells in three different experiments. (G) Control or shCLIC4 cells were incubated with SiR-actin and live imaged. Quantification of actin intensity at the cell cortex and furrows are shown. Data shown are means and standard deviations of individual cell intensities from randomly picked cells in three different experiments. Scale bars in A,C,D,G: 10 μm. Scale bar in B: 100 μm.

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Staining, shRNA, Control, Time-lapse Microscopy, Derivative Assay, Incubation

Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 4. An optogenetic targeting of CLIC4 to the mitochondria delays cell division. (A) Schematic of Mitotrap used in this experiment. In all cases cells expressing endogenously tagged GFP–CLIC4 were co-transfected with Cry2-GFP-VHH and CIB-Tom20 plasmids and pulsed with a 488 nm laser to re-target GFP–CLIC4 at the mitochondria. (B) Interphase cell exposed to 488 nm to activate Mitotrap. Mitochondria are labeled in red and endogenous GFP–CLIC4 in green. (C,D) Still images from time-lapse microscopy where Mitotrap was activated. Arrows point to blebs or cytokinesis failure induced by GFP–CLIC4 Mitotrap. Scale bars: 100 μm. (E) Quantification of time required for cells to complete mitotic cell division in control and Mitotrapped cells. Data shown are the means and standard deviations.

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Expressing, Transfection, Labeling, Time-lapse Microscopy, Control

Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 5. Loss of CLIC4 results in decreases in NMMIIA and -IIB at cortex and furrow. (A) HeLa cells expressing GFP–CLIC4 were fixed and stained with anti-NMMIIB antibodies. (B–E) Control (B,D) or shCLIC4 (C,E) HeLa cells were fixed and stained with either anti-NMMIIA (B,C) or anti-NMMIIB (D,E) antibodies. Arrows point to ingressing furrow during anaphase. (F) Quantification of NMMIIA or NMMIIB at the furrow or at the poles of the anaphase cell. The data shown are the means and standard deviations of 20–40 randomly picked cells from three different experiments. Scale bars in A–E: 10 μm.

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Expressing, Staining, Control

Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 7. CLIC4 interacts with MST4 and affects its localization during anaphase. (A) Putative candidates revealed via immunoprecipitation of GFP–CLIC4 followed by mass spectrometry. (B) Glutathione bead pulldown assay using HeLa cell lysate and either GST only or GST–CLIC4. Coomassie staining showing equal loading as well as quality of the recombinant proteins used in the pulldown assay. (C) HeLa cells were fixed and stained with phalloidin–Alexa 568 and anti-MST4 antibodies. Arrow points to MST4 at the cleavage furrow. (D) Control or shCLIC4 HeLa cells were fixed and stained with anti-MST4 antibody. Arrows point to the cleavage furrow. (E) Quantification of MST4 enrichment in the furrow in control, shCLIC4, siAnillin and shCLIC4/siAnillin cells. Data shown are from four individual experiments, horizontal bar indicates the mean. n is the total number of cells quantified. Scale bars in C,D: 10 µm.

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Immunoprecipitation, Mass Spectrometry, Staining, Recombinant, Control

Journal: Journal of cell science

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

doi: 10.1242/jcs.241117

Figure Lengend Snippet: Fig. 8. MST4 is required for phospho-ezrin recruitment to cytokinetic furrow. (A) Triton X-100 lysates from HeLa control cells or cells transfected with MST4 siRNA were analysed by western blotting with anti-MST4 and anti-tubulin antibodies. (B) Quantification of the enrichment of phospho-ezrin at the furrow. Data shown are the means and standard deviations, and n indicates the number of cells analysed. (C,D) Control HeLa cells (C) or cells transfected with MST4 siRNA (D) were fixed and stained with anti-phospho-ezrin (green) and anti-tubulin (red) antibodies. Scale bars: 5 µm. (E) Schematic representation of the proposed role for CLIC4 during cytokinesis.

Article Snippet: Optogenetics for Mitotrap and FRET analysis For the RhoA activation and GFP–CLIC4 recruitment assay, cells expressing GFP-tagged endogenous CLIC4 were transfected with Cry2PHR-mCHRhoA (42959; Addgene).

Techniques: Control, Transfection, Western Blot, Staining