Journal: bioRxiv

Article Title: Self-assembly of CIP4 drives actin-mediated asymmetric pit-closing in clathrin-mediated endocytosis

doi: 10.1101/2022.11.21.517438

Figure Lengend Snippet: A. The endogenous amount of CIP4, FBP17, and Synd2 in Cos7 cells was examined using western blotting. The total cell lysate of Cos7 cells expressing EGFP-fused CIP4, FBP17, or Synd2 was subjected to western blotting using anti-CIP4, -FBP17, or -Synd2 antibody, respectively. The same lysate was also blotted with an anti-GFP antibody for reference. The amount of β-actin was detected with an anti-β-actin antibody as an internal control. B. The endogenous amount of CIP4, FBP17, and Synd2 obtained from western blotting is shown in (A). Data from the three independent experiments are summarized. Statistical analysis was performed using Student’s t-test, α = 0.05; ****: P < 0.0001. Data are presented as mean ± standard deviation from three independent experiments as a relative value to that of CIP4. C. Knockdown efficiency of CIP4, FBP17, and Synd2 in Cos7 cells. Total cell lysate of non-transfected Cos7 cells and Cos7 cells transfected with siRNA for the control (luciferase), CIP4, FBP17, or Synd2 was prepared 48 h after the transfection and then subjected to the western blot analysis using anti-CIP4, FBP17, or Synd2 antibodies. β-actin was detected as an internal control. D. Additional examples of the time-lapse HS-AFM images obtained from Cos7 cells transfected with siRNA against CIP4, Synd2, or FBP17. Details of the experimental conditions and data presentations are the same as those in . E. The frequency of CCP formation in non-transfected Cos7 cells and Cos7 cells transfected with siRNA for the control (luciferase), CIP4, FBP17, or Synd2 was quantified and summarized. Statistical analysis was performed using Student’s t-test, α = 0.05. There was no significant differences between any two groups. Data are presented as mean ± standard deviation from four independent experiments. F. Additional examples of the correlative imaging of HS-AFM and confocal laser scanning microscopy (CLSM). The fluorescent (FL) intensity of EGFP-fused CLTB and mCherry-fused CIP4 and the maximum membrane height at the CCP area were plotted against time. Time 0 was defined as the time when the pit completely closed and is indicated with a black dotted line. Other details of the experimental conditions and data presentations are the same as those in . G. Additional examples of the time-lapse superresolution SIM images obtained from Cos7 cells expressing EGFP-fused CLTB and mCherry-fused CIP4. Details of the experimental conditions and data presentations are the same as those in .

Article Snippet: Superresolution structure illumination microscopy (SIM) was performed using Elyra 7 with Lattice SIM (Zeiss, Oberkochen, Germany).

Techniques: Western Blot, Expressing, Control, Standard Deviation, Knockdown, Transfection, Luciferase, Imaging, Confocal Laser Scanning Microscopy, Membrane

Journal: bioRxiv

Article Title: Self-assembly of CIP4 drives actin-mediated asymmetric pit-closing in clathrin-mediated endocytosis

doi: 10.1101/2022.11.21.517438

Figure Lengend Snippet: CIP4 is necessary for the asymmetric bulge. A. Time-lapse fluorescence images obtained from Cos7 cells expressing EGFP-fused CLTB and mCherry-fused CIP4, FBP17, or Synd2. Images were acquired every 5 s. Image size: 1.0 × 1.0 μm 2 . Time 0 was defined as the time when the clathrin signal disappeared. Representative images are presented here, and additional images are provided in . B. A summary of the assembly profile of the F-BAR domain proteins. The times when the fluorescence signal appeared and disappeared at the CCP area are defined as “start” and “end,” respectively, and are plotted (N = 20, for each condition). Time 0 was defined as the time when the clathrin signal disappeared. Statistical analysis was performed using Student’s t-test, α = 0.05; ***: P < 0.001; ****: P < 0.0001. Error bars represent the standard deviation. C. (Left) Time-lapse HS-AFM images obtained from Cos7 cells transfected with siRNA for CIP4, FBP17, or Synd2. The representative images are presented here. Additional cases are shown in . Images were taken every 10 s. Time 0 was defined as the time point when the pit completely closed on the HS-AFM image. The membrane bulge is indicated by arrowheads and the height information of the AFM image is presented using a color bar. Image size: 1.0 × 1.0 μm 2 . (Right) The ratio of three different closing patterns in non-transfected Cos7 cells (N = 98) and the cells transfected with siRNA for the control (luciferase) (N = 60), CIP4 (N = 80), FBP17 (N = 60), or Synd2 (N = 60). D. (Left) Correlative imaging of HS-AFM and CLSM. Cos7 cells expressing EGFP-fused CLTB and mCherry-fused CIP4 were imaged. Images were taken every 10 s. Time 0 was defined as the time point when the pit completely closed on the HS-AFM image. The membrane bulge is indicated by arrowheads and the height information of the AFM image is presented using a color bar. Image size: 1.0 × 1.0 μm 2 . The representative images are presented here. Additional cases are shown in . (Right) A summary of the assembly profile of CIP4 and the membrane bulge formation. The time points when the membrane bulge and the fluorescence signal of CIP4 appeared and disappeared at the CCP area were defined as “start” and “end” points, respectively, and are plotted (N=16). Time 0 was defined as the time point of the complete pit closing and indicated with a black dotted line. Statistical analysis was performed using Student’s t-test, α = 0.05; n.s., not significant (P > 0.05). Error bars represent the standard deviation. E. Time-lapse superresolution SIM images obtained from Cos7 cells expressing EGFP-fused CLTB and mCherry-fused CIP4. Images were taken every second and displayed every 2 s. Image size: 0.5 × 0.5 μm 2 . The fluorescent intensity of CIP4 and the x-y distance between the centroids of clathrin and CIP are plotted against time (bottom). Time 0 was defined as the time when the clathrin signal disappeared. The representative images are presented here. Additional cases are shown in . F. The histogram of the x-y distance between the centroids of the clathrin and CIP4 fluorescence spots within the first 5 s of CIP4 assembly (N = 160, from 32 CMEs). Please also refer to and .

Article Snippet: Superresolution structure illumination microscopy (SIM) was performed using Elyra 7 with Lattice SIM (Zeiss, Oberkochen, Germany).

Techniques: Fluorescence, Expressing, Standard Deviation, Transfection, Membrane, Control, Luciferase, Imaging