Structural inhibition of dynamin-mediated membrane fission by endophilin
- University of Geneva, Switzerland
- University of California, United States
- University of Utah, United States
- Chan Zuckerberg Biohub, United States
- Yale University School of Medicine, United States
- Howard Hughes Medical Institute, Yale University School of Medicine, United States
- Swiss National Centre for Competence in Research Programme Chemical Biology, Switzerland
Figures
Figure 1 with 1 supplement
Endophilin inhibits dynamin-mediated constriction and fission.
(A, B) Visualization of tubule fission using membrane sheets assay. We injected GTP at t = 0 s on tubules generated by dynamin (A) or dynamin and endophilin (4x) (B). (C) Scheme of bead rotation …
Figure 1—figure supplement 1
Colocalization of dynamin and endophilin and GTPase assay.
(A) Colocalization of endophilin ATTO 565 and dynamin ATTO 488 on tubules generated from membrane sheets. Bar, 5 microns (B) Malachite green GTPase assay, values are normalized to the positive …
Figure 2 with 1 supplement
Endophilin reduces fission rate, fission efficiency and dynamin density.
(A) Tube pulling setup. A membrane tube is pulled from a GUV aspirated in a micropipette. Protein is injected using a second pipette. (B) Endophilin constructs used for the experiments. (C) Confocal …
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Figure 2—source data 1
Dynamin fluorescence intensities for three different endophilin/dynamin ratios.
- https://doi.org/10.7554/eLife.26856.006
Figure 2—figure supplement 1
Endophilin intensity on tube increases with increased endophilin concentration in solution.
Quantification of endophilin signal intensity on the same tubes as those whose dynamin signal was measured for Figure 2K. The indicated values are raw integrals of the fluorescence peaks obtained by …
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Figure 2—figure supplement 1—source data 1
Quantification of endophilin signal intensity on the same tubes as those whose dynamin signal was measured for Figure 2K.
- https://doi.org/10.7554/eLife.26856.007
Figure 3 with 1 supplement
Endophilin can interleave between turns of a dynamin oligomer.
CryoEM of coated tubules of (A) endophilin alone (B) dynamin (GMPPCP) or (C) the endophilin-dynamin co-complex (GDP). Black circles on the cryoEM micrographs (A-C) delineate example particle …
Figure 3—figure supplement 1
1-start and 2-start helices formed by Dynamin-1 with the non-hydrolyzable GTP analog GMPPCP.
(A) CryoEM 2D class average of a dynamin one-start helix, versus (B) a two-start start helix assembled around lipid tubules and in presence of GMPPCP. Stalk to stalk distances were ~13.9 nm and ~14.0…
Figure 4
Endophilin overexpression blocks endocytic pits in vivo.
(A) TIRFM images of genome-edited SK-MEL-2 cells overexpressing endophilinA2-TagRFP. Purple arrows, transfected cells; green arrows, non-transfected cells. Inset: Magnification of white box; left, …
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Figure 4—source data 1
Dynamin event durations for non-transfected (NT) and endophilinA2-TagRFP overexpressing cells.
- https://doi.org/10.7554/eLife.26856.011
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Figure 4—source data 2
Quantification of transferrin fluorescence in non-transfected cells, cells overexpressing endophilinA2-TagRFP, cells with high (>10,000) and low (<10,000) endophilinA2-TagRFP levels, and cells co-overexpressing dynamin2 and endophilinA2-GFP.
- https://doi.org/10.7554/eLife.26856.012
Tables
Table 1
Data collection parameters
https://doi.org/10.7554/eLife.26856.013| Dataset | Endophilin | Dynamin GMPPCP | Co-complex GDP |
|---|---|---|---|
| Microscope | TF30 Polara | TF30 Polara | TF30 Polara |
| Detector | K2 Summit | K2 Summit | K2 Summit |
| Collection | UCSFimage4 | SerialEM | SerialEM |
| Pixel size (Å) | 1.22 | 2.49 | 1.22 |
| Exposure (sec) | 6.0 | 8.0 | 8.0 |
| Total Dose (e-/Å2) | 40 | 20 | 40 |
| Micrographs | 204 | 2006 | 1660 |
| Motion Correction | MotionCorr | MotionCor2 | MotionCor2 |
| Defocus Range (μm) | 0.6–2.6 | 0.3–5.0 | 0.8–2.8 |
| Particles contributing to class averages in Figure 3 | 2047 | 2766 | 19,662 |
Additional files
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Source code file 1
Matlab code for fitting of cumulative fission probability data for Figure 2E and H.
The code determines the variables a and τ based on the experimental data, for a fit to a*(1-exp(-t/τ)).
- https://doi.org/10.7554/eLife.26856.014
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Source code file 2
Matlab code for fitting of cumulative fission probability data for Figure 2L.
The code determines the variables a and τ based on the experimental data, for a fit to a*(1-exp(-t/τ)).
- https://doi.org/10.7554/eLife.26856.015
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Transparent reporting form
- https://doi.org/10.7554/eLife.26856.016
