Fig 1.
DRP1 ring- and spiral-like structures by negative-stain TEM.
Negative-stained TEM images of DRP1 in the absence of nucleotide (A) or in the presence of GDP (B), GMP-PNP (C), or GTP (D). Scale bar = 50 nm).
Fig 2.
3D structure of human GTP-bound DRP1 ring-like oligomers.
(A) A representative cryo-EM CCD image of GTP-bound human DRP1. The inserts show examples of top, side and intermediate views (Scale bar = 30 nm). (B) The two most characteristic class averages showing the DRP1 -GTP rings on its side and as seen from the top. The contrast has been inverted for clarity purposes in respect to (A) (Scale bar = 30 nm). (C) Isosurface 3D representation of the electron density map of GTP-bound DRP1 ring-like structures by single particle image analysis. The map displayed by Chimera has been radially coloured. The estimated resolution according to the Fourier shell correlation of 0.5 is ~25Å (Scale bar = 30 nm).
Fig 3.
Localization of the C-terminus of DRP1 using Ni-NTA Gold labeling.
(A) TEM image of DRP1-GTP rings labelled with Nanogold-NTA (black arrows) and negatively stained with NanoVan (Scale bar = 50 nm). (B) Cryo-TEM image of DRP1-GTP rings labelled with Nanogold (white arrowheads). The rings are shown in the top (i & ii), oblique (iii) and side views (iv & v) (Scale bar = 30 nm). (C) Radially-coloured isosurface representation of the 3D electron density map of the DRP1-GTP rings labelled with Nanogold (Scale bar = 10 nm). (D) Reconstructed monomer extracted from the boxed area of the Nanogold-bound DRP1-GTP ring (C) was rotated 90° about the axis to show the monomer map (Scale bar = 5 nm). (E), (F), (G) Isosurface representation of the Nanogold-bound DRP1-GTP rings reconstructed from the tomogram. (E) and (F) show 45° rotation about the y- axis (Scale bar = 10 nm).
Fig 4.
Domains assignment to the electron density map of the GTP-bound DRP1 ring-like oligomers.
(A) Electron density map of a monomer segmented from the map (small box (C)). The monomer is shown as two contour levels to emphasize the globular shape of the top domain and the elongated shape of the bottom domain (Scale bar = 5 nm). (B) The high-resolution structures of human dynamin GTPase domain (pdb id: 3SNH, shown in green as a cartoon representation) and the human MxA stalk Middle-Effector Domain (residues 366−636, pdb id: 3LJB, shown in dark blue and light blue as a cartoon representation) were docked into the EM map of a segmented monomer (shown in grey chicken wire) using Chimera. The EM map contains additional densities that cannot be accounted for by the GTPase and the MID/GED domains. These are likely to correspond to the position of the BSE and B-insert domains. For size estimation only, we have introduced the PH domain of dynamin (residues 518−631, pdb id: 3SNH) (Scale bar = 5 nm). (C) EM map of GTP-bound DRP1 in an oblique view (Scale bar = 30 nm). (D) The domains in (B) are shown for the three adjacent monomers (dashed box (C)) (Scale bar = 5 nm).
Fig 5.
3D Analysis of GMP-PNP-bound DRP1 helices.
(A−B) TEM images of DRP1-GMP-PNP helices after negative staining with NanoVan. The mean pitch of the helices is 13.7 nm (yellow markers; Scale bar = 30 nm). (C) Image represents the average of 20 slices from the tomogram of DRP1-GMP-PNP helix in cryo-ET. The contrast has been inverted for viewing purposes. Protein density is shown in white. Black arrows show the helical turns (Scale bar = 15 nm). (D) A filtered-traced helix traced from the cryo-tomogram of a DRP1-GMP-PNP helix (grey chicken wire). The position of the monomers with an ideal helix of 30 nm in diameter and a pitch of 13.7 nm (Red dots; Scale bar = 15 nm). (E), (F), (G) Isosurface representation of Nanogold-bound DRP1-GMP-PNP helices reconstructed from the tomogram with (E) rotated 90° about the y-axis; (F) rotated 90° about the x- axis, and (G) cross-sectional view showing the gold labelled inside the helix. The map displayed by chimera has been low pass filtered to ~30Å (Scale bar = 30 nm).
Fig 6.
Binding DRP1 to mitochondria in the presence of GMP-PNP by negative-stain EM.
(A) Negative-stain TEM image of mitochondria (red arrows) and mitochondria tubules (blue arrows) in the presence of DRP1-GMP-PNP (Scale bar = 100 nm). (B) TEM image shows three turns of the helices forming the mitochondria tubules (black arrows) (Scale bar = 30 nm). (C) Negative-stain TEM image of mitochondria control (without DRP1). (Scale bar = 100 nm).
Fig 7.
Model showing the mechanism of DRP1 in mitochondrial fission.
DRP1 monomers or dimers are recruited from the cytosol by recruitment factors including Mff and Mid49. They assemble into spiral/rings shaped oligomers of 30 nm outer diameter and inner diameter of 20 nm. As the thickness of both inner and outer mitochondria membranes are 7 nm [67], assembly of DRP1 onto the mitochondria would simultaneously constrict the mitochondria in such a way that four independent membranes would not be able to exist as separate units inside the DRP1 rings (Stage C; orange area).