Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites
- Geisel School of Medicine at Dartmouth, United States
- The University of Iowa, United States
Figures
Figure 1 with 2 supplements
Mitochondrial association of Drp1 puncta.
(A) Time-lapse image of region of gDrp1-U2OS cell transiently expressing mito-BFP and processed to remove background GFP (described in Figure 1—figure supplement 2A). Arrows denote variety of Drp1 …
Figure 1—figure supplement 1
Characterization of stable GFP-Drp1 cell line (gDrp1-U2OS cells).
(A) Western blot of control U2OS cells or gDrp1-U2OS cells showing expression level of GFP-Drp1 and endogenous Drp1 with varying amount of extract loaded (0.5, 0.75 and 1.0x from left to right). (B) …
Figure 1—figure supplement 2
Image processing for GFP-Drp1 intensity in gDrp1-U2OS cells.
(A) Example of image processing for analysis of Drp1 dynamics. Images were threshold-adjusted by subtracting background Drp1 in the cytosol using a rolling ball algorithm (radius = 2 pixels or 0.304 …
Figure 2 with 1 supplement
Maturation of mitochondrially-bound Drp1 puncta.
(A) Example of Drp1 maturation events (arrowheads), followed by mitochondrial fission (63 s). Time in sec. Scale bar, 1 μm (Video 2). (B) Quantification of mitochondrial Drp1 merging rate, defined …
Figure 2—figure supplement 1
Drp1 maturation events.
(A) Multiple Drp1 merging events, followed by fission at a looped junction (arrow at 36 s) (Video 3). (B) Drp1 merging event, followed by splitting of the punctum. Three dim Drp1 puncta merge to …
Figure 3 with 1 supplement
Drp1 puncta motility and relationship to mitochondrial fission.
(A) Example of motile Drp1 punctum (yellow arrow) engaging in fission at 60 s, and a stationary punctum (white arrowhead). Lower panel maps tracks of these puncta. Time in sec. Scale bar, 1 μm (Video…
Figure 3—figure supplement 1
Correlation between mitochondrial length and fission, and Drp1 punctum persistence on mitochondrial ends after mitochondrial fission.
(A) Analysis of mitochondrial lengths for mitochondria not undergoing fission (“Non-dividing”, left), and mitochondria undergoing fission (“Dividing mitochondria, middle) over a 10 min imaging …
Figure 4 with 1 supplement
Drp1 and mitochondrial diameter by live-cell 3D-SIM.
(A) 3D-SIM image of region of a live gDrp1-U2OS cell transiently expressing Tom20-mCherry. Yellow arrowhead, Drp1 punctum engaged in fission. Green arrowhead, non-productive punctum. White …
Figure 4—figure supplement 1
3D-SIM imaging of Drp1 and mitochondria.
(A) Two close-up examples of non-productive stationary puncta, showing constriction of OMM (left panel) in the absence of fission. (B) Additional example of mitochondrial fission event from 3D-SIM …
Figure 5 with 1 supplement
Drp1 motility by live-cell 3D-SIM.
(A) Motile Drp1 punctum on a stationary mitochondrion (yellow arrow) and stationary Drp1 punctum on a motile mitochondrion (white arrow). White arrowhead indicates stationary Drp1 punctum on …
Figure 5—figure supplement 1
(A) Additional example of motile Drp1 punctum from 3D-SIM (similar to Figure 5A).
Yellow arrow indicates the motile Drp1 punctum moving along the mitochondrion. White arrowhead indicates stationary Drp1 punctum. Time in sec. Scale bar, 1 μm (Video 10). (B) Velocity quantification …
Figure 6 with 4 supplements
Ionomycin treatment induces actin polymerization, Drp1 maturation and mitochondrial fission.
gDrp1-U2OS cells transiently transfected with mApple-F-tractin and mito-blue plasmids. (A) Time course of changes in Drp1 oligomer and actin filaments (judged by changes in GFP and mApple signal …
Figure 6—figure supplement 1
Suppression of INF2 or myosin IIA decreases fission rate in un-stimulated conditions.
(A) Western blots for INF2 (left) or myosinIIA (right) using lysates from siRNA-treated cells (tubulin as loading control). (B) Quantification of mitochondrial fission rates in U2OS cells …
Figure 6—figure supplement 2
Actin filament enrichment with Drp1 puncta at fission sites in un-stimulated conditions.
(A) Example of actin filaments enriching with Drp1 punctum prior to fission in gDrp1-U2OS cell transiently transfected with mApple-Ftractin and mito-BFP. Upper panel, mitochondrial signal only; …
Figure 6—figure supplement 3
Comparison of mitochondrial fission rates in mitoRed labeled versus mitoBFP labeled cells.
Quantification was based on 11 ROIs from 7 DMSO treated, mitoRed labeled cells over 25.4 min; 9 ROIs from 7 Ionomycin treated, mitoRed labeled cells over 24.3 min; 11 ROIs from 10 DMSO treated, …
Figure 6—figure supplement 4
Larger field of super resolution Airyscan images, including the region shown in Figure 6D.
Actin filaments accumulate at three mitochondrial constriction sites (red arrows) prior to Drp1 maturation (yellow arrowheads). Two of the constriction sites do not undergo fission in this time …
Figure 7
Inhibition of actin, INF2 or myosin IIA reduces ionomycin-induced Drp1 maturation.
(A,B) gDrp1-U2OS cells were transiently transfected with mitochondrial matrix marker, mito-BFP, and actin filament marker, mApple-F-tractin. Cells were treated with 0, 0.1, 0.2, 1 and 2 μM LatA (or …
Figure 8 with 1 supplement
Drp1 binds to actin filaments.
(A) Co-sedimentation assay in which Drp1 (1.3 μM) is incubated with indicated concentration of pre-polymerized actin (concentrations indicate total actin) for 1 hr, then centrifuged at >100,000 ×g …
Figure 8—figure supplement 1
Binding of Drp1 to actin filaments by co-sedimentation assay.
(A) Example of an SDS-PAGE gel from a high-speed co-sedimentation assay, where 1.3 μM Drp1 was incubated with the indicated concentration of actin filaments for 1 hr at 23°C. Samples were …
Figure 9
Actin fialments stimulate Drp1 GTP hydrolysis synergistically with Mff.
(A) GTPase assays containing 1 μM Drp1 in the presence or absence of 0.5 or 1 μM actin (pre-polymerized for 1 hr) for 5 min before GTP addition (250 μM). N = 6 experiments. (B) GTPase assays …
Figure 10
Model for assembly of fission-productive Drp1 on mitochondria.
Step 1: recruitment. Drp1 units are in equilibrium between cytosol and OMM, possibly binding to OMM receptors such as Mff, MiD49, MiD51, or Fis1, or to cardiolipin. We suggest that several distinct …
Videos
Video 1
Left: confocal time-lapse of Drp1 dynamics in gDrp1-U2OS cell transiently expressing mito-BFP; Right: mitochondrially-associated Drp1 puncta pseudo-colored in blue.
Time lapse was taken in single z-plane every 3 s. Time min:sec. Bar, 2 μm (Figure 1A).
Video 2
Confocal time-lapse of Drp1 maturation in gDrp1-U2OS cell transiently expressing mCherry-mito7 (red).
Time lapse was taken in single z-plane every 3 s. Time min:sec. Bar, 1 μm (Figure 2A).
Video 3
confocal time-lapse of multiple Drp1 merging events, followed by fission at a looped junction in gDrp1-U2OS cell transiently expressing mCherry-mito7 (red).
Time lapse was taken in single z-plane every 3 s. Time min:sec. Bar, 1 μm (Figure 2—figure supplement 1A).
Video 4
Confocal live cell image of Drp1 merging event, followed by splitting of the punctum in gDrp1-U2OS cell transiently expressing mCherry-mito7 (red).
Time lapse was taken in single z-plane every 3 s. Time min:sec. Bar, 1 μm (Figure 2—figure supplement 1B).
Video 5
Airyscan time-lapse of Drp1 maturation in gDrp1-U2OS cell (GFP in green) transiently expressing mCherry-mito-7 (red).
Time lapse was taken in single z-plane in dorsal region of cells every 7 s. Time min:sec. Bar, 1 μm (Figure 2C).
Video 6
Confocal time-lapse of Drp1 movement along mitochondrion in gDrp1-U2OS cell transiently expressing mito-BFP (red).
Mitochondrially-bound Drp1 puncta were followed by Trackmate. Time lapse was taken in single z-plane every 1.5 s. Time min:sec. Bar, 1 μm (Figure 3A).
Video 7
3D-SIM time-lapse of Drp1-mediated mitochondrial fission in gDrp1-U2OS cell transiently expressing Tom20-mCherry (red).
Time lapse was taken every 4.5 s. Time min:sec. Bar, 1 μm (Figure 4A,E).
Video 8
3D-SIM live cell image of Drp1-mediated mitochondrial fission in gDrp1-U2OS cell transiently expressing Tom20-mCherry (red).
Time lapse was taken every 4.5 s. Time min:sec. Bar, 1 μm (Figure 4—figure supplement 1B).
Video 9
3D-SIM time-lapse of Drp1 movement along mitochondrion in stable gDrp1-U2OS cell transiently expressing Tom20-mCherry (red).
Time lapse was taken every 4.5 s. Time min:sec. Bar, 1 μm (Figure 5A).
Video 10
3D-SIM live cell image of Drp1 movement along mitochondrion in gDrp1-U2OS cell transiently expressing Tom20-mCherry (red).
Time lapse was taken every 4.5 s. Time min:sec. Bar, 1 μm (Figure 5—figure supplement 1A).
Video 11
Confocal live cell image of mitochondrial fission in an un-stimulated U2OS cell transiently expressing mApple-F-tractin and mito-BFP.
Time lapse was taken in single z-plane in dorsal region of cells to avoid massive actin based structures every 3 s. Time min:sec. Bar, 1 μm (Figure 6—figure supplement 1A).
Video 12
Airyscan time-lapse of mitochondrial fission in response to Ionomycin treatment in gDrp1-U2OS cell (GFP in blue) transiently expressing mApple-F-tractin (green) and mito-BFP (red).
Time lapse was taken in single z-plane in dorsal region of cells every 18 s. Time min:sec. Bar, 1 μm (Figure 6D).
Video 13
TIRF microscopy time-lapse GFP-Drp1 (270 nM) was added to TAMRA-actin filaments (1 μM, 20% TAMRA initially).
Actin filaments were polymerized for 10 min prior to GFP-Drp1 addition. Dual-color simultaneous images were collected every 1 s. Scale bar, 2 μm. 307 frames played at 75 ms frame rate (13.3-fold …
Video 14
TIRF microscopy time-lapse showing filament bundling.
GFP-Drp1 (270 nM) was added to TAMRA-actin filaments (1 μM, 20% TAMRA initially). Actin filaments were polymerized for 10 min prior to GFP-Drp1 addition. Dual-color simultaneous images were …
Video 15
TIRF microscopy time-lapse showing filament bundling.
GFP-Drp1 (2 μM) was added to TAMRA-actin filaments (1 μM, 20% TAMRA initially). Actin filaments were polymerized for 10 min prior to GFP-Drp1 addition. Images were collected every 2 s. Scale bar, 2 …
