1. Volume 4, Issue 5, Pages 815-826 (November 1999)
C. elegans Dynamin-Related Protein DRP-1 Controls Severing of the Mitochondrial Outer Membrane 
Arnaud M. Labrousse, Mauro D. Zappaterra, Daniel A. Rube, Alexander M. van der Bliek 
Molecular Cell 
Volume 4, Issue 5, Pages (November 1999)
DOI: /S (00)

2. Figure 1 Expression Pattern of C. elegans drp-1
(A) The drp-1 gene is expressed in neurons, as shown by X-gal staining of a worm containing a drp-1::NLS::β-galactosidase construct. In this construct, the drp-1 gene promoter was fused in frame with a nuclear localization signal and β-galactosidase coding sequences. The lines point to two of the ventral nerve cord cells. This worm also shows strong staining in the head (left), which reflects high levels of expression around the nerve ring, and strong staining in the preanal ganglion (right).
(B) The drp-1 gene is expressed in muscle cells. The construct is the same as in (A), but this animal shows a different staining pattern due to mosaicism of the extrachromosomal array. The lines point to two of the body wall muscle cells. Their identities are apparent in other animals, because leakage of X-gal into the cytoplasm reveals the characteristic shapes of body wall muscles. In addition, this animal shows expression in pharyngeal muscles (head region), in vulval muscles (middle region), and anal muscles (tail region).
(C) The drp-1 gene is expressed in intestinal cells. The lines point to two of the intestinal cells. The intestines have pairs of cells that enclose the intestinal lumen. The nuclei are larger than the nuclei of other somatic cells because they are polyploid.
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2 Effects of drp-1 RNAi on Oogenesis and Viability
(A) The effect of drp-1 RNAi was assessed by monitoring the viability of eggs laid by injected worms every 12 hr. The plot shows the percentage survival of progeny from wild-type animals (open circles) or ced-3(n1286) animals (closed circles) injected with drp-1 RNAi. As a negative control, buffer was injected into wild-type animals (open triangles) or ced-3(n1286) animals (closed triangles). As a positive control, dyn-1 RNAi (C. elegans dynamin) was injected into wild-type animals (closed diamonds). The drp-1 RNAi data are averages of three independent experiments in which 18 wild-type or ced-3 worms were injected and then tracked for 3 days. The buffer and dyn-1 data represent single experiments. Eggs were deemed viable if the larvae developed to the L4 stage. RNAi also affects the number of eggs. Wild-type worms laid on average 270 eggs when injected with buffer, 81 eggs when injected with drp-1 RNAi, and 27 eggs when injected with dyn-1 RNAi. Those numbers were 259, 104, and 18 for ced-3 worms.
(B) Nuclei and mitochondria are arranged in an orderly pattern in the distal segment of the gonad of a wild-type C. elegans hermaphrodite. This worm was grown for 2 days on NGM agar with rhodamine 6G to stain mitochondria (red). The gonad was then dissected and nuclear DNA was stained with Hoechst (green). The nuclei line the perimeter of the gonad tube. Each nucleus is surrounded by mitochondria.
(C) drp-1 RNAi disrupted the distribution of mitochondria in the distal segment of the gonad of the injected worm. Hermaphrodites were injected with RNAi and grown for 2 days on NGM agar with rhodamine 6G to stain mitochondria (red). Their gonads were then dissected, and nuclear DNA was stained with Hoechst (green). The mitochondria formed large clusters. Injection of buffer did not affect the distributions of mitochondria or nuclei (data not shown). Scale bar = 5 μm.
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3
Mutant DRP-1 Disrupts Mitochondrial Morphology in C. elegans Muscle Cells
(A) Organelle markers were expressed in C. elegans body wall muscles using the myo-3 gene promoter. The markers that we used show: cytoplasm (GFP alone), nuclei (nuclear localization signal fused to GFP and β-galactosidase), ER (signal peptide fused to the N terminus of GFP and an ER retention signal fused to the C terminus), and mitochondria (mitochondrial leader sequence fused to GFP). Scale bar = 5 μm.
(B) The effect of mutant DRP-1 on mitochondrial morphology was detected in transgenic animals using mito::GFP expressed with the myo-3 promoter. Muscle cells without transgenic DRP-1 usually have tubular mitochondria that run parallel with the body axis (wt). Overexpression of DRP-1(K40A) causes the mitochondrial matrix to collapse into blebs (+K40A). Overexpression of DRP-1(V43F) also induces blebs, but these are often connected (+V43F). Overexpression of DRP-1(T61A) and antisense drp-1 cDNA causes mitochondria to collapse into blebs (+T61A and +antisense), similar to the blebs that were observed with DRP-1(K40A). Scale bar = 5 μm.
(C) The endoplasmic reticulum in body wall muscles is unaffected by DRP-1(K40A), while the mitochondria in those same cells are severely affected by DRP-1(K40A). The ER of C. elegans body wall muscles was labeled with YFP, and mitochondria are labeled with CFP. Separate images of mitochondria and ER are shown along with a merged image with ER in green and mitochondria in red. The panels on the left show a portion of a muscle cell without transgenic DRP-1. The ER is a fine net of thin tubules, which occasionally coalesce into small blebs. The panels on the right show that ER morphology is unaffected by DRP-1(K40A), but there are some large holes to accommodate large mitochondrial blebs. Scale bar = 5 μm.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4
Different Effects of Mutant and Wild-Type DRP-1 on the Mitochondrial Outer Membrane and the Mitochondrial Matrix
(A) Mitochondria in a muscle cell without transgenic DRP-1 (wt) have the normal tubular morphology. The mitochondrial outer membrane was detected with TOM70:YFP expressed with the myo-3 promoter. The mitochondrial matrix was detected with mito::CFP. The short tubes of mitochondrial matrix (red) are enveloped by mitochondrial outer membrane (green).
(B–E′) The overexpression of mutant DRP-1 causes the mitochondrial matrix to collapse into blebs (red), as seen in previous pictures. The mitochondrial outer membrane marker showed that these blebs were invariably connected to each other by thin tubes of outer membrane (green). In some cells, the nuclei were also stained (n). (B) shows a cell with DRP-1(V43F) (+V43F). (C) shows a cell with DRP-1(T61A) (+T61A). (D) shows a cell with antisense DRP-1 (+antisense). (E and E′) show cells with DRP-1(K40A) (+K40A).
(F and F′) Separate images obtained with the mitochondrial matrix marker in cells expressing DRP-1(K40A). These images correspond to the red in (E) and (E′). (E) shows an example of a cell in which the matrix blebs appear disconnected. (E′) shows an example of a cell in which matrix blebs have thin tubular connections. Approximately 20% of cells with DRP-1(K40A) have the latter phenotype.
(G and G′) Separate images obtained with the mitochondrial outer membrane marker in cells expressing DRP-1(K40A). These images, which correspond to the green in (F) and (F′), show that the outer membrane forms connections between the blebs.
(H and I) Mitochondria are excessively fragmented in cells that overexpressed wild-type DRP-1. (H) shows mitochondria labeled with the mitochondrial outer membrane marker in a cell with no transgenic DRP-1 (wt). (I) shows mitochondria in a cell that overexpresses wild-type DRP-1 (+wt DRP-1). These mitochondria are fragmented into many small spheres. Scale bar = 5 μm.
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5
Boundaries of the Mitochondrial Compartments Delineated by Photobleaching
(A, B, and C) The connections between mitochondrial matrix compartments was investigated with FLIP (fluorescence loss in photobleaching), which effectively drains contiguous compartments of fluorescent mito::GFP. Each cycle consists of bleaching a small area and then scanning a larger area to determine the effect of bleaching. The bleached areas are indicated by boxes. Fluorescence intensities were measured in the areas indicated by arrowheads. (A) shows images obtained with wild-type cells after 0, 2, or 5 min of bleaching cycles. Note that one mitochondrial tubule in the lower half of the pictures is also bleached, indicating that this particular segment is contiguous with mitochondria in the upper half of the picture (arrowhead 3). (B) shows the bleaching of cells with DRP-1(V43F). (C) shows the bleaching of cells with DRP-1(K40A). These images are representative of 5 experiments with wild-type cells, 16 with DRP-1(V43F), and 7 with DRP-1(K40A). Scale bar = 2 μm.
(D, E, and F) The fluorescence intensities of areas indicated by arrowheads in (A), (B), and (C) were determined after each bleaching cycle. The intensities of the specific areas relative to their intensities at 0 min were plotted against time. (D) shows plots obtained with wild-type cells. (E) shows plots obtained with DRP-1(V43F). (F) shows plots obtained with DRP-1(K40A). The rate at which fluorescence is lost from a specific area indicates how well that area is connected to the bleached area (boxes in [A], [B], and [C]).
Molecular Cell 1999 4, DOI: ( /S (00) )

7. Figure 6
Effect of Mutant DRP-1 on Mitochondrial Segregation in the M Lineage
(A and B) These panels show M cells at the one-cell stage. The pictures show the midsections of L1 larvae, expressing the mitochondrial outer membrane marker (TOM70::GFP) under control of the hlh-8 promoter. The left panel shows the distribution of mitochondria in the M cell of an animal without transgenic DRP-1 (A). The right panel shows that mitochondria are collapsed into a cluster in an M cell that overexpresses DRP-1(K40A) (B). The positions of the nuclei are indicated by (n).
(C and D) These panels show M cells at the two-cell stage (approximately 5 hr after hatching). The left panel shows the midsection of a worm without transgenic DRP-1 (C). The mitochondria are clearly divided between the two daughters of the M cell. The right panel shows the midsection of a worm overexpressing DRP-1(K40A) in the M lineage (D). Most of the mitochondria are distributed between the two daughter cells. These two cells are connected by a strand of mitochondrial outer membrane. This strand was observed in almost all cells expressing DRP-1(K40A), but never in wild-type worms.
Molecular Cell 1999 4, DOI: ( /S (00) )

8. Figure 7
Colocalization of GFP::DRP-1 with Mitochondrial Scission Events
(A and B) The distribution of wild-type DRP-1 was detected with GFP fused to the N terminus of wild-type DRP-1 (green). The mitochondrial markers are shown in red. The mitochondrial matrix was detected with mito::CFP (A), and the mitochondrial outer membrane was detected with TOM70::CFP (B). GFP::DRP-1 was concentrated in small patches that were invariably localized to mitochondria. Many of the GFP:DRP-1 spots were localized to places where the mitochondrial outer membrane was constricted (B). Scale bar = 2 μm.
(C and D) Mitochondrial scission was observed by time lapse photography of muscle cells labeled with a mitochondrial matrix marker (red) and GFP::DRP-1 (green). Scission was induced by incubating the worms with PMA (C) or by raising the temperature from 15°C to 25°C (D). Muscle cells were photographed at the indicated times. Arrows show where scission is about to occur or has just occurred. The left arrow in (D) shows a spot that disappears after scission. This arrow also serves as a reference point to show movement of the fluorescent spot indicated by the right arrow. Scale bar = 2 μm.
Molecular Cell 1999 4, DOI: ( /S (00) )