Uncoupled mitochondria quickly shorten along their long axis to form indented spheroids, instead of rings, in a fission-independent manner

Abstract

Loss of mitochondrial membrane potential (ΔΨm) triggers dramatic structural changes in mitochondria from a tubular to globular shape, referred to as mitochondrial fragmentation; the resulting globular mitochondria are called swelled or ring/doughnut mitochondria. We evaluated the early period of structural changes during the ΔΨm loss-induced transformation after carbonyl cyanide m-chlorophenyl hydrazine (CCCP) administration using a newly developed correlative microscopic method combined with fluorescence microscopic live imaging and volume electron microscopy. We found that most mitochondria changed from a tubular shape to a globular shape without fusion or fission and typically showed ring shapes within 10 min after CCCP exposure. In contrast, most ring mitochondria did not have a true through hole; rather, they had various indents, and 47% showed stomatocyte shapes with vase-shaped cavities, which is the most stable physical structure without any structural support if the long tubular shape shortens into a sphere. Our results suggested that loss of ΔΨm triggered collapse of mitochondrial structural support mechanisms.

Figure 1

Confocal fluorescence and transmission electron microscopic (TEM) images of mitochondria in MEFs and HeLa cells 10 min after treatment with 10 μM CCCP. Confocal fluorescence images (a) of MEFs labelled with Su9 and HeLa cells labelled with GFP-PDHA showed typical tubular mitochondria before administration of CCCP (a1, a2, a5, and a6). After CCCP treatment, most mitochondria showed small globular shapes in lower-magnification images (a3 and a7) and ring shapes in higher-magnification images (a4, a8) in MEFs and HeLa cells. TEM observations of CCCP-treated cells (b) showed distinct U-, C-, and ring-shaped mitochondria in both cell types 10 min after administration. Each mitochondrion had an intact mitochondrial membrane and cristae structures. Some mitochondria had single or multiple lumina (b5 and b6). No differences were observed between cell lines. Scale bars, 0.5 μm (b) and 2 μm (a).

Figure 2

Time-lapse images of the mitochondrial transformation after CCCP administration observed by confocal microscopy. The time-lapse observations were started before administration, and the time elapsed after administration is indicated under each photograph. The series in (a) shows acute morphological changes in mitochondria from slender tubes to a ring-like structure within 1 min. The series in (b–f) shows that the ring-shape originated from the midpoint of mitochondrial tubes and that the diametre of the ring increased gradually. Scale bar, 2 μm.

Figure 3

Confocal fluorescence and TEM images of mitochondria in Drp1-knockout Su9-RFP MEFs 10 min after treatment with 10 μM CCCP. Confocal fluorescence images (a,b,c,d) in Drp1-KO MEFs labelled with Su9 showed typical tubular mitochondria before administration of CCCP at lower and higher magnification (a,c). After CCCP treatment, most mitochondria showed small globular and ring shapes at a lower magnification (b) and higher magnification (d). TEM observations of CCCP-treated Drp1-KO MEFs (e–j) showed U-, C-, and ring-shaped mitochondria 10 min after administration. Scale bars,10 μm for a and b and 2 μm for c and d, and 0.2 μm for e–j.

Figure 4

Schematic representation of 3D volume CLEM combined with live imaging. We developed a new technique to observe mitochondrial transformation after uncoupling in the acute stage. Cells were fixed during time lapse observation on a grid-marked 35-mm dish and then embedded in resin. The specimens were immersed in toluene to remove the dish. Subsequently, the cellular shape on the undersurface of resin-embedded cells was observed by SEM at a high acceleration voltage for relocation. The same region in light microscopy and electron microscopy analyses was reconstructed by FIB-SEM tomography at a high spatial resolution.

Figure 5

Correlative observation and interpretation of the structure of CCCP-treated mitochondria in MEFs using live imaging combined with 3D-CLEM. The last frame of live imaging of mitochondria after CCCP treatment showed the globular and ring-shaped mitochondria (red square in a1), which transformed from tube-shaped mitochondria. The whole frame of live imaging is shown in Supplemental Movie 7. Identical mitochondria were reconstructed by FIB-SEM tomography, and the same area is displayed in a2. The volume rendered view of the mitochondrion from the flipped direction shows the cup-shaped feature (a3), and some cristae were observed in cross-sections of the volume (a4). (b,c) Serial slice images of the volume data from different directions. Both discoidal mitochondria and vase-like stomatocyte-shaped mitochondria 10 min after CCCP treatment showed a ring shape in fluorescence microscopy (5d,5e). Two ring-shaped mitochondria (red squares) were selected under fluorescence microscopy, and identical mitochondria were reconstructed using the 3D-CLEM method based on FIB-SEM tomography. One showed a discoidal shape but did not have a through hole or invagination of the cytosol (d). The cross-section through the equator plane showed an erythrocyte-like biconcave shape and a very thin matrix at section 149, almost in contact with the opposite side of the membrane (arrow in section 149). The other (e) shows a large lumen connected to the cytosol through a small orifice (e, arrowhead). The translucent surface view shows the stomatocyte shape, also referred to as the mitochondrial spheroid structure (e), which also had a thin matrix portion (arrow in section 473) in the deepest region of the invagination. This shape was categorised as a “vase” in this work. Scale bar, 2 μm for CLSM images, 1 μm for others.

Figure 6

Mitochondrial shape 10 min after CCCP treatment evaluated by FIB-SEM tomography. Fifty-three randomly selected mitochondria (of a total of 226) from MEFs are shown (a). The shape of the mitochondria was categorised into seven groups, with adjacent images showing solid and translucent views of the surface rendering to observe invaginations. The proportion belonging to each group is shown in chart (b), and most mitochondria showed a vase shape (123/226). Structural characteristics of the orifice diameter (c) and the thinnest thickness of the mitochondrial matrix (d) in vase-shaped mitochondria are shown in histograms. Scale bar, 1 μm.

Figure 7

Three-dimensional interactions between the mitochondrial lumen and endoplasmic reticulum (ER) through the orifice in CCCP-treated vase-shaped mitochondria. In serial cross-sections by FIB-SEM (a), the endoplasmic reticulum occasionally appeared in the lumen of vase-shaped mitochondria induced by CCCP, and the ER frequently contacted the outer membrane of mitochondrial invaginations (arrows). The 3D reconstruction showed that the ER rose from the cytosol, entered the lumen of the mitochondria through the orifice, and spread into the lumen (b). Similar mitochondria with ER-associated lumina were observed in 14.6% of vase-shaped mitochondria. Scale bar, 1 μm.

Figure 8

True shape of spherical mitochondria observed after CCCP treatment. Ring-shaped mitochondria were observed in fluorescent microscopy, as shown in the upper row, 10 min after CCCP treatment. Multiple void-like regions (or holes in rings) were sometimes observed. According to our observations, 1–3 lumina were present in TEM photomicrographs (middle row).