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. 2008 Aug 29;283(35):24128-35.
doi: 10.1074/jbc.M802996200. Epub 2008 Jul 1.

VSports注册入口 - Mitochondrial Extrusion through the cytoplasmic vacuoles during cell death

Akihito Nakajima  1 , Hidetake Kurihara, Hideo Yagita, Ko Okumura, Hiroyasu Nakano
Affiliations

VSports注册入口 - Mitochondrial Extrusion through the cytoplasmic vacuoles during cell death

VSports手机版 - Akihito Nakajima et al. J Biol Chem. .

Abstract (VSports手机版)

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing VSports手机版. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology. .

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Figures

FIGURE 1.
FIGURE 1.
Fragmented mitochondria are engulfed by the cytoplasmic vacuoles and extruded from c-Flip-/- MEFs during cell death. A and B, c-Flip-/- MEFs stably expressing GFP-COX IV were unstimulated (A) or stimulated with TNFα for 60 min (B). Scale bars, 25 μm. C and D, c-Flip-/- MEFs were stimulated with TNFα for 60 (C) or 90 (D) min and analyzed by transmission electron microscopy. The enlarged images of the boxed areas are shown in the right panels. The arrowheads and arrows indicate extruded and fragmented mitochondria, respectively. Scale bars, 500 nm. E, the percentages of cells showing typical apoptosis, necrosis, and atypical apoptosis characterized by numerous cytoplasmic vacuoles and mitochondrial extrusion were calculated by counting randomly selected areas (total 100-200 cells/sample). Three independently prepared samples were counted and are presented as the means ± S.D. F, c-Flip-/- MEFs were stimulated with TNFα for 90 min and then analyzed by immunoelectron microscopy using anti-COX IV antibody, followed by 10-nm colloidal gold-conjugated secondary antibody. Scale bar, 200 nm.
FIGURE 2.
FIGURE 2.
A genotoxic stress-inducing agent does not elicit mitochondrial extrusion in c-Flip-/- MEFs. c-Flip-/- MEFs were stimulated with cisplatin for 16 h and analyzed by transmission electron microscopy. The enlarged image of the boxed area is shown in the right panel. N, nucleus. M, mitochondria. Scale bars, 2 μm.
FIGURE 3.
FIGURE 3.
TNFα-induced mitochondrial extrusion, but not mitochondrial fragmentation, is suppressed in the presence of cytochalasin D or paclitaxel. A and B, c-Flip-/- MEFs were stimulated with TNFα for 60 min in the presence of cytochalasin D (A) or paclitaxel (B) and analyzed by transmission electron microscopy. The enlarged images of the boxed areas are presented in the right panels. N, nucleus. M, mitochondria. Scale bars, 2 μm. C, TNFα-induced caspase 3 activities are not inhibited in the presence of cytochalasin D or paclitaxel. c-Flip-/- MEFs were stimulated with TNFα for 60 min in the absence or presence of cytochalasin D or paclitaxel, and caspase 3 activities were measured by using fluorogenic substrates. The results are presented as the means ± S.D. of triplicate samples.
FIGURE 4.
FIGURE 4.
The caspase-dependent pathway plays a crucial role in mitochondrial extrusion. A, c-Flip-/- MEFs were stimulated with TNFα for 60 min in the presence of Z-VAD-fmk and analyzed by transmission electron microscopy. Mitochondria showing normal structures are shown in the boxed area. N, nucleus. M, mitochondria. Scale bars, 2 μm. B, wild-type, c-Flip-/- MEFs, and c-Flip-/- c-FLIPL MEFs were stimulated with TNFα for 90 min, and caspase 3 activities were measured by using fluorogenic substrates. The results are presented as the means ± S.D. of triplicate samples. C and D, wild-type and c-Flip-/- c-FLIPL MEFs were stimulated as in B and analyzed by transmission electron microscopy. N, nucleus. E, c-Flip-/- MEFs were unstimulated (thin lines) or stimulated (bold lines) with TNFα in the absence or presence of Z-VAD-fmk or butylated hydroxyanisole for 2 h, and then the cells were labeled with CM-H2DCFDA and analyzed by flow cytometry.
FIGURE 5.
FIGURE 5.
Plasma membranes are involved in vacuole formation. A, c-Flip-/- MEFs were stimulated with TNFα for 90 min and analyzed by transmission electron microscopy. The red arrowheads indicate extruded mitochondria. Scale bar, 100 nm. B, c-Flip-/- MEFs were unstimulated or stimulated with TNFα for 90 min. Then the cells were stained with FM1-43FX (green) and MitoTracker (red). The white arrowheads indicate colocalization of the plasma membrane and mitochondria. Scale bars, 10 μm. C, c-Flip-/- MEFs were stimulated and analyzed as in A. The electron microscopical images corresponding to normal (top panel), typical apoptosis characterized by membrane blebbing (middle panel), and atypical apoptosis containing numerous cytoplasmic vacuoles (bottom panel) are shown. Scale bars, 1 μm.
FIGURE 6.
FIGURE 6.
Autophagy does not play a major role in mitochondrial extrusion. A, c-Flip-/- MEFs were unstimulated or stimulated with TNFα for 90 min. Then the cells were fixed and immunostained with anti-LC3 antibody (green), and the nuclei were stained with Hoechst 33258 (blue). N, nucleus. Scale bar, 10 μm. B, c-Flip-/- MEFs were untreated or treated with TNFα, 3-MA, or TNFα plus 3-MA for 90 min. The cell lysates were analyzed by immunoblotting (IB) with anti-LC3 antibody. The arrows indicate LC3-I and LC3-II. The equal loading of the samples was verified by Western blotting with anti-tubulin antibody. The molecular mass markers are shown on the left. C, c-Flip-/- MEFs were stimulated with TNFα plus 3-MA for 90 min and analyzed by transmission electron microscopy. The enlarged image of the red box is presented in the right panel. The red arrowheads indicate extruded mitochondria. Scale bar, 1 μm. D, c-Flip-/- MEFs were untreated or treated as in B, and caspase 3 activities were measured by using fluorogenic substrates. The results are presented as the means ± S.D. of triplicate samples. E, c-Flip-/- MEFs were stimulated as in A. Then the cells were stained with LysoTracker (green) and MitoTracker (red). Scale bars, 10 μm. F, c-Flip-/- MEFs were stimulated as in A. Then the cells were fixed and immunostained with anti-Lamp1 (green) and anti-COX IV (red) antibodies. Scale bars, 10 μm.
FIGURE 7.
FIGURE 7.
Fragmented mitochondria are engulfed in the cytoplasmic vacuoles and extruded from hepatocytes after anti-Fas antibody injection. The mice were injected with PBS or anti-Fas antibody and sacrificed at 4 h after injection. A, histological analysis of the livers. The sections were stained with toluidine blue (×100). B and C, ultrastructural analysis of the liver. Normal and fragmented mitochondria are shown in the red boxes (B). The enlarged image of the boxed area is shown in C. The arrowheads and arrow indicate fragmented and extruded mitochondria, respectively. N, nucleus. S, sinusoid. Scale bars, 500 nm. D, release of COX IV in the sera of mice injected with anti-Fas antibody. The sera were collected at 4 h after injection and analyzed by immunoblotting (IB) with anti-COX IV (upper panel) and anti-mouse Ig (lower panel) antibodies. IgH and IgL indicate Ig heavy and light chains, respectively. The numbers indicate individual mice. The arrow indicates COX IV. The asterisks indicate nonspecific bands. The molecular mass markers are shown on the left. E, GM130, a cis-Golgi marker does not present in the sera. The same sera used in D were analyzed by Western blotting with anti-GM130 antibody. As a positive control, total lysates of the liver were applied to the same gel (L). The numbers indicate individual mice. The arrow indicates GM130. The asterisks indicate nonspecific bands. The molecular mass markers are shown on the left.
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