Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.     

Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.     

The BH3‐only Bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms

Opa1 modulates mitochondrial fusion, cristae structure and apoptosis. The relationships between these functions and autosomal dominant optic atrophy, caused by mutations in Opa1, are poorly defined. We show that Bnip3 interacts with Opa1, leading to mitochondrial fragmentation and apoptosis. Fission is due to inhibition of Opa1‐mediated fusion and is counteracted by Opa1 in an Mfn1‐dependent manner. Bnip3–Opa1 interaction is necessary to trigger Opa1 complex disruption in a Bax‐ and/or Bak‐dependent manner, ultimately leading to apoptosis. Our results uncover a direct link between Opa1 on the inner mitochondrial membrane and the apoptotic machinery on the outer membrane that modulates fusion and cristae structure by separate mechanisms. These findings might help to unravel optic atrophy aetiology as retinal ganglion cells are particularly prone to hypoxia, an inductor of Bnip3 expression.     

Structural and mechanistic basis of Parl activity and regulation

The mitochondrial rhomboid protease Parl governs apoptosis, morphology, metabolism and might be implicated in Parkinson's disease, but the structural basis of its activity and complex regulation remain unknown. We report the discovery of γ-cleavage, a proteolytic event on the loop connecting the first transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the protease. This cleavage disrupts the '1+6' structure that defines every mitochondrial rhomboid and generates a new form of Parl, PROD (Parl-rhomboid-domain). Structure-function analysis of Parl suggests that γ-cleavage could be implicated in eliminating Parl proteolytic activity, and structural modeling of PROD reveals structural conservation with the bacterial rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based mechanism of catalysis, Parl appears to use a conserved mitochondrial rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis. This work provides unexpected insights into the structural determinants regulating Parl stability and activity in vivo, and reveals a complex cascade of proteolytic events controlling the function of the protease in the mitochondrion.     

The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.     

Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts

During human pregnancy, placental trophoblasts differentiate and syncytialize into syncytiotrophoblasts that sustain progesterone production [1]. This process is accompanied by mitochondrial fragmentation and cristae remodeling [2], two facets of mitochondrial apoptosis, whose molecular mechanisms and functional consequences on steroidogenesis are unclear. Here we show that the mitochondria-shaping protein Optic atrophy 1 (Opa1) controls efficiency of steroidogenesis. During syncytialization of trophoblast BeWo cells, levels of the profission mitochondria-shaping protein Drp1 increase, and those of Opa1 and mitofusin (Mfn) decrease, leading to mitochondrial fragmentation and cristae remodeling. Manipulation of the levels of Opa1 reveal an inverse relationship with the efficiency of steroidogenesis in trophoblasts and in mouse embryonic fibroblasts where the mitochondrial steroidogenetic pathway has been engineered. In an in vitro assay, accumulation of cholesterol is facilitated in the inner membrane of isolated mitochondria lacking Opa1. Thus, Opa1-dependent inner membrane remodeling controls efficiency of steroidogenesis.     

Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila

Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. Our results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.     

Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis

The endoplasmic reticulum (ER) can elicit proapoptotic signalling that results in transmission of Ca(2+) to the mitochondria, which in turn stimulates recruitment of the fission enzyme DRP1 to the surface of the organelle. Here, we show that BH3-only BIK activates this pathway at the ER in intact cells, resulting in mitochondrial fragmentation but little release of cytochrome c to the cytosol. The BIK-induced transformations in mitochondria are dynamic in nature and involve DRP1-dependent remodelling and opening of cristae, where the major stores of cytochrome c reside. This novel function for DRP1 is distinct from its recognized role in regulating mitochondrial fission. Selective permeabilization of the outer membrane with digitonin confirmed that BIK stimulation results in mobilization of intramitochondrial cytochrome c. Of note, BIK can cooperate with a weak BH3-only protein that targets mitochondria, such as NOXA, to activate BAX by a mechanism that is independent of DRP1 enzyme activity. When expressed together, BIK and NOXA cause rapid release of mobilized cytochrome c and activation of caspases.     

Normal mitochondrial dynamics requires rhomboid-7 and affects Drosophila lifespan and neuronal function

In addition to being energy generators, mitochondria control many cellular processes including apoptosis. They are dynamic organelles, and the machinery of membrane fusion and fission is emerging as a key regulator of mitochondrial biology. We have recently identified a novel and conserved mitochondrial rhomboid intramembrane protease that controls membrane fusion in Saccharomyces cerevisiae by processing the dynamin-like GTPase, Mgm1, thereby releasing it from the membrane. The genetics of mitochondrial membrane dynamics has until now focused primarily on yeast. Here we show that in Drosophila, the mitochondrial rhomboid (Rhomboid-7) is required for mitochondrial fusion during fly spermatogenesis and muscle maturation, both tissues with unusual mitochondrial dynamics. We also find that mutations in Drosophila optic atrophy 1-like (Opa1-like), the ortholog of yeast mgm1, display similar phenotypes, suggesting a shared role for Rhomboid-7 and Opa1-like, as with their yeast orthologs. Loss of human OPA1 leads to dominant optic atrophy, a mitochondrial disease leading to childhood onset blindness. rhomboid-7 mutant flies have severe neurological defects, evidenced by compromised signaling across the first visual synapse, as well as light-induced neurodegeneration of photoreceptors that resembles the human disease. rhomboid-7 mutant flies also have a greatly reduced lifespan.     

Loss of mitofusin 2 promotes endoplasmic reticulum stress

The outer mitochondrial membrane GTPase mitofusin 2 (Mfn2) is known to regulate endoplasmic reticulum (ER) shape in addition to its mitochondrial fusion effects. However, its role in ER stress is unknown. We report here that induction of ER stress with either thapsigargin or tunicamycin in mouse embryonic fibroblasts leads to up-regulation of Mfn2 mRNA and protein levels with no change in the expression of the mitochondrial shaping factors Mfn1, Opa1, Drp1, and Fis1. Genetic deletion of Mfn2 but not Mfn1 in mouse embryonic fibroblasts or cardiac myocytes in mice led to an increase in the expression of the ER chaperone proteins. Genetic ablation of Mfn2 in mouse embryonic fibroblasts amplified ER stress and exacerbated ER stress-induced apoptosis. Deletion of Mfn2 delayed translational recovery through prolonged eIF2α phosphorylation associated with decreased GADD34 and p58(IPK) expression and elevated C/EBP homologous protein induction at late time points. These changes in the unfolded protein response were coupled to increased cell death reflected by augmented caspase 3/7 activity, lactate dehydrogenase release from cells, and an increase in propidium iodide-positive nuclei in response to thapsigargin or tunicamycin treatment. In contrast, genetic deletion of Mfn1 did not affect ER stress-mediated increase in ER chaperone synthesis or eIF2α phosphorylation. Additionally, ER stress-induced C/EBP homologous protein, GADD34, and p58(IPK) induction and cell death were not affected by loss of Mfn1. We conclude that Mfn2 but not Mfn1 is an ER stress-inducible protein that is required for the proper temporal sequence of the ER stress response.     

Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency

Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle‐specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF‐κB activation, and enhanced expression of pro‐inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF‐κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell‐autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF‐κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF‐κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.     

Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.     

MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria

In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles which form a reticulum around the cell periphery. To determine the mechanism by which mitochondrial shape is established and maintained, we screened yeast mutants for those defective in mitochondrial morphology. One of these mutants, mmm1, is temperature- sensitive for the external shape of its mitochondria. At the restrictive temperature, elongated mitochondria appear to quickly collapse into large, spherical organelles. Upon return to the permissive temperature, wild-type mitochondrial structure is restored. The morphology of other cellular organelles is not affected in mmm1 mutants, and mmm1 does not disrupt normal actin or tubulin organization. Cells disrupted in the MMM1 gene are inviable when grown on nonfermentable carbon sources and show abnormal mitochondrial morphology at all temperatures. The lethality of mmm1 mutants appears to result from the inability to segregate the aberrant-shaped mitochondria into daughter cells. Mitochondrial structure is therefore important for normal cell function. Mmm1p is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytosol. We propose that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton.     

线粒体分裂、融合与细胞凋亡

线粒体是高度动态变化的细胞器,其在细胞内不断分裂、融合并形成网状结构。线粒体的分裂和融合是由多种蛋白质精确调控完成的。Drp1／Dnm1p,Fis1／Fis1p,Caf4p和Mdv1p参与线粒体分裂的调控;Mfn1／2／Fzo1p控制线粒体外膜的融合,而Mgm1p／OPA1则参与线粒体内膜的融合。在细胞凋亡过程中线粒体片段化,网状结构被破坏,线粒体嵴发生重构,抑制这一过程可以部分抑制细胞色素c的释放和细胞凋亡。线粒体形态对于细胞维持正常生理代谢和机体发育起着重要的作用,一旦出现障碍会导致严重的疾病。     

Caspase-Cleaved Tau Impairs Mitochondrial Dynamics in Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by the presence of aggregates of tau protein. Tau truncated by caspase-3 (D421) or tau hyperphosphorylated at Ser396/S404 might play a role in the pathogenesis of AD. Mitochondria are dynamic organelles that modify their size and function through mitochondrial dynamics. Recent studies have shown that alterations of mitochondrial dynamics affect synaptic communication. Therefore, we studied the effects of pathological forms of tau on the regulation of mitochondrial dynamics. We used primary cortical neurons from tau(?/?) knockout mice and immortalized cortical neurons (CN1.4) that were transfected with plasmids containing green fluorescent protein (GFP) or GFP with different tau forms: full-length (GFP-T4), truncated (GFP-T4C3), pseudophosphorylated (GFP-T42EC), or both truncated and pseudophosphorylated modifications of tau (GFP-T4C3-2EC). Cells expressing truncated tau showed fragmented mitochondria compared to cells that expressed full-length tau. These findings were corroborated using primary neurons from tau(?/?) knockout mice that expressed the truncated and both truncated and pseudophosphorylated forms of tau. Interestingly, mitochondrial fragmentation was accompanied by a significant reduction in levels of optic atrophy protein 1 (Opa1) in cells expressing the truncated form of tau. In addition, treatment with low concentrations of amyloid-beta (Aβ) significantly reduced mitochondrial membrane potential, cell viability, and mitochondrial length in cortical cells and primary neurons from tau(?/?) mice that express truncated tau. These results indicate that the presence of tau pathology impairs mitochondrial dynamics by reducing Opa1 levels, an event that could lead to mitochondrial impairment observed in AD.     

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1‐like protein

Dynamin 1‐like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.     

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin‐related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin‐related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1‐knockout cells. Re‐activation of Opa1 and Mitofusin 1 in Drp1‐knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.     

A proteomic screen with Drosophila Opa1-like identifies Hsc70-5/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis

Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission processes. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. Here, we show Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with ‘mitochondria-shaping’ proteins like Mitochondrial assembly regulatory factor (Marf) and Drosophila Mitofilin (dMitofilin). To gain an insight into Opa1-like's network, we delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (Hsc70-5) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of hsc70-5 in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in hsc70-5 RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing hsc70-5 RNAi. Taken together, we have identified a novel interaction between Hsc70-5/Mortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.