Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.     

SIRT1: Regulation of longevity via autophagy

Recent studies have emphasized the importance of SIRT1, a mammalian homolog of Sir2 longevity factor, in the regulation of metabolism, cellular survival, and organismal lifespan. The signaling network interacting with SIRT1 continues to expand as does the number of functions known to be regulated by SIRT1. Autophagy is also an emerging field in longevity studies. Autophagocytosis is a housekeeping mechanism cleaning cells from aberrant and dysfunctional molecules and organelles. The extension of lifespan has been linked to the efficient maintenance of autophagic degradation, a process which declines during aging. Interestingly, recent observations have demonstrated that SIRT1 regulates the formation of autophagic vacuoles, either directly or indirectly through a downstream signaling network. We will examine the signaling pathways linking SIRT1 to the regulation of autophagic degradation. The interactions of SIRT1 with the FoxO and p53 signaling can also regulate both the autophagic degradation and lifespan extension emphasizing the key role of autophagy in the regulation of lifespan.     

Autophagy and signaling: their role in cell survival and cell death

Macroautophagy is a vacuolar, self-digesting mechanism responsible for the removal of long-lived proteins and damaged organelles by the lysosome. The discovery of the ATG genes has provided key information about the formation of the autophagosome, and about the role of macroautophagy in allowing cells to survive during nutrient depletion and/or in the absence of growth factors. Two connected signaling pathways encompassing class-I phosphatidylinositol 3-kinase and (mammalian) target of rapamycin play a central role in controlling macroautophagy in response to starvation. However, a considerable body of literature reports that macroautophagy is also a cell death mechanism that can occur either in the absence of detectable signs of apoptosis (via autophagic cell death) or concomitantly with apoptosis. Macroautophagy is activated by signaling pathways that also control apoptosis. The aim of this review is to discuss the signaling pathways that control macroautophagy during cell survival and cell death.     

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.     

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.     

To die or not to die: that is the autophagic question

Macroautophagy (commonly referred to as autophagy) is the process by which intact organelles and/or large portions of the cytoplasm are engulfed within double-membraned autophagic vacuoles for degradation. Whereas basal levels of autophagy ensure the physiological turnover of old and damaged organelles, the massive accumulation of autophagic vacuoles may represent either an alternative pathway of cell death or an ultimate attempt for cells to survive by adapting to stress. The activation of the autophagic pathway beyond a certain threshold may promote cell death directly, by causing the collapse of cellular functions as a result of cellular atrophy (autophagic, or type II, cell death). Alternatively, autophagy can lead to the execution of apoptotic (type I) or necrotic (type III) cell death programs, presumably via common regulators such as proteins from the Bcl-2 family. On the other hand, limited self-eating can provide cells with metabolic substrates to meet their energetic demands under stressful conditions, such as nutrient deprivation, or favor the selective elimination of damaged (and potentially dangerous) organelles. In these instances, autophagy operates as a pro-survival mechanism. The coordinate regulation of these opposite effects of autophagy relies upon a complex network of signal transducers, most of which also participate in non-autophagic signaling cascades. Thus, autophagy occupies a crucial position within the cell's metabolism, and its modulation may represent an alternative therapeutic strategy in several pathological settings including cancer and neurodegeneration. Here, we present a general outline of autophagy followed by a detailed analysis of organelle-specific autophagic pathways and of their intimate connections with cell death.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.     

Distribution of electric charges and concanavalin A binding sites on autophagic vacuoles and lysosomes in mouse hepatocytes

The distributions of electric charges and Concanavalin A binding sites in autophagic vacuoles and lysosomes in mouse hepatocytes were studied by utilizing a frozen ultrathin section labeling method with cationized ferritin (CF) or anionized ferritin and ferritin-conjugated Concanavalin A (Con A-F) as visual probes. Our observations revealed that the inner surface of the autophagic vacuole membrane has more anionic sites (CF binding) than other organelle membranes. This suggests that if the limiting membranes of autophagic vacuoles originate from preexisting membranes, such membranes must undergo structural and compositional alternation during the formation of the autophagic vacuoles. In contrast to CF, Con A-F showed no distinct binding to the membranes of autophagic vacuoles, but the contents of vacuoles displayed varying Con A-F binding, depending on the stage of the autophagic process. Increased binding was seen in more mature autophagic vacuoles. Since lysosomes showed a preferential accumulation of Con A-F particles, molecules with Con A-F binding sites in autophagic vacuoles may be of lysosomal origin. Con A-F distribution varied from lysosome to lysosome in the same cell, indicating heterogeneity of lysosomal contents. These results suggest that ferritin-conjugated lectin labeling methods applied to frozen, ultrathin section are a useful new approach in analyzing the natural history of autophagic vacuoles and the heterogeneity of lysosomes.     

Loss of Pten,a tumor suppressor,causes the strong inhibition of autophagy without affecting LC3 lipidation

1Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [14C]-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microsopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.     

c-myc induces autophagy in rat 3Y1 fibroblast cells

The proto-oncogene c-myc is a multifunctional gene that regulates cell division, cell growth, and apoptosis. Here we report a new function of c-myc: induction of autophagy. Autophagy is a bulk degradation system for intracellular proteins. Autophagy proceeds with characteristic morphologies, which begins with the formation of a double-membrane structure called the autophagosome surrounding a portion of the cytoplasm, after which its outer membrane then fuses with the lysosomal membrane to become an autolysosome. Autophagosomes and autolysosomes are generally called autophagic vacuoles. When c-Myc protein was overexpressed in rat 3Y1 fibroblasts or when the chimeric protein c-MycER was activated by estrogen, the number of autophagic vacuoles in cells increased significantly. The formation of autophagic vacuoles induced by c-Myc was completely blocked by a specific inhibitor of autophagosome formation, 3-methyladenine. A c-Myc mutant lacking Myc Box II induced neither apoptosis nor oncogenic transformation, but still stimulated autophagy. An inhibitor of caspases suppressed apoptosis but not autophagy. These results suggest that the autophagy caused by c-myc is not due to the apoptosis or tumorigenesis induced by c-myc. Taken together, our results suggest that the induction of autophagy is a novel function of c-myc.     

Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death

Autophagic cell death is characterized by the accumulation of vacuoles in physiological and pathological conditions. However, its molecular event is unknown. Here, we show that Atg5, which is known to function in autophagy, contributes to autophagic cell death by interacting with Fas-associated protein with death domain (FADD). Down-regulation of Atg5 expression in HeLa cells suppresses cell death and vacuole formation induced by IFN-gamma. Inversely, ectopic expression of Atg5 using adenoviral delivery induces autophagic cell death. Deletion mapping analysis indicates that procell death activity resides in the middle and C-terminal region of Atg5. Cells harboring the accumulated vacuoles triggered by IFN-gamma or Atg5 expression become dead, and vacuole formation precedes cell death. 3-Methyladenine or expression of Atg5(K130R) mutant blocks both cell death and vacuole formation triggered by IFN-gamma, whereas benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk) inhibits only cell death but not vacuole formation. Atg5 interacts with FADD via death domain in vitro and in vivo, and the Atg5-mediated cell death, but not vacuole formation, is blocked in FADD-deficient cells. These results suggest that Atg5 plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD.     

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.     

Loss of Pten, a tumor suppressor, causes the strong inhibition of autophagy without affecting LC3 lipidation

(1)Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [(14)C-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microscopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.     

Autophagy gene disruption reveals a non-vacuolar cell death pathway in Dictyostelium

Types of cell death include apoptosis, necrosis, and autophagic cell death. The latter can be defined as death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Are autophagy and vacuolization causes, consequences, or side effects in cell death with autophagy? Would control of autophagy suffice to control this type of cell death? We disrupted the atg1 autophagy gene in Dictyostelium discoideum, a genetically tractable model for developmental autophagic vacuolar cell death. The procedure that induced autophagy, vacuolization, and death in wild-type cells led in atg1 mutant cells to impaired autophagy and to no vacuolization, demonstrating that atg1 is required for vacuolization. Unexpectedly, however, cell death still took place, with a non-vacuolar and centrally condensed morphology. Thus, a cell death mechanism that does not require vacuolization can operate in this cell death model showing conspicuous vacuolization. The revelation of non-vacuolar cell death in this protist by autophagy gene disruption is reminiscent of caspase inhibition revealing necrotic cell death in animal cells. Thus, hidden alternative cell death pathways may be found across kingdoms and for diverse types of cell death.     

Formation and fate of ethionine-induced cytoplasmic crystalloids in rat parotid acinar cells.

The formation and fate of cytoplasmic crystalloids in rat parotid acinar cells were investigated during ethionine intoxication and recovery. By day 3 of ethionine treatment, acinar cells had numerous autophagic vacuoles containing recognizable secretory granules and fragments of rough endoplasmic reticulum. By day 5, immature crystalloids were present in many of the autophagic vacuoles, and as the crystalloids matured, a 7-nm periodicity became apparent. Crystalloids were never observed in the Golgi saccules or in any other organelle associated with secretory granule formation. When ethionine treatment was stopped, the acinar cells rapidly returned to their normal morphology. The majority of the crystalloids and autophagic vacuoles were lost from the cells during the first two to three days of recovery. At this time annulate lamellae were present intracellularly, and macrophages, many containing crystalloids, were associated with the basal surface of the acinar cells. These results indicate that the cytoplasmic crystalloids are formed in autophagic vacuoles, and do not represent an abnormal secretory product. Additiontionally, during recovery crystalloids may be removed from the acinar cells by interaction with macrophages. The sequence of autophagic vacuole formation, development of crystalloids, macrophage infiltration and phagocytosis of acinar cell debris appears to be a non-specific response of the rat parotid gland to cellular injury occurring in a variety of experimental and pathological conditions.     

Regulation and role of autophagy in mammalian cells

The recent period has witnessed progress in the understanding of the lysosomal autophagic pathway. The discovery of a family of genes conserved from yeast to humans, and involved in the formation of autophagosomes, has unraveled new protein-conjugation systems and has shed light on the importance of autophagy in physiology and pathophysiology. The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of autophagy during cell death. As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) as well as intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of autophagy. The mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals. In addition, autophagy, which is characterized by a flux of membrane from the formation of the autophagosome to the fusion with the lysosome, is regulated by GTPases, similarly to the vesicular transport along the exocytic/endocytic pathway. The aim of the present review is to give an overview of autophagy and to discuss its regulation by activators and effectors of mTOR and GTPases.     

A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells

Cancer develops when molecular pathways that control the fine balance between proliferation, differentiation, autophagy and cell death undergo genetic deregulation. The prospects for further substantial advances in the management of colorectal cancer reside in a systematic genetic and functional dissection of these pathways in tumor cells. In an effort to evaluate the impact of p38 signaling on colorectal cancer cell fate, we treated HT29, Caco2, Hct116, LS174T and SW480 cell lines with the inhibitor SB202190 specific for p38alpha/beta kinases. We report that p38alpha is required for colorectal cancer cell homeostasis as the inhibition of its kinase function by pharmacological blockade or genetic inactivation causes cell cycle arrest, autophagy and cell death in a cell type-specific manner. Deficiency of p38alpha activity induces a tissue-restricted upregulation of the GABARAP gene, an essential component of autophagic vacuoles and autophagosomes, whereas simultaneous inhibition of autophagy significantly increases cell death by triggering apoptosis. These data identify p38alpha as a central mediator of colorectal cancer cell homeostasis and establish a rationale for the evaluation of the pharmacological manipulation of the p38alpha pathway in the treatment of colorectal cancer.     

Biogenesis of multilamellar bodies via autophagy

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.     

盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。