In praise of M. Anselmier who first used the term “autophagie” in 1859

A Google search for the combined terms “de Duve, autophagy, 1963” will reveal over 45,000 hits, most of them referring to the idea that the term autophagy was coined by the brilliant Christian de Duve on the sidelines of a symposium on lysosomes that took place in 1963. However, the first use of the term “autophagy” actually took place a century earlier.     

Autophagy revisited: a conversation with Christian de Duve

The word "autophagy" was invented by Christian de Duve, the discoverer of lysosomes, who also initiated the first experiments that provided clear biochemical proof of the involvement of lysosomes in this process. I recently had an opportunity to speak with de Duve and some of his former coworkers about their reminiscences of these events, and am pleased to share what they had to say with the readers of this journal, many of whom may not be familiar with this historical background to their field of interest.     

Autophagy revisited: A conversation with Christian de Duve

The word "autophagy" was invented by Christian de Duve, the discoverer of lysosomes, who also initiated the first experiments that provided clear biochemical proof of the involvement of lysosomes in this process. I recently had an opportunity to collect de Duve's reminiscences of these events, as well as those of some of his former coworkers, and am pleased to share them with the readers of this journal, many of whom may not be familiar with this historical background to their field of interest.     

自噬的前世今生

自噬是细胞通过溶酶体（或液泡）分解自身组分以达到维持细胞内正常生理活动及稳态的一种细胞代谢过程。自噬作为一种在真核生物中保守存在的细胞通路,与人类的疾病与健康息息相关。2016年,诺贝尔生理学或医学奖颁发给为自噬通路研究做出过卓越贡献的日本生物学家大隅良典（Yoshinori Ohsumi）。本文其一旨在通过介绍自噬及自噬相关基因的发现细节,带领读者了解自噬被发现和阐释的历程;其二旨在通过介绍自噬起始的相关机制及自噬与疾病的联系,引导读者对于自噬生理功能有更深入的理解;最后本文还提出了一些自噬领域目前尚待进一步研究的方向,供读者参考。     

Historical landmarks of autophagy research

The year of 2013 marked the 50th anniversary of C de Duve''s coining of the term “autophagy” for the degradation process of cytoplasmic constituents in the lysosome/vacuole. This year we regretfully lost this great scientist, who contributed much during the early years of research to the field of autophagy. Soon after the discovery of lysosomes by de Duve, electron microscopy revealed autophagy as a means of delivering intracellular components to the lysosome. For a long time after the discovery of autophagy, studies failed to yield any significant advances at a molecular level in our understanding of this fundamental pathway of degradation. The first breakthrough was made in the early 1990s, as autophagy was discovered in yeast subjected to starvation by microscopic observation. Next, a genetic effort to address the poorly understood problem of autophagy led to the discovery of many autophagy-defective mutants. Subsequent identification of autophagy-related genes in yeast revealed unique sets of molecules involved in membrane dynamics during autophagy. ATG homologs were subsequently found in various organisms, indicating that the fundamental mechanism of autophagy is well conserved among eukaryotes. These findings brought revolutionary changes to research in this field. For instance, the last 10 years have seen remarkable progress in our understanding of autophagy, not only in terms of the molecular mechanisms of autophagy, but also with regard to its broad physiological roles and relevance to health and disease. Now our knowledge of autophagy is dramatically expanding day by day. Here, the historical landmarks underpinning the explosion of autophagy research are described with a particular focus on the contribution of yeast as a model organism.     

Secretory autophagy holds the key to lysozyme secretion during bacterial infection of the intestine

In 2013, Dr. Lora Hooper and colleagues described the induction of antibacterial macroautophagy/autophagy in intestinal epithelial cells as a cytoprotective host defense mechanism against invading Salmonella enterica serovar Typhimurium (S. Typhimurium). Canonical autophagy functions in a primarily degradative capacity to safeguard cells and ensure survival during stress conditions, including pathogen infection. In contrast, secretory autophagy has emerged as an alternative nondegradative mechanism for cellular trafficking and unconventional protein secretion. More recently, a study by Bel et al. from Dr. Hooper's lab describes how intestinal Paneth cells exploit the endoplasmic reticulum (ER) stress response to release antibacterial lysozyme through secretory autophagy in response to S. Typhimurium infection.     

Microautophagy in mammalian cells: Revisiting a 40-year-old conundrum

The term microautophagy was first used in 1966 by de Duve and Wattiaux1 and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen (“multivesicular lysosomes”). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we consider the current state of knowledge about microautophagic processes. We examine some of the main characteristics of microautophagy in yeast with a view to assessing their relevance for our understanding of microautophagy in mammalian cells.     

Microautophagy in mammalian cells: revisiting a 40-year-old conundrum

The term microautophagy was first used in 1966 by de Duve and Wattiaux and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen ("multivesicular lysosomes"). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we consider the current state of knowledge about microautophagic processes. We examine some of the main characteristics of microautophagy in yeast with a view to assessing their relevance for our understanding of microautophagy in mammalian cells.     

There is more to autophagy than induction: regulating the roller coaster

Considerable attention has been paid to the topic of autophagy induction. In part, this is because of the potential for modulating this process for therapeutic purposes. Of course we know that induced autophagy can also be problematic—for example, when trying to eliminate an established tumor that might be relying on autophagy for its own cytoprotective uses. Accordingly, inhibitory mechanisms have been considered; however, the corresponding studies have tended to focus on the pathways that block autophagy under noninducing conditions, such as when nutrients are available. In contrast, relatively little is known about the mechanisms for inhibiting autophagy under inducing conditions. Yet, this type of regulation must be occurring on a routine basis. We know that dysregulation of autophagy, e.g., due to improper activation of Beclin 1 leading to excessive autophagy activity, can cause cell death.¹ Accordingly, we assume that during starvation or other inducing conditions there must be a mechanism to modulate autophagy. That is, once you turn it on, you do not want to let it continue unchecked. But how is autophagy downregulated when the inducing conditions still exist?Key words: Atg1, autophagosome, flux, lysosome, macroautophagy, phagophore, regulation, stress, TOR, Ulk1, vacuoleOne possibility for downregulating macroautophagy is suggested by Tom Neufeld’s lab, which showed that p70S6 kinase is a positive regulatory factor for autophagy in the Drosophila fat body.² Accordingly, inhibition of TOR activity ultimately results in decreased p70S6K function, which in turn downregulates autophagy. An alternate suggestion from Fred Meijer and Patrice Codogno is that p70S6K acts in part by negatively regulating the class I PtdIns 3-kinase.³ In this scenario, when TOR is inhibited the decrease in p70S6K activity results in the eventual reactivation of the class I PtdIns3K, which then stimulates TOR and downregulates autophagy.Further insight into this question is provided by a relatively recent study from Adi Kimchi’s lab. The conserved protein DAP1 is an mTOR substrate that inhibits macroautophagy. In nutrient-rich conditions, active mTORC1 inhibits DAP1 so that the latter has no effect on autophagy. The inhibition of mTORC1 during nutrient starvation results in the dephosphorylation and activation of DAP1, and the subsequent inhibition of macroautophagy, which limits the magnitude of autophagy-dependent degradation.^4,5Another mechanism of regulation is indicated in studies by Li Yu and colleagues who showed that autophagy is downregulated through mTOR reactivation in an autophagy-dependent manner that requires protein degradation in autolysosomes.⁶ This negative feedback mechanism provides another simple means of self-regulation whereby the nutrient levels within the cell dictate whether autophagy needs to be maintained or shut down. A study described in this issue of the journal provides further support for this mechanism, demonstrating that autophagy can be downregulated during starvation in yeast.⁷ Shin and Huh found that TOR activity is recovered during prolonged starvation, and that this again depends on autophagy (see Fig. 1 in the Autophagic Flux section, p. 803). These studies suggest that autophagy may cycle on and off repeatedly during starvation as nutrient supplies are consumed and then resupplied, ensuring that autophagy is maintained at optimal, and not excessive, levels. The latter mechanism, however, cannot explain how autophagy is regulated during other types of stress, suggesting that multiple control systems are involved.In closing, we introduce a new category of papers and a new section to the journal that we are calling Resource and Autophagic Flux, respectively. Resource papers will provide information that may be useful to the autophagy community, but that may not have specific mechanistic information, such as may occur with large-scale screens. For example, in this issue, see the paper from Marja Jäättelä’s lab that describes the use of a human kinome siRNA library to identify new kinases that regulate macroautophagy. Finally, we have chosen the name Autophagic Flux for the new section because it encompasses the full spectrum of the autophagic process. The schematic summary in Figure 1 of that section highlights the paper by Shin and Huh that we mention here. Our intention is to provide schematic highlights of most of the research papers in the Autophagic Flux section, providing readers with a quick overview and summary of the key point(s) of the study. We hope you find this useful; we welcome feedback.     

Regulation and Function of Autophagy during Cell Survival and Cell Death

Autophagy is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. Autophagy promotes cell survival by elimination of damaged organelles and proteins aggregates, as well as by facilitating bioenergetic homeostasis. Although autophagy has been considered a cell survival mechanism, recent studies have shown that autophagy can promote cell death. The core mechanisms that control autophagy are conserved between yeast and humans, but animals also possess genes that regulate autophagy that are not present in yeast. These regulatory differences may be explained by the need to control autophagy in a cell context-specific manner in multicellular animals, such as during cell survival and cell death. Autophagy was thought to be a bulk cytoplasmic degradation mechanism, but recent studies have shown that specific cargo is recruited for degradation. This suggests the possibility that either cell survival or death may be regulated by selective autophagic clearance of cytoplasmic material. Here we summarize the mechanisms that regulate autophagy and how they may contribute to cell survival and death.Autophagy (self-eating) is an evolutionarily conserved catabolic process that is used to deliver cytoplasmic materials, including organelles and proteins, to the lysosome for degradation. Three types of autophagy have been described, including macroautophagy, microautophagy, and chaperone-mediated autophagy (Mizushima and Komatsu 2011). Although macroautophagy involves the fusion of the double membrane autophagosome and lysosomes, microautophagy is poorly understood and thought to involve direct uptake of material by the lysosome via a process that appears similar to pinocytosis. By contrast, chaperone-mediated autophagy is a biochemical mechanism to import proteins into the lysosome; it depends on a signature sequence and interaction with protein chaperones. Here we will focus on macroautophagy (hereafter called autophagy) because of our knowledge of this process in cell survival and cell death.Autophagy was likely first observed when electron microscopy was used to observe “dense bodies” containing mitochondria in mouse kidneys (Clark 1957). Five years later, it was reported that rat hepatocytes exposed to glucagon possessed membrane-bound vesicles that were rich in mitochondria and endoplasmic reticulum (Ashford and Porter 1962). Almost simultaneously, it was shown that these membrane-bound vesicles contained lysosomal hydrolases (Novikoff and Essner 1962). In 1965 de Duve coined the term “autophagy” (Klionsky 2008).The delivery of cytoplasmic material to the lysosome by autophagy involves membrane formation and fusion events (Fig. 1). First an isolation membrane, also known as a phagophore, must be initiated from a membrane source known as the phagophore assembly site (PAS). de Duve suggested that the smooth endoplasmic reticulum could be the source of autophagosome membrane (de Duve and Wattiaux 1966), and subsequent studies have supported this possibility (Dunn 1990; Axe et al. 2008). Although controversial, mitochondria and plasma membrane could also supply membranes for the formation of the autophagosomes under different conditions (Hailey et al. 2010; Ravikumar et al. 2010). The elongating isolation membrane surrounds cargo that is ultimately enclosed in the double membrane autophagosome. Once the autophagosome is formed, it fuses with lysosomes (known as the vacuole in yeasts and plants) to form autolysosomes in which the cargo is degraded by lysosomal hydrolases. At this stage lysosomes must reform so that subsequent autophagy may occur (Yu et al. 2010).Open in a separate windowFigure 1.Macroautophagy (autophagy) delivers cytoplasmic cargo to lysosomes for degradation, and involves membrane formation and fusion. The isolation membrane is initiated from a membrane source known as the from the phagophore assembly site (PAS). The isolation membrane surrounds cargo, including organelles and proteins, to form a double membrane autophagosome. Autophagosomes fuse with lysosomes to form autolysosomes in which the cargo is degraded by lysosomal hydrolases.     

Inhibiting autophagy by shRNA knockdown

A glance through Autophagy or any other journal in this field shows that it is very common to block autophagy by RNA interference-based knockdown of ATG mRNAs in mammalian cell lines. Our lab’s experience is that this approach can easily make for failed experiments because good knockdown of even essential autophagy regulators does not necessarily mean you will get good inhibition of autophagy, and, over time, cells can find ways to circumvent the inhibitory effects of the knockdown.     

The intense gravitational attraction of autophagy

In the course of my work as Autophagy editor, I try to gauge the overall patterns of interest in autophagy research. Not surprisingly, the number of papers associated with this topic has increased steadily. However, that trend provides only one glimpse into the way interest in this field has been changing—that the number of people working on autophagy has expanded. Perhaps not surprisingly, the number of different research areas that now include autophagy studies is also increasing. Thus, I decided to carry out an informal, imprecise analysis of the number of different journals (presumably reflecting in part the number of topics) that include papers on autophagy.     

I think autophagy controls the death of my cells: What do I do to get my paper published?

Many people are studying how autophagy intersects with cell death. While most of those studies relate to autophagy acting as a protective mechanism (e.g., to block apoptosis), many papers conclude that autophagy is a death mechanism, and there is a widespread belief that autophagy (in most, but not all cases, we are talking about macroautophagy) can both kill and protect cells depending on the circumstances. Not surprisingly therefore, many of the papers submitted to Autophagy study the relationship between autophagy and cell death.     

Tension-induced autophagy

Impairment of autophagy in patients and animal models severely affects mechanically strained tissues such as skeletal muscle, heart, lung and kidney, leading for example to muscle dystrophy, cardiomyopathy and renal injury. However, the reason for this high reliance on autophagy remained largely elusive. Recent work in our lab now provides a possible explanation. We identified chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in mammalian cells.     

Dr. Haifan Lin. Interviewed by Han Lee and Rachel Rosenstein

Dr. Haifan Lin is professor of Cell Biology at Yale University, where he studies the mechanism of stem cell self-renewal in fruit flies, mice, and human cancer cells. Recently named director of the Yale Stem Cell Center, Dr. Lin has made seminal contributions to the stem cell field, most notably his demonstration of the stem cell niche theory using the fruit fly model, his discovery of the PIWI/AGO gene family that is essential for stem cell division in diverse organisms, and his recent finding of a group of small RNAs called PIWI-interacting, or piRNAs, which may play a crucial role in stem cell proliferation and germline development. Dr. Lin’s work on piRNAs was recognized by Science Magazine as a top scientific breakthrough of 2006. Recently, the Lin lab has begun exploring the role of these molecules in stem cell division and oncogenesis.     

Sharpening the focus; the business of epithelial cell biology. An interview with Chris Potten by David Pearton

Dr Chris Potten is a singularly influential figure in the field of epithelial biology. His contributions have been seminal and include the introduction of the epidermal proliferative unit and of the concept of epidermal stem cells. With around 400 scientific papers and reviews to his credit as well as two books, he has certainly made his mark. His contributions have been recognised by the award of the Curie medal and recently the Weiss medal for radiation biology. Dr. Potten graciously agreed to be interviewed for this Special Issue of The International Journal of Developmental Biology. This interview was conducted via e-mail during June -August 2003.     

Opening new doors in autophagy research: Patrice Codogno

Patrice Codogno (Fig. 1), one of the associate editors of Autophagy since it was established, is well known in the autophagy field, and has played a particularly important role in France, serving as the first president of Club Francophone de l'AuTophaGie (CFATG). Patrice's research career spans from the predominantly biochemical analyses that were commonly used in the 1980s to the molecular studies that are the primary focus of many labs currently studying autophagy today. Anyone who has met Patrice knows that he is modest, which means his contributions to autophagy and to promoting the careers of scientists globally, are underappreciated. In addition, there is a fun-loving side to Patrice that is often hidden to the casual observer, and it is time to share some of his personality and thoughts with the rest of the autophagy community.     

Lipid droplet–mediated ER homeostasis regulates autophagy and cell survival during starvation

Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.     

The paradox of autophagy and its implication in cancer etiology and therapy

Autophagy is a cellular self-catabolic process in which cytoplasmic constituents are sequestered in double membrane vesicles that fuse with lysosomes where they are degraded. As this catabolic activity generates energy, autophagy is often induced under nutrient limiting conditions providing a mechanism to maintain cell viability and may be exploited by cancer cells for survival under metabolic stress. However, progressive autophagy can be cytotoxic and autophagy can under certain settings substitute for apoptosis in induction of cell death. Moreover, loss of autophagy is correlated with tumorigenesis and several inducers of autophagy are tumor-suppressor genes. Thus, the relation of autophagy to cancer development is complex and depends on the genetic composition of the cell as well as on the extra-cellular stresses a cell is exposed to. In this review we describe the intricate nature of autophagy and its regulators, particularly those that have been linked to cancer. We discuss the multifaceted relation of autophagy to tumorigenesis and highlight studies supporting a role for autophagy in both tumor-suppression and tumor-progression. Finally, various autophagy-targeting therapeutic strategies for cancer treatment are presented. This review is dedicated to the memory of Dr. Avner Eisenberg 1953–2004.     

Autophagy and Neuronal Cell Death in Neurological Disorders

Autophagy is implicated in the pathogenesis of major neurodegenerative disorders although concepts about how it influences these diseases are still evolving. Once proposed to be mainly an alternative cell death pathway, autophagy is now widely viewed as both a vital homeostatic mechanism in healthy cells and as an important cytoprotective response mobilized in the face of aging- and disease-related metabolic challenges. In Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis, and other diseases, impairment at different stages of autophagy leads to the buildup of pathogenic proteins and damaged organelles, while defeating autophagy’s crucial prosurvival and antiapoptotic effects on neurons. The differences in the location of defects within the autophagy pathway and their molecular basis influence the pattern and pace of neuronal cell death in the various neurological disorders. Future therapeutic strategies for these disorders will be guided in part by understanding the manifold impact of autophagy disruption on neurodegenerative diseases.Soon after the discovery of lysosomes by de Duve in the 1950s, electron microscopists recognized the presence of cytoplasmic organelles within membrane-limited vacuoles (Clark 1957) and observed what appeared to be the progressive breakdown of these contents (Ashford and Porter 1962). Proposing that “prelysosomes” containing sequestered cytoplasm matured to autolysosomes by fusion with primary lysosomes, de Duve and colleagues (de Duve 1963; de Duve and Wattiaux 1966) named this process “autophagy” (self-eating). Neurons, as cells particularly rich in acid phosphatase-positive lysosomes, were a preferred model in the initial investigations of autophagy. Early studies of pathologic states such as neuronal chromatolysis (Holtzman and Novikoff 1965; Holtzman et al. 1967) linked neurodegenerative phenomena to robust proliferation of autophagic vacuoles (AVs) and lysosomes. Although de Duve appreciated the importance of lysosomes for maintaining cell homeostasis, he was especially intrigued with their potential as “suicide bags” capable of triggering cell death by releasing proteases into the cytoplasm. Despite some support for this notion (Brunk and Brun 1972), the concept was not significantly embraced until many decades later. Instead, for many years, lysosomes and autophagy were mainly considered to perform cellular housekeeping and to scavenge and clean up debris during neurodegeneration in preparation for regenerative processes. The connection between autophagy and neuronal cell death reemerged in the 1970s from observations of Clarke and colleagues, who presented evidence that the developing brain deployed autophagy as a form of programmed neuronal cell death during which autophagy was massively up-regulated to eliminate cytoplasmic components, at once killing the neuron and reducing its cell mass for easy removal. Self-degradation was suggested as a more efficient elimination mechanism than apoptosis, which requires a large population of phagocytic cells and access of these cells to the dying region (Baehrecke 2005). Indeed, the best evidence for this process is in the context of massive cell death, as in metamorphosis and involutional states (Das et al. 2012).Clarke proposed that autophagic cell death (ACD)—type 2 programmed cell death (PCD)—could be a relatively common alternative route to death distinct from apoptosis—type 1 PCD (Clarke 1990)—or caspase-independent cell death—type 3 PCD (Fig. 1). The distinguishing features of ACD are marked proliferation of AVs and progressive disappearance of organelles but relative preservation of cytoskeletal and nuclear integrity until late in the process (Schweichel and Merker 1973; Hornung et al. 1989). In this original concept of ACD or type 2 PCD, death is achieved by autophagic digestion of organelles and essential regulatory molecules and elimination of death inhibitory factors (Baehrecke 2005). With the advent of the molecular era of autophagy research in the 1990s, it became possible to verify the most important implication of ACD, namely, that the death could be prevented by inhibiting autophagy genetically or pharmacologically. Meanwhile, reports of prominent lysosomal/autophagic pathology in Alzheimer’s disease (AD) (Cataldo et al. 1997; Nixon et al. 2000, 2005) and other neuropathic states (Anglade et al. 1997; Rubinsztein et al. 2005) raised important questions about whether autophagy pathology signifies a prodeath program or an attempt to maintain survival—a critical question for any potential therapy based on autophagy modulation. In this article, we will examine evidence for the various neuroprotective roles of autophagy and review our current understanding of how specific stages of autophagy may become disrupted and influence the neurodegenerative pattern seen in major adult-onset neurological diseases. We will particularly focus on how neurons regulate the balance between prosurvival autophagy and well-established cell death mechanisms in making life or death decisions.Open in a separate windowFigure 1.Neuronal cell death: three general morphological types of dying cells in the developing nervous system, as initially classified by Schweichel and Merker (1973) and later Clarke (1990). (A,B) Type 1 (“apoptotic”) cell death: (A) A neuron, from the brain of a postnatal day 6 mouse pup, in the middle of apoptotic degeneration showing cell shrinkage, cytoplasmic condensation, ruffled plasma membrane, and a highly electron-dense nucleus. Endoplasmic reticulum (ER) is still recognizable and some are dilated. A small number of autophagic vacuoles (AVs) can be seen (arrows). (B) A late-stage apoptotic neuron displaying electron-dense chromatin balls (CB), each surrounded by a small amount of highly condensed cytoplasm. (Panel from Yang et al. 2008; reprinted, with permission, from the American Association of Pathologists and Bacteriologists.) (C) Type 2 (“autophagic”) cell death: a deafferented isthmo-optic neuron in developing chick brain after uptake of horseradish peroxidase to highlight (electron dense) endocytic and autophagic compartments. The cell death pattern features pyknosis, abundant AVs, and sometimes dilated ER and mitochondria. (Panel from Hornung et al. 1989; reproduced, with permission, from John Wiley & Sons) (D) Type 3 (“cytoplasmic, nonlysosomal”) cell death: a motoneuron displaying markedly dilated rough ER, Golgi, and nuclear envelope, late vacuolization, and increased chromatin granularity. (Panel from Chu-Wang and Oppenheim 1978; reproduced, with permission, from John Wiley & Sons) Scale bars, 1 µm (A,B); 2 µm (C,D).