A systems biology approach to learning autophagy

With its relevance to our understanding of eukaryotic cell function in the normal and disease state, autophagy is an important topic in modern cell biology; yet, few textbooks discuss autophagy beyond a two- or three-sentence summary. Here, we report an undergraduate/graduate class lesson for the in-depth presentation of autophagy using an active learning approach. By our method, students will work in small groups to solve problems and interpret an actual data set describing genes involved in autophagy. The problem-solving exercises and data set analysis will instill within the students a much greater understanding of the autophagy pathway than can be achieved by simple rote memorization of lecture materials; furthermore, the students will gain a general appreciation of the process by which data are interpreted and eventually formed into an understanding of a given pathway. As the data sets used in these class lessons are largely genomic and complementary in content, students will also understand first-hand the advantage of an integrative or systems biology study: No single data set can be used to define the pathway in full-the information from multiple complementary studies must be integrated in order to recapitulate our present understanding of the pathways mediating autophagy. In total, our teaching methodology offers an effective presentation of autophagy as well as a general template for the discussion of nearly any signaling pathway within the eukaryotic kingdom.     

The viral killer system in yeast: from molecular biology to application

Since the initial discovery of the yeast killer system almost 40 years ago, intensive studies have substantially strengthened our knowledge in many areas of biology and provided deeper insights into basic aspects of eukaryotic cell biology as well as into virus-host cell interactions and general yeast virology. Analysis of killer toxin structure, synthesis and secretion has fostered understanding of essential cellular mechanisms such as post-translational prepro-protein processing in the secretory pathway. Furthermore, investigation of the receptor-mediated mode of toxin action proved to be an effective means for dissecting the molecular structure and in vivo assembly of yeast and fungal cell walls, providing important insights relevant to combating infections by human pathogenic yeasts. Besides their general importance in understanding eukaryotic cell biology, killer yeasts, killer toxins and killer viruses are also becoming increasingly interesting with respect to possible applications in biomedicine and gene technology. This review will try to address all these aspects.     

Teaching systems biology: an active-learning approach

With genomics well established in modern molecular biology, recent studies have sought to further the discipline by integrating complementary methodologies into a holistic depiction of the molecular mechanisms underpinning cell function. This genomic subdiscipline, loosely termed "systems biology," presents the biology educator with both opportunities and obstacles: The benefit of exposing students to this cutting-edge scientific methodology is manifest, yet how does one convey the breadth and advantage of systems biology while still engaging the student? Here, I describe an active-learning approach to the presentation of systems biology. In graduate classes at the University of Michigan, Ann Arbor, I divided students into small groups and asked each group to interpret a sample data set (e.g., microarray data, two-hybrid data, homology-search results) describing a hypothetical signaling pathway. Mimicking realistic experimental results, each data set revealed a portion of this pathway; however, students were only able to reconstruct the full pathway by integrating all data sets, thereby exemplifying the utility in a systems biology approach. Student response to this cooperative exercise was extremely positive. In total, this approach provides an effective introduction to systems biology appropriate for students at both the undergraduate and graduate levels.     

Pathways that regulate autophagy and their role in mediating tumor response to treatment

In addition to their role in cellular homeostasis, pathways that regulate autophagy affect both tumorigenesis and tumor response to treatment. Therefore, understanding the regulation of autophagy in treated cancer cells is relevant to the discovery of molecular targets for the development of anti-cancer drugs. Our recent report points to radiation-induced inactivation of the mTOR pathway as an underlying mechanism of radiation-induced autophagy in the human breast cancer cell line MCF-7. Most importantly, radiation-induced inactivation of this pathway was detrimental to cell survival and was associated with reversal of mitochondrial ATPase activity and mitochondrial hyperpolarization, decreased level of eukaryotic initiation factor 4G (eIF4G) and increased phosphorylation of p53. Future analysis of the interrelationship among these events and the role each of them plays in cell survival following radiation will increase our ability to employ the mTOR pathway in anti-cancer therapy.     

That which does not degrade you makes you stronger

Several years ago, an explosion of research into pathogens and autophagy showed that viruses have a wide variety of relationships to this conserved homeostatic pathway. Often, autophagy acts as a host defense mechanism, degrading viruses before they can escape the host cell, and, as such, autophagy is suppressed or avoided by those viruses. A subset of viruses, however, induces and subverts the autophagic machinery to promote their own replication. Many of these viruses inhibit the degradative step in the autophagic pathway, presumably to prevent degradation of cytosolic virions before they exit the cell. Recently, we published a study showing that poliovirus (PV), a well-studied model virus, induces true autophagic degradation. The remainder of our study provided surprising clues about the role of autophagy in promoting virus production. The purpose of this punctum is to discuss the significance of our findings to a general understanding of the autophagic pathway and its relationship to a common class of cellular pathogens.     

Autophagy: eating for good health

A renaissance in the autophagy field has illuminated many areas of biology, and infectious disease is no exception. By identifying key components of this broadly conserved membrane traffic pathway, yeast geneticists generated tools for microbiologists and immunologists to explore whether autophagy contributes to host defenses. As a result, autophagy is now recognized to be another barrier confronted by microbes that invade eukaryotic cells. Mounting evidence also indicates that autophagy equips cells to deliver cytosolic Ags to the MHC class II pathway. By applying knowledge of the autophagy machinery and exploiting microbes as genetic probes, experimentalists can now examine in detail how this ancient membrane traffic pathway contributes to these and other mechanisms critical for infection and immunity.     

Evolutionary cell biology: functional insight from “endless forms most beautiful”

In animal and fungal model organisms, the complexities of cell biology have been analyzed in exquisite detail and much is known about how these organisms function at the cellular level. However, the model organisms cell biologists generally use include only a tiny fraction of the true diversity of eukaryotic cellular forms. The divergent cellular processes observed in these more distant lineages are still largely unknown in the general scientific community. Despite the relative obscurity of these organisms, comparative studies of them across eukaryotic diversity have had profound implications for our understanding of fundamental cell biology in all species and have revealed the evolution and origins of previously observed cellular processes. In this Perspective, we will discuss the complexity of cell biology found across the eukaryotic tree, and three specific examples of where studies of divergent cell biology have altered our understanding of key functional aspects of mitochondria, plastids, and membrane trafficking.The field of cell biology has made tremendous strides in understanding eukaryotic cells, especially animals and yeast. Concurrently, evolutionary biology has opened up a window to the origins of our species and the genes that define us. Though these fields have intersected conceptually for decades, a recent movement is explicitly uniting these two fields into the discipline of evolutionary cell biology with great success (Brodsky et al., 2012 ; Lynch et al., 2014 ) and, we argue here, potentially an even greater future. One drive behind this movement is to harness the comparative approach of evolutionary biology and apply it to questions of cellular origins and cellular function. This approach has yielded beautiful insight into animal cellular function from mitotic spindle dynamics (Helmke and Heald, 2014 ) to glycosylation machinery (Varki, 2006 ). However, expanding the scope of investigation to organisms beyond fungi and animals to span eukaryotic diversity has allowed for discoveries that force us to adjust some fundamental ideas of how eukaryotic organelles work, and why.     

Autophagy and antigen presentation

CD4(+) T cells co-ordinate adaptive immunity and are required for immunological memory establishment and maintenance. They are thought to primarily recognize extracellular antigens, which are endocytosed, processed by lysosomal proteases and then presented on major histocompatibility complex (MHC) class II. However, recent studies have demonstrated that viral, tumour and autoantigens can gain access to this antigen presentation pathway from within cells by autophagy. This review will discuss the autophagic pathways that contribute to endogenous MHC class II antigen processing. Furthermore, potential characteristics of autophagy substrates, qualifying them to access these pathways, and regulation of autophagy will be considered. Finally, I will suggest how antigen presentation after autophagy might contribute to immune surveillance of infected and transformed cells.     

Active Interaction Mapping as a tool to elucidate hierarchical functions of biological processes

Increasingly, various ‘omics data are contributing significantly to our understanding of novel biological processes, but it has not been possible to iteratively elucidate hierarchical functions in complex phenomena. We describe a general systems biology approach called Active Interaction Mapping (AI-MAP), which elucidates the hierarchy of functions for any biological process. Existing and new ‘omics data sets can be iteratively added to create and improve hierarchical models which enhance our understanding of particular biological processes. The best datatypes to further improve an AI-MAP model are predicted computationally. We applied this approach to our understanding of general and selective autophagy, which are conserved in most eukaryotes, setting the stage for the broader application to other cellular processes of interest. In the particular application to autophagy-related processes, we uncovered and validated new autophagy and autophagy-related processes, expanded known autophagy processes with new components, integrated known non-autophagic processes with autophagy and predict other unexplored connections.     

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.     

Yeast viral killer toxins: lethality and self-protection

Since the discovery of toxin-secreting killer yeasts more than 40 years ago, research into this phenomenon has provided insights into eukaryotic cell biology and virus-host-cell interactions. This review focuses on the most recent advances in our understanding of the basic biology of virus-carrying killer yeasts, in particular the toxin-encoding killer viruses, and the intracellular processing, maturation and toxicity of the viral protein toxins. The strategy of using eukaryotic viral toxins to effectively penetrate and eventually kill a eukaryotic target cell will be discussed, and the cellular mechanisms of self-defence and protective immunity will also be addressed.     

Pathways that Regulate Autophagy and their Role in Mediating Tumor Response to Treatment

Addenda to:

Rapamycin-Sensitive Pathway Regulates Mitochondrial Membrane Potential, Autophagy and Survival in Irradiated MCF-7 Cells

Paglin S, Lee N-Y, Nakar C, Fitzgerald M, Plotkin J, Deuel B, Hackett N, McMahill M, Sphicas E, Lampen N and Yahalom J.

Cancer Res 2005; 65:11061-70.

In addition to their role in cellular homeostasis, pathways that regulate autophagy affect both tumorigenesis and tumor response to treatment. Therefore, understanding regulation of autophagy in treated cancer cells is relevant to discovery of molecular targets for development of anti-cancer drugs. Our recent report points to radiation-induced inactivation of mTOR pathway as an underlying mechanism of radiation-induced autophagy in the human breast cancer cell line MCF-7. Most importantly, radiation-induced inactivation of this pathway was detrimental to cell survival and was associated with reversal of mitochondrial ATPase activity and mitochondrial hyperpolarization, decreased level of eukaryotic initiation factor 4G (eIF4G) and increased phosphorylation of p53. Future analysis of the interrelationship among these events and the role each of them plays in cell survival following radiation will increase our ability to employ the mTOR pathway in anti-cancer therapy.     

New insights into the function of Atg9

Autophagy is a lysosomal degradation pathway that is essential for cellular homeostasis. Identification of more than 30 autophagy related proteins including a multi-spanning membrane protein, Atg9, has increased our understanding of the molecular mechanisms involved in autophagy. Atg9 is required for autophagy in several eukaryotic organisms although its function is unknown. Recently, we identified a novel interacting partner of mAtg9, p38 MAPK interacting protein, p38IP. We summarise recent data on the role of Atg9 trafficking in yeast and mammalian autophagy and discuss the role of p38IP and p38 MAPK in regulation of mAtg9 trafficking and autophagy.     

自噬的检测方法及评述

细胞自噬是广泛存在于真核细胞内的一种降解途径,在机体发育过程中、在生理和病理状况下都起重要作用。近年,自噬成为热点研究领域,但少有论文深入分析不同检测方法得来的数据在研究中的不同意义,说明不同检测方法所获的结果的优缺点和适用条件。本文拟对哺乳动物细胞自噬检测方法进行归类、介绍及评述,旨在为研究工作中自噬检测方法的选择和研究结果的解释起帮助作用。     

Live imaging and single-cell analysis reveal differential dynamics of autophagy and apoptosis

Autophagy is induced by many cytotoxic stimuli but it is often unclear whether, under specific conditions, autophagy plays a prosurvival or a prodeath role. To answer this critical question we developed a novel methodology that employs automated live microscopy and image analysis to measure autophagy and apoptosis simultaneously in single cells. We used this approach to perform a systems-level analysis of pathway dynamics for both autophagy and apoptosis. We found that induction of autophagy in response to different stimuli is uniformly unimodal; in contrast, cells induce apoptosis in an all-or-none bimodal fashion. By tracking the fate of single cells we found that autophagy precedes apoptosis, and that within the same population apoptosis is delayed in cells that mount a stronger autophagy response. Inhibition of autophagy by knocking down ATG5 promoted apoptosis, thus confirming that autophagy plays a protective role. We anticipate that our single-cell approach will be a powerful tool for gaining a quantitative understanding of the complex regulation of autophagy, its influence on cell fate decisions and its relationship with other cellular pathways.     

IL4 (interleukin 4) induces autophagy in B cells leading to exacerbated asthma

Allergic asthma is a common airway inflammatory disease in which B cells play important roles through IgE production and antigen presentation. SNP (single nucleotide polymorphism) analysis showed that Atg (autophagy-related) allele mutations are involved in asthma. It has been demonstrated that macroautophagy/autophagy is essential for B cell survival, plasma cell differentiation and immunological memory maintenance. However, whether B cell autophagy participates in asthma pathogenesis remains to be investigated. In this report, we found that autophagy was enhanced in pulmonary B cells from asthma-prone mice. Autophagy deficiency in B cells led to attenuated immunopathological symptoms in asthma-prone mice. Further investigation showed that IL4 (interleukin 4), a key effector Th2 cytokine in allergic asthma, was critical for autophagy induction in B cells both in vivo and in vitro, which further sustained B cell survival and enhanced antigen presentation by B cells. Moreover, IL4-induced autophagy depended on JAK signaling via an MTOR-independent, PtdIns3K-dependent pathway. Together, our data indicate that B cell autophagy aggravates experimental asthma through multiple mechanisms.     

细胞自噬在植物碳氮营养中作用的研究进展

自噬（autophagy）是真核生物细胞通过形成自噬体,回收利用胞内物质,维持细胞健康的高通量亚细胞降解途径。随着酵母和动物自噬研究的深入,植物自噬也受到越来越多的关注。近期的研究揭示了植物自噬的基本机制及其生理意义,也发现了植物特有的自噬形式与自噬相关基因。该文主要综述了自噬在植物碳、氮营养中的作用。     

Eating the enemy within: autophagy in infectious diseases

Autophagy is emerging as a central component of antimicrobial host defense against diverse viral, bacterial, and parasitic infections. In addition to pathogen degradation, autophagy has other functions during infection such as innate and adaptive immune activation. As an important host defense pathway, microbes have also evolved mechanisms to evade, subvert, or exploit autophagy. Additionally, some fungal pathogens harness autophagy within their own cells to promote pathogenesis. This review will highlight our current understanding of autophagy in infection, focusing on the most recent advances in the field, and will discuss the potential implications of these studies in the design of anti-infective therapeutics.     

Autophagy: a barrier or an adaptive response to cancer

Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity. The involvement of proteins with properties of tumor suppressor and oncogenic properties at different steps of the pathway implies that autophagy must be considered in tumor progression. Autophagy as a stress response mechanism protects cancer cells from low nutrient supply or therapeutic insults. Autophagy is also involved in the elimination of cancer cells by triggering a non-apoptotic cell death program, suggesting a negative role in tumor development. These two aspects of autophagy will be discussed in this review.