Using enhanced-mitophagy to measure autophagic flux

Macroautophagy (hereafter termed autophagy) is a cellular membrane-trafficking process that functions to deliver cytoplasmic constituents to lysosomes for degradation. Autophagy operates at basal levels to turn over damaged and misfolded proteins and it is the only process for the turnover of organelles. The process is therefore critically important for the preservation of cellular integrity and viability. Autophagy is also highly adaptable and the rate and cargoes of autophagy can be altered to bring about desired cellular responses to intracellular and environmental cues, disease states and a spectrum of pharmaceutical drugs. As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations. To date, the majority of assays to monitor autophagy either measure changes in a parameter of the process at a set point in time or use markers/tracers to monitor flow of membrane-bound proteins from one point in the process to another. As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process. We describe here an assay to measure autophagic cargo degradation by engineering cells to degrade mitochondria en masse. We show that this ‘enhanced-mitophagy’ assay can be used to measure differences in the rate of autophagy between different cells or in response to agents which are known to promote or inhibit autophagic flux. We consider therefore that this assay will prove to be a valuable resource for investigations in which autophagy is considered important and is believed to be modulated

DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions

Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death

Characterisation of the role of DRAM-related TMEM150 proteins in cancer cell survival, cell death and autophagy

Autophagy, a cellular recycling mechanism, and apoptosis, a regulated mode of cell death, are two fundamental cellular processes that contribute to carcinogenesis and tumour growth as well as treatment sensitivity and resistance. The protein encoded by DRAM1, a p53-responsive gene, has previously been described as an autophagy and apoptosis modulator downstream of p53 activation. Furthermore, a family of DRAM1-related proteins has been uncovered by in silico analysis. Of the 5 members of this protein family, only DRAM1 and DRAM-2 had previously been tested for their roles in cell death and autophagy. Much less was known about the remaining three DRAM-family members TMEM150A/B/C (termed DRAM-5/-3/-4 by us for ‘DRAM-related/associated member-5/-3/-4’) and their potential roles in autophagy, cell death or cell survival in cancer cells. In this project, we therefore aimed to test whether these DRAM-family proteins could modulate autophagy, cell death or cell survival in in vitro cancer cell line systems. We used both retroviral, constitutive overexpression systems and CRISPR/Cas9-mediated gene disruption systems to study the effect of TMEM150 overexpression or TMEM150 ablation on these processes. In summary, we found that none of the TMEM150 genes were induced by p53, but starvation conditions increased TMEM150A and C transcript levels in some conditions. Moderate changes in TMEM150 protein levels showed no dramatic effect on cell growth and survival. Of the three TMEM150 proteins, only TMEM150B affected autophagy, while TMEM150A and C did not modulate autophagic processes in any of the assays performed. Moreover, we show that TMEM150B overexpression can improve cellular survival under glucose deprived conditions, while none of the other DRAM-family proteins seems capable of doing so. Additionally, serum or amino acid starvation did not show parallel effects. Lastly, we show that the influence of TMEM150B on autophagic processes is uncoupled from its ability to modulate survival in glucose-starved cells. Taken all together, with this work we provide an initial characterisation of the TMEM150 proteins, which may lay a foundation for future, expanded studies on the cellular functions of the DRAM-family

Another DRAM involved in autophagy and cell death

Macroautophagy (hereafter referred to as autophagy) is controlled by a number of core proteins that are critical for all autophagy responses. In addition, a number of autophagy regulators have been found that are not critical for macroautophagy per se, but which play roles in regulating autophagy in either selective situations or in response to specific stimuli. In a recent study, we reported the initial characterization of a new autophagy regulator encoded by TMEM150B that is related to the Damage-Regulated Autophagy Modulator, DRAM1. We have termed this factor DRAM3 for DRAM-Related/Associated Member 3. Interestingly, like DRAM1, DRAM3 regulates both autophagy and cell death, but we found these two functions of the protein are not intrinsically connected

Lysosomal proteins in cell death and autophagy

Nearly 60 years ago, lysosomes were first described in the laboratory of Christian de Duve, a discovery that significantly contributed to him being awarded a share of the 1974 Nobel Prize in Physiology or Medicine for elucidating ‘the structural and functional organization of the cell’. Initially thought of as a simple waste degradation facility of the cell, these organelles recently emerged as signalling centres with connections to major cellular processes. This review provides an overview of the many roles of lysosomal proteins in two of these processes: cell death and autophagy. We discuss both resident lysosomal proteins as well those that temporarily associate with lysosomes to influence autophagy and cell death pathways. Particular focus is given to studies in mammalian cells and in vivo systems

DRAM-4 and DRAM-5 are compensatory regulators of autophagy and cell survival in nutrient-deprived conditions

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic material to lysosomes for degradation. The process preserves cellular integrity by removing damaged cellular constituents and can promote cell survival by providing substrates for energy production during hiatuses of nutrient availability. The process is also highly responsive to other forms of cellular stress. For example, DNA damage can induce autophagy and this involves up-regulation of the Damage-Regulated Autophagy Modulator-1 (DRAM-1) by the tumor suppressor p53. DRAM-1 belongs to an evolutionarily conserved protein family, which has five members in humans and we describe here the initial characterization of two members of this family, which we term DRAM-4 and DRAM-5 for DRAM-Related/Associated Member 4/5. We show that the genes encoding these proteins are not regulated by p53, but instead are induced by nutrient deprivation. Similar to other DRAM family proteins, however, DRAM-4 principally localizes to endosomes and DRAM-5 to the plasma membrane and both modulate autophagy flux when over-expressed. Deletion of DRAM-4 using CRISPR/Cas-9 also increased autophagy flux, but we found that DRAM-4 and DRAM-5 undergo compensatory regulation, such that deletion of DRAM-4 does not affect autophagy flux in the absence of DRAM-5. Similarly, deletion of DRAM-4 also promotes cell survival following growth of cells in the absence of amino acids, serum, or glucose, but this effect is also impacted by the absence of DRAM-5. In summary, DRAM-4 and DRAM-5 are nutrient-responsive members of the DRAM family that exhibit interconnected roles in the regulation of autophagy and cell survival under nutrient-deprived conditions