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Human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause in vitro caspase-independent cell death, accompanied by previously undescribed cytoplasmic vacuolization with abnormal fusion of various types of endosomal-lysosomal organelles. Yeasts are unicellular eukaryotes widely used as a biological model to study fundamental cellular processes. In particular, yeast models of cell death are useful due to the similarity of regulated cell death mechanisms between yeast and humans. This, together with the functional similarity of yeast vacuoles and the lysosomal/endosomal compartment of mammalian cells, suggests yeasts to be a promising model to study molecular mechanisms of 3Cpro action. Here, we expressed 3Cpro and its mutant inactivated form (3Cmut) in Saccharomyces cerevisiae using pYES2 vector. It was shown that expression of 3Cpro, but not 3Cmut, caused cells to lose the ability to proliferate, but maintained viability, thus indicating the enzyme exerts a cytostatic effect. In addition, using vacuolar dyes and light and electron microscopy, it was found that 3Cpro causes vacuole distortion and fragmentation, accompanied by the abnormal vacuolar staining. In summary, the effects of the 3Cpro on yeast cells resemble the response of mammalian cells. Therefore, the experimental system based on S. cerevisiae can be used for further studies of 3Cpro action.

Damaged, dysfunctional, or excess mitochondria are removed from cells via a selective form of macroautophagy termed mitophagy. The clearance of mitochondria during mitophagy is mediated by double-membrane vesicles called autophagosomes, which encapsulate mitochondria that have been tagged for mitophagic removal before delivering them to lysosomes for degradation. A variety of different mitophagy pathways exist that differ in their mechanisms of initiation but share a common pathway of autophagosome formation. Autophagosome biogenesis is regulated by a number of autophagy factors which translocate from the cytosol to spatially defined focal points (foci) on the mitochondrial surface after mitophagy has been initiated. The functional analysis of autophagosome biogenesis requires the use of microscopy-based techniques which assess the recruitment of autophagy factors to mitophagic foci representing autophagosome formation sites. Here, we describe a routine method for the quantitative 3D analysis of mitophagic foci in PINK1/Parkin mitophagy immunofluorescence samples through the application of object-based image analysis (OBIA) to 3D confocal imaging datasets. The approach enables unbiased high-throughput characterisation of autophagosome biogenesis during mitophagy.

The sole proteases of the macroautophagy/autophagy machinery, the ATG4s, contribute to autophagosome formation by cleaving Atg8-family protein members (LC3/GABARAPs) which enables Atg8-family protein lipidation and de-lipidation. Our recent work reveals that ATG4s can also promote phagophore growth independently of their protease activity and of Atg8-family proteins. ATG4s and their proximity partners including ARFIP2 and LRBA function to promote trafficking of ATG9A to mitochondria during PINK1-PRKN mitophagy. Through the development of a 3D electron microscopy framework utilizing FIB-SEM and artificial intelligence (termed AIVE: Artificial Intelligence-directed Voxel Extraction), we show that ATG4s promote ER-phagophore contacts during the lipid-transfer phase of autophagosome biogenesis, which requires ATG2B and ATG9A to support phagophore growth. We also discovered that ATG4s are not essential for removal of Atg8-family proteins from autolysosomes, but they can function as deubiquitinase-like enzymes to counteract the conjugation of Atg8-family proteins to other proteins, a process that we have termed ATG8ylation (also known as LC3ylation). These discoveries demonstrate the duality of the ATG4 family in driving autophagosome formation by functioning as both autophagy proteases and trafficking factors, while simultaneously raising questions about the putative roles of ATG8ylation in cell biology.

Current models of selective autophagy dictate that autophagy receptors, including Optineurin and NDP52, link cargo to autophagosomal membranes. This is thought to occur via autophagy receptor binding to Atg8 homologs (LC3/GABARAPs) through an LC3 interacting region (LIR). The LIR motif within autophagy receptors is therefore widely recognised as being essential for selective sequestration of cargo. Here we show that the LIR motif within OPTN and NDP52 is dispensable for Atg8 recruitment and selectivity during PINK1/Parkin mitophagy. Instead, Atg8s play a critical role in mediating ubiquitin-independent recruitment of OPTN and NDP52 to growing phagophore membranes via the LIR motif. The additional recruitment of OPTN and NDP52 amplifies mitophagy through an Atg8-dependent positive feedback loop. Rather than functioning in selectivity, our discovery of a role for the LIR motif in mitophagy amplification points toward a general mechanism by which Atg8s can recruit autophagy factors to drive autophagosome growth and amplify selective autophagy., Selective autophagy receptors are thought to selectively recruit Atg8 positive membranes to cargo via their LIR motif. Here, the authors show the LIR motifs in OPTN and NDP52 are dispensable for selectivity, functioning instead to recruit additional receptors and amplify mitophagy.

SummaryThe sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The Atg4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like Atg8 family (LC3/GABARAPs). We make the unexpected discovery that human Atg4s can directly promote autophagosome formation independently of their protease activity and of Atg8 family processing. High resolution structures of phagophores generated with artificial intelligence-directed 3D electron microscopy reveal a role for the Atg4 family in promoting phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. Atg4 proximity interaction networks stimulated by PINK1/Parkin mitophagy are consistent with roles for Atg4s in protein/vesicle transport and lipid modification. We also show that Atg8 removal during autophagosome maturation does not depend on Atg4 de-lipidation activity as previously thought. Instead, we find that Atg4s can disassemble Atg8-protein conjugates, revealing a role for Atg4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the Atg4 family beyond the Atg8 lipidation axis and provide an AI driven framework for high-throughput 3D electron microscopy.

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon β expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase–dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Parkinson’s disease is diagnosed upon the presentation of motor symptoms, resulting from substantial degeneration of dopaminergic neurons in the midbrain. Prior to diagnosis, there is a lengthy prodromal stage in which non-motor symptoms, including olfactory deficits (hyposmia), develop. There is limited information about non-motor impairments and there is a need for directed research into these early pathogenic cellular pathways that precede extensive dopaminergic death in the midbrain. The protein tau has been identified as a genetic risk factor in the development of sporadic PD. Tau knockout mice have been reported as an age-dependent model of PD, and this study has demonstrated that they develop motor deficits at 15-months-old. We have shown that at 7-month-old tau knockout mice present with an overt hyposmic phenotype. This olfactory deficit correlates with an accumulation of α-synuclein, as well as autophagic impairment, in the olfactory bulb. This pathological feature becomes apparent in the striatum and substantia nigra of 15-month-old tau knockout mice, suggesting the potential for a spread of disease. Initial primary cell culture experiments have demonstrated that ablation of tau results in the release of α-synuclein enriched exosomes, providing a potential mechanism for disease spread. These alterations in α-synuclein level as well as a marked autophagy impairment in the tau knockout primary cells recapitulate results seen in the animal model. These data implicate a pathological role for tau in early Parkinson’s disease. Electronic supplementary material The online version of this article (10.1186/s40478-018-0560-y) contains supplementary material, which is available to authorized users.

Functional mitochondria are critically important for the maintenance of cellular integrity and survival. Mitochondrial dysfunction is a major contributor to neurodegenerative diseases including Parkinson's disease (PD). Two gene products mutated in familial Parkinsonism, PINK1 and Parkin, function together to degrade damaged mitochondria through a selective form of autophagy termed mitophagy. PINK1 accumulates on the surface of dysfunctional mitochondria where it simultaneously recruits and activates Parkin's E3 ubiquitin ligase activity. This forms the basis of multiple signaling events that culminate in engulfment of damaged mitochondria within autophagosomes and degradation by lysosomes. This review discusses the molecular signals of PINK1/Parkin mitophagy and the ubiquitin code that drives not only Parkin recruitment and activation by PINK1 but also the downstream signaling events of mitophagy.

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.

Bacterial viruses are among the most numerous biological entities within the human body. These viruses are found within regions of the body that have conventionally been considered sterile, including the blood, lymph, and organs. However, the primary mechanism that bacterial viruses use to bypass epithelial cell layers and access the body remains unknown. Here, we used in vitro studies to demonstrate the rapid and directional transcytosis of diverse bacteriophages across confluent cell layers originating from the gut, lung, liver, kidney, and brain. Bacteriophage transcytosis across cell layers had a significant preferential directionality for apical-to-basolateral transport, with approximately 0.1% of total bacteriophages applied being transcytosed over a 2-h period. Bacteriophages were capable of crossing the epithelial cell layer within 10 min with transport not significantly affected by the presence of bacterial endotoxins. Microscopy and cellular assays revealed that bacteriophages accessed both the vesicular and cytosolic compartments of the eukaryotic cell, with phage transcytosis suggested to traffic through the Golgi apparatus via the endomembrane system. Extrapolating from these results, we estimated that 31 billion bacteriophage particles are transcytosed across the epithelial cell layers of the gut into the average human body each day. The transcytosis of bacteriophages is a natural and ubiquitous process that provides a mechanistic explanation for the occurrence of phages within the body., IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria. They cannot infect eukaryotic cells but can penetrate epithelial cell layers and spread throughout sterile regions of our bodies, including the blood, lymph, organs, and even the brain. Yet how phages cross these eukaryotic cell layers and gain access to the body remains unknown. In this work, epithelial cells were observed to take up and transport phages across the cell, releasing active phages on the opposite cell surface. Based on these results, we posit that the human body is continually absorbing phages from the gut and transporting them throughout the cell structure and subsequently the body. These results reveal that phages interact directly with the cells and organs of our bodies, likely contributing to human health and immunity.

Bacterial viruses are among the most numerous biological entities within the human body. These viruses are found within regions of the body that have conventionally been considered sterile, including the blood, lymph, and organs. However, the primary mechanism that bacterial viruses use to bypass epithelial cell layers and access the body remains unknown. Here, we used in vitro studies to demonstrate the rapid and directional transcytosis of diverse bacteriophages across confluent cell layers originating from the gut, lung, liver, kidney, and brain. Bacteriophage transcytosis across cell layers had a significant preferential directionality for apical-to-basolateral transport, with approximately 0.1% of total bacteriophages applied being transcytosed over a 2-h period. Bacteriophages were capable of crossing the epithelial cell layer within 10 min with transport not significantly affected by the presence of bacterial endotoxins. Microscopy and cellular assays revealed that bacteriophages accessed both the vesicular and cytosolic compartments of the eukaryotic cell, with phage transcytosis suggested to traffic through the Golgi apparatus via the endomembrane system. Extrapolating from these results, we estimated that 31 billion bacteriophage particles are transcytosed across the epithelial cell layers of the gut into the average human body each day. The transcytosis of bacteriophages is a natural and ubiquitous process that provides a mechanistic explanation for the occurrence of phages within the body. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria. They cannot infect eukaryotic cells but can penetrate epithelial cell layers and spread throughout sterile regions of our bodies, including the blood, lymph, organs, and even the brain. Yet how phages cross these eukaryotic cell layers and gain access to the body remains unknown. In this work, epithelial cells were observed to take up and transport phages across the cell, releasing active phages on the opposite cell surface. Based on these results, we posit that the human body is continually absorbing phages from the gut and transporting them throughout the cell structure and subsequently the body. These results reveal that phages interact directly with the cells and organs of our bodies, likely contributing to human health and immunity.

Current autophagy models suggest that Atg8 family LC3/GABARAP proteins are essential mediators of autophagosome biogenesis. Nguyen et al. exploit CRISPR/Cas9-generated knockouts of the LC3 or GABARAP subfamilies, or both subfamilies, to show that Atg8s are dispensable for autophagosome biogenesis but essential for autophagosome–lysosome fusion., Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members divided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome–lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome–lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.

Live-cell correlative light and electron microscopy permits the visualization of ultrastructure details associated with dynamic biological processes. On the optical level, fluorescence microscopy can be further combined with functional studies of intracellular processes and manipulation of biological samples using laser light. However, the major challenge is to relocate intracellular compartments in three dimensions after the sample has undergone an extensive EM sample preparation process. Here, we describe a detailed protocol for live-cell CLEM that provides easy guidance for 3D relocalization. Based on the use of the novel polymer film TOPAS as direct imaging substrate, we provide a setup that uses highly visible toner particles for tracking the region of interest in 2D and fiducial markers for the 3D relocation of intracellular structures. An example is given where a single mitochondria is targeted by laser microirradiation in live-cell fluorescence microscopy. After relocating the same structure in 3D in serial EM sections, the changes to the mitochondrial ultrastructure are observed by TEM. The method is suitable for correlation of live-cell microscopy of cells and can be performed using any inverted optical microscope.

Live-cell correlative light and electron microscopy permits the visualization of ultrastructure details associated with dynamic biological processes. On the optical level, fluorescence microscopy can be further combined with functional studies of intracellular processes and manipulation of biological samples using laser light. However, the major challenge is to relocate intracellular compartments in three dimensions after the sample has undergone an extensive EM sample preparation process. Here, we describe a detailed protocol for live-cell CLEM that provides easy guidance for 3D relocalization. Based on the use of the novel polymer film TOPAS as direct imaging substrate, we provide a setup that uses highly visible toner particles for tracking the region of interest in 2D and fiducial markers for the 3D relocation of intracellular structures. An example is given where a single mitochondria is targeted by laser microirradiation in live-cell fluorescence microscopy. After relocating the same structure in 3D in serial EM sections, the changes to the mitochondrial ultrastructure are observed by TEM. The method is suitable for correlation of live-cell microscopy of cells and can be performed using any inverted optical microscope.

Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.

Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.