Projects Fulvio Reggiori

Autophagy

Autophagy is a catabolic transport route conserved among all eukaryotes and it is required for the rapid degradation of large portions of the cytoplasm, protein aggregates, excess or damaged organelles and invading pathogens. It has long been known that this pathway is essential to generate an internal pool of nutrients that allows cells surviving starvation periods. Recent studies, however, have revealed that autophagy participates in a multitude of other cellular processes including cell differentiation and development, degradation of aberrant structures, lifespan extension, type II programmed cell death, innate and adaptive immunity. As a result, this pathway plays a relevant role in the pathophysiology of neurodegenerative, cardiovascular, chronic inflammatory, muscular and autoimmune diseases, and some malignancies. Conversely, a deficiency in this protective transport route leads to illnesses such as sporadic breast, ovarian and prostate cancers, and cardiomyophaties. The elucidation of the molecular mechanism of autophagy is therefore vital to understand the regulation and contribution of this pathway in all these physiological and pathological situations.

The basic mechanism of autophagy is the sequestration of the cargo that has to be degraded by autophagosomes (Figure 1). These large double-membrane vesicles are formed by expansion and sealing of a small cisterna known as the phagophore or isolation membrane. Once complete, they first fuse with endosomes to form amphisomes and then with lysosomes to deliver their cargo into the hydrolytic interior of this compartment for degradation. The central actors of this highly regulated pathway are the autophagy (Atg) proteins, which mediate the formation of the phagophore and its expansion into an autophagosome (Figure 1). Upon induction of autophagy, Atg proteins associate to form the so-called phagophore assembly site (PAS) or pre-autophagosomal structure, the precursor structure of the phagophore. Despite the identification of the Atg proteins, the molecular mechanism that directs formation of the sequestering vesicles remains largely unknown.

Figure-1-(F.Reggiori)

Figure 1. The mechanism of autophagy (simplified).
The cargo destined for degradation is sequestered into a double-membrane vesicle called an autophagosome. Once the sequestration process is completed, the autophagosome fuses with the mammalian lysosome or the plant and yeast vacuole. During the fusion event, the external lipid bilayer of the autophagosome becomes part of the lysosome/vacuole limiting membrane while the internal vesicle, termed an autophagic body, is liberated into the interior of this organelle where, together with the cargo, it is degraded in basic metabolites by resident hydrolases.

The molecular mechanism of autophagosome formation in the yeast Saccharomyces cerevisiae

The model organism used in our studies on the molecular mechanism of autophagy is the yeast Saccharomyces cerevisiae, a unicellular eukaryote very suitable for either genetic or biochemical experimental approaches, which we combine with fluorescence and electron microscopy techniques to unveil how the complex formation of autophagosomes is regulated and accomplished.
Despite the important progress made in understanding autophagy, the study of the precise function of the Atg proteins as well as the mechanism of this pathway has been hampered by the lack of information regarding the membrane dynamics during the autophagosome formation process. To gain insights into this crucial question, we have studied the biogenesis of the yeast PAS by combining a recently developed immuno-electron microscopy (IEM) protocol [1] with yeast genetics. We have focused our attention on the transmembrane Atg9 because intrinsically associated with lipid bilayers and therefore this protein has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary to the formation and expansion of nascent autophagosomes. We have shown that the PAS originates from Atg9-positive clusters of vesicles and tubules (Figure 2) that we have called Atg9 reservoirs [2, 3]. These compartments are a new organelle derived from the secretory pathway that often is adjacent to mitochondria [2, 3]. Translocation of one or more Atg9 reservoirs in proximity to the vacuole, together with the successive recruitment of other Atg proteins, generates the PAS [2]. Hence, fusion of the tubulo-vesicular membranes of the Atg9 reservoirs leads to the formation of the phagophore necessary for the subsequent biogenesis of a double-membrane vesicle. Thus, our results suggest the de novo formation of the PAS from vesicles and tubules and highlight the crucial role of Atg9 in this process.

Figure-2

Figure 2. The ultrastructure of the Atg9 reservoirs
[2]. Wild-type cells expressing Atg9-GFP grown in reach medium were processed for IEM [1]. Cryo-sections were immuno-gold labelled for GFP. M, mitochondria; MVB, multivesicular bodies; V, vacuole. Bar 200 nm.

In a different work, we have studied the role of the Golgi complex in autophagy and have determined that in yeast, activation of Arf1 and Arf2 GTPases by Sec7, Gea1 and Gea2 is essential for this catabolic process [4]. The two main events catalyzed by these components, the biogenesis of COPI- and clathrin-coated vesicles, however, do not play a critical role in autophagy. Analysis of the sec7 strain under starvation conditions has revealed that the autophagy machinery is correctly assembled and the precursor the phagophore is normally formed. Though, the expansion of the phagophore into an autophagosome is severely impaired [4]. Our data have thus shown that the Golgi complex plays a crucial role in supplying the lipid bilayers necessary for the biogenesis of double-membrane vesicles possibly through a new class of transport carriers or a new mechanism [4, 5].
Currently, the laboratory is also investigating other aspects of the molecular mechanism and regulation of autophagy.

Coronaviruses, autophagy and the ERAD tuning

The long-term objective of the laboratory is to understand the contribution of autophagy in medically-relevant physiological and pathological situations in humans.
We have recently worked on coronavirus (CoVs) infection and the interrelationship of these viruses with the autophagy machinery in partnership with the laboratory of Xander de Haan (Virology Division, Utrecht University, the Netherlands) and that of Maurizio Molinari (Institute of Biomedical Research, Bellinzona, Switzerland). CoV are positive-stranded RNA viruses, the relevance of which has considerably increased due to the recent emerging of the SARS-CoV. Once inside the host cell, they induce the formation of double-membrane vesicles (DMVs) on the surface of which they anchor their RNA transcription/replication complexes. These DMVs resemble autophagosomes and it has been suggested that genes involved in autophagy could play a role in DMV formation. Using the mouse hepatitis virus (MHV) as a model for the CoV infection cycle, we have found that MHV-induced DMVs are associated with the non-lipidated form of LC3/Atg8, a protein involved in autophagy [6]. Although LC3 depletion blocks DMV formation and severely impairs MHV replication, the autophagy machinery is not required for MHV infection. The ER-associated degradation (ERAD) tuning mediates the rapid turnover of chaperones such as EDEM1 and OS-9 by transporting them to the lysosome through vesicles also coated with non-lipidated LC3. We have discovered that during MHV infection, EDEM1 and OS-9 are inside the DMVs and fail to be degraded [6]. Our data have revealed the cellular pathway hijacked by CoV, e.g. the ERAD tuning, and this discovery has opened new therapeutic avenue for the treatment of infections caused by this family of viruses. We are currently trying to identify the MHV proteins that induce DMVs formation and determine with which ERAD tuning components they interact by using biochemical and cell biological approaches [7]. Our investigations are providing an alternative strategy to elucidate fundamental aspects of the mechanism of DMV formation but also isolate components of the ERAD tuning, the mechanism of which remains largely unknown.
This, however, is not the only aspect of CoV infection that we have investigated. In particular, by performing a time-course experiment in which the MHV-induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)-electron microscopy, we have unveiled and characterized at the ultrastructural level the subcellular manipulations implemented by CoV in order to efficiently replicate and assemble in host cells [8]. With our approach we were able to confirm the appearance of 6, previously reported, membranous structures during the course of a complete infection cycle. These structures include the DMVs, convoluted membranes (CMs) and virions but also the more enigmatic large virion-containing vacuoles (LVCVs), tubular bodies (TBs) and cubic membrane structures (CMSs) (Figure 3). We have characterized the LVCVs, TBs and CMSs, and found that the CoV-induced structures appear in a strict order. By combining these data with quantitative analyses on viral RNA, protein synthesis and virion release, this study generates an integrated molecular and ultrastructural overview of CoV infection [8]. In particular, it provides insights in the role of each CoV-induced structure and reveals that LVCVs are ERGIC/Golgi compartments that expand to accommodate an increasing production of viral particles. We are currently working on other aspects of CoV life cycle, in particular on the mechanism used by these viruses to generate their replicative DMVs.

Figure-3

Figure 3. Ultrastructure of membranous structures induced by MHV in host cells [8]. HeLa-CEACAM1a cells inoculated with MHV-A59 were fixed at 8h p.i. and processed for conventional electron microscopy. (A, B) DMVs are cytoplasmic double-membrane vesicles (arrows) that frequently possess an invagination. DMVs are often found clustered together in close proximity of a small network of membranes, the CMs (arrowheads). The inset in panel B shows a magnification of two DMVs to highlight the two lipid bilayers characterizing these vesicles. (C) Newly made virions (arrows) present in the lumen of the Golgi complex. (D) LVCVs are large circular organelles with a diameter of approximately 450-750 nm that contain numerous virions in their interior. In addition, viral particles can be observed that are assembling at the limiting membrane of this structure by invagination and successive pinching off (arrows). (E) The TBs (arrow) are ball of wool-like membranous rearrangements with a diameter of approximately 300-650 nm that appear to be continuous with the ER. (F) CMSs are extended (up to 850 nm in length), geometrical and highly organized conformations, which are often seen connected to a swollen ER (arrowhead). ER, endoplasmic reticulum; G, Golgi complex; M, mitochondria; PM, plasma membrane; L, lysosome. White bar, 500 nm; black bar, 200 nm.

References
[1] Griffith J, Mari M, De Mazière A, Reggiori F (2008), A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast S. cerevisiae, Traffic, 9, 1060-1072.
[2] Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F (2010), An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis, J Cell Biol, 190, 1005-1022.
[3] Mari M, Reggiori F (2010), Atg9 reservoirs, a new organelle of the yeast endomembrane system? Autophagy, 6, 1221-1223.
[4] van der Vaart A, Griffith J, Reggiori F (2010), Exit from the Golgi is required for the expansion of the autophagosomal phagophore in the yeast Saccharomyces cerevisiae, Mol Biol Cell, 21, 2270–2284.
[5] van der Vaart A, Reggiori F (2010), The Golgi complex as a source for yeast autophagosomal membranes, Autophagy, 6, 800-801.
[6] Reggiori F, Monastyrska I, Verheije MH, Calì' T, Ulasli M, Bianchi S, Bernasconi R, de Haan CAM, Molinari M (2010), Coronaviruses hijack LC3-I-positive EDEMosome membranes for replication, Cell Host Microbe, 7, 500-508.
[7] de Haan CAM, Molinari M, Reggiori F (2010), Autophagy-independent LC3 function in vesicular traffic, Autophagy, 6, 994-996.
[8] Ulasli M, Verheije MH, de Haan CAM, Reggiori F (2010), Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus, Cell Microbiol, 12, 844-861.

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