Autophagy in the test tube: In vitro reconstitution of aspects of autophagosome biogenesis

Autophagy is a versatile recycling pathway that delivers cytoplasmic contents to lysosomal compartments for degradation. It involves the formation of a cup-shaped membrane that expands to capture cargo. After the cargo has been entirely enclosed, the membrane is sealed to generate a double-membrane-enclosed compartment, termed the autophagosome. Depending on the physiological state of the cell, the cargo is selected either specifically or non-specifically. The process involves a highly conserved set of autophagy-related proteins. Reconstitution of their action on model membranes in vitro has contributed tremendously to our understanding of autophagosome biogenesis. This review will focus on various in vitro techniques that have been employed to decipher the function of the autophagic core machinery

Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease

Reconstituting Autophagy Initiation from Purified Components

International audienceThe hallmark of macroautophagy is the de novo generation of a membrane structure that collects cytoplasmic material and delivers it to lysosomes for degradation. The nucleation of this precursor membrane, termed phagophore, involves the coordinated assembly of the Atg1-kinase complex and the recruitment of Atg9 vesicles. The latter represents one important membrane source in order to produce phagophores in vivo. We explain how the process of phagophore nucleation can be reconstituted from purified components in vitro. We describe the assembly of the ~500 kDa pentameric Atg1-kinase complex from its purified subunits. We also explain how Atg9-donor vesicles are generated in vitro to study the interaction of Atg9 and Atg1-kinase complexes by floatation experiments