We observed local recruitment of autophagic and pre-autophagic markers at E-Syts domains of the cortical ER during autophagy initiation. Indeed, we show here that stress situations that induce autophagy lead as well to ER-PM contact site mobilization, highlighting a connection between ER-PM tethering and the autophagy machinery. Because both ER and PM have been directly associated with autophagy regulation and because ER tethering could be important for membrane remodelling, we hypothesized that the ER cooperates with plasma membrane during the very first steps of the autophagosome biogenesis via the establishment of ER-PM specialized contact sites. In higher eukaryotes, three ER-localized proteins, the extended synaptotagmins (E-Syts 1, 2 and 3), play crucial roles in tethering the ER to the PM and are thus considered as key regulators, as well as precise markers, of ER-PM tethering zones (Giordano et al, 2013). Notably, ER–mitochondria contact sites actively participate in autophagosome biogenesis via the regulation of PI3KC3 complex (Hamasaki et al, 2013).ĮR-PM contact sites are important for lipid metabolism and transport, notably of phosphoinositides, and these domains have the potential to affect membrane trafficking and signalling events that occur at the PM (Stefan et al, 2013). There is growing evidence that close appositions between the ER and the membranes of virtually all other organelles play major roles in cell physiology (Helle et al, 2013). The ER is a dynamic and complex membranous network that extends throughout the cell impacting a multiplicity of cellular functions (Friedman & Voeltz, 2011). The phagophore then elongates and is close to form a mature autophagosome that will latter fuse with the lysosome.Īlthough the ER membrane requirement is well established, other membrane sources, like the Golgi apparatus, endosomes, the mitochondria and the plasma membrane (PM), have been proposed to participate, directly, indirectly or partially, in autophagosome biogenesis (Molino et al, 2017), from phagophore generation to growth of the organelle (Ravikumar et al, 2010a, b Rubinsztein et al, 2012). The PI3P pool engaged in autophagosome biogenesis is synthesized by the class 3 PI3kinase complex (PI3KC3), comprised of VPS34, VPS15, ATG14L, Beclin1, and regulating adaptors, such as VMP1, NRBF2 and Ambra1, and is dependent on ULK1 complex signalling (Nascimbeni et al, 2017). Autophagy initiates with biogenesis of a pre-autophagosomal double-membrane structure, termed the phagophore, which emanates from the omegasome, a subdomain of the endoplasmic reticulum (ER) membrane positive for PI3P (phosphatidyl-inositol-3-phosphate) and PI3P-binding proteins (Axe et al, 2008). The biogenesis of autophagosome is orchestrated by multiple signalling pathways and complexes that regulate membrane dynamics that contain autophagy-related (ATG) proteins. Autophagy is initiated by the formation of a specific double-membrane organelle called the autophagosome. Macro-autophagy (hereafter referred to as autophagy) is a highly regulated intracellular degradation pathway necessary for cellular homeostasis (Boya et al, 2013).
These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis.
Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear.
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