Membrane Contact Sites in Autophagy

Abstract

Eukaryotes utilize different communication strategies to coordinate processes between different cellular compartments either indirectly, through vesicular transport, or directly, via membrane contact sites (MCSs). MCSs have been implicated in lipid metabolism, calcium signaling and the regulation of organelle biogenesis in various cell types. Several studies have shown that MCSs play a crucial role in the regulation of macroautophagy, an intracellular catabolic transport route that is characterized by the delivery of cargoes (proteins, protein complexes or aggregates, organelles and pathogens) to yeast and plant vacuoles or mammalian lysosomes, for their degradation and recycling into basic metabolites. Macroautophagy is characterized by the de novo formation of double-membrane vesicles called autophagosomes, and their biogenesis requires an enormous amount of lipids. MCSs appear to have a central role in this supply, as well as in the organization of the autophagy-related (ATG) machinery. In this review, we will summarize the evidence for the participation of specific MCSs in autophagosome formation, with a focus on the budding yeast and mammalian systems.

Defining the EM-signature of successful cell-transfection

“…This single ultrastructural feature strongly suggests that the elaborated ER around them is ’protecting’ or ’enclosing’ the freshly-released Lipofectamine, or at least is attempting to do so. In this and many other ways, this ER- elaboration reminds us of early forms of autophagocytosis (Biazik et al, 2017; Biazik et al, 2015; Fengsrud et al, 1995; Gudmundsson et al, 2022; Hayashi-Nishino et al, 2009; Kovacs et al, 1982; Park et al, 2020; Yla-Anttila et al, 2009; Zwilling and Reggiori, 2022) and strongly suggest that the cell is attempting to encapsulate or ’insulate’ itself from the Lipofectamine- droplets that are emerging from the breaking endosomes. That this ’insulation’ is partially successful is most notably indicated by the absence of any ribosome-decoration of the Lipofectamine, at this early stage (cf., Fig.4, upper panels).…”

“…However, we should also stress that despite the obvious extensiveness and persistence of these ER-elaborations, we have never observed, within them, the formation of any of the classical "isolation membranes" that structurally define autophagocytosis (Biazik et al, 2017; Biazik et al, 2015; Fengsrud et al, 1995; Gudmundsson et al, 2022; Hayashi-Nishino et al, 2009; Kovacs et al, 1982; Park et al, 2020; Yla-Anttila et al, 2009; Zwilling and Reggiori, 2022). So, despite the obvious impression that the ER is attempting to ’wall off’ or ’shore up’ the breaking endosomes, the lipoplexes that are thereby released into the cytoplasm must not recruit LC3 or activate any of the rest of the normal cellular autophagocytosis-program.…”

How Membrane Contact Sites Shape the Phagophore

“…From the first electron micrographs of phagophores amid other organelles in fixed cells, our understanding of the structure, scope, and function of MCSs in autophagy has by now advanced tremendously due to the integration of biochemical, imaging, and structural studies. In addition to the contact sites discussed here, going forward, it will be interesting to explore how metabolic conditions and various types of cargo influence the established contact sites and their functional role (Zwilling and Reggiori, 2022). Systematic screenings involving many cell types and conditions might help to reveal the underlying principles and mechanisms explaining the variable involvement of different organelles in autophagosome biogenesis.…”

“…Moreover, there are nonphagophore MCSs with potential roles in autophagy, such as the ER-mitochondria and ER-plasma membrane MCSs that regulate local PI3P synthesis (Böckler and Westermann, 2014;Hamasaki et al, 2013;Nascimbeni et al, 2017). The role of these nonphagophore MCSs has been reviewed elsewhere (Kohler et al, 2020;Zwilling and Reggiori, 2022). Here, we will focus in detail on the three best-characterized MCSs of the phagophore, with the vacuole, ER, and LDs.…”

“…For room temperature TEM, chemically or cryo-fixed samples are embedded in resin and cut into thin sections which can then be used for 2D imaging or 3D tomography. The advantages of the method include the relatively high throughput and the possibility to reconstruct larger 3D volumes by combining data from adjacent sections, showcased in a study providing detailed maps of the ER structure and its contacts in S. cerevisiae ( West et al, 2011 ).…”