Sphingolipids are structural lipid components of cell membranes, including membrane of
Sphingolipids are structural lipid components of cell membranes, including membrane of organelles, such as mitochondria or endoplasmic reticulum, playing a role in signal transduction as well as in the transport and intermixing of cell membranes. vacuoles. In particular, 960201-81-4 manufacture fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses revealed that this ganglioside interacts with phosphatidylinositol 3-phosphate and can be detected in immature autophagosomes in association with LC3-II as well as in autolysosomes associated with LAMP1. Hence, it appears as a structural component of autophagic flux. Accordingly, we found that autophagy was significantly impaired by knocking down ST8SIA1/GD3 synthase (ST8 -N-acetyl-neuraminide -2,8-sialyltransferase 1) or by altering sphingolipid metabolism with fumonisin B1. Interestingly, exogenous administration of GD3 ganglioside was capable of reactivating the autophagic process inhibited by fumonisin B1. Altogether, these results suggest that gangliosides, via their molecular interaction with autophagy-associated molecules, could be recruited to autophagosome and contribute to morphogenic remodeling, e.g., to changes of membrane curvature and fluidity, finally leading to mature autolysosome formation. suppressed autophagosome biogenesis through a mechanism that was both independent of the ATG12CATG5 and ATG8Cphosphatidylethanolamine conjugates as well as the formation of phagophores.18 However, a defined role for lipid rafts or individual gangliosides in regulating autophagy induction as well as autophagosome biogenesis and/or maturation is unclear. In this study, we analyzed whether gangliosides play a role in autophagosome morphogenic remodeling leading to autophagosome formation. To do this, we decided to use well established but untransformed cell models (primary human fibroblasts and mouse 960201-81-4 manufacture embryo fibroblasts), where GM3, GD3 and GD1a gangliosides are constitutively expressed. 19 Results Autophagy induction in primary fibroblasts We preliminarily analyzed autophagy triggering. Common cellular stresses known to induce autophagy include amino acid starvation, glucose withdrawal, and ER stress. To trigger 960201-81-4 manufacture autophagic machinery in our experimental model we used a classical autophagic stimulus, such as amino acid starvation (HBSS) and a pharmacological stimulus able to induce ER stress, tunicamycin (TNC). As shown in Figure?1A (left panel), flow cytometry analysis of cells stained with Cyto-ID Autophagy detection kit showed a significant (< 0.01) increase of green 960201-81-4 manufacture fluorescence emission in cells starved in HBSS or treated with TNC in comparison with untreated control cells. These data were also confirmed by using a specific antibody able to recognize cleaved form of LC3 (Fig.?1A, right panel). Flow cytometry results were confirmed by western blot analysis, which revealed a band corresponding to LC3-II either after cell starvation or in cells treated with TNC (Fig.?1B). Figure?1. Autophagy induction in primary fibroblasts. (A) Semiquantitative flow cytometry analysis of autophagy performed in primary human fibroblasts untreated (full gray curves), starved 18 h with HBSS medium (dashed lines) or treated 18 h with ... Engagement of sphingolipids in the autophagic process Starting from the observation that sphingolipids have already been hypothesized to be related to autophagy in cancer cells,20 we first investigated their possible involvement in autophagic progression by impairing their biosynthesis. For this purpose, we pretreated primary human fibroblasts 960201-81-4 manufacture with SIRT3 fumonisin B1 (FB1), an inhibitor of sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis, before the autophagic stimulus. Flow cytometry analysis after cell staining with anti-LC3-II antibodies clearly showed that cell pretreatment with FB1 significantly inhibited autophagy either induced by HBSS or by TNC after 4 or 18 h (Fig.?2A). We therefore analyzed LC3 expression by western blot after autophagy triggering, in the presence or in the absence of FB1, under the same experimental conditions (Fig.?2B). Our results confirmed that in the presence of FB1 the conversion of soluble LC3-I to lipid-membrane-bound LC3-II, considered as the prototypical marker of autophagy, was significantly lower (Fig.?2B, see densitometric analysis). Similar results were obtained in experiments performed with mouse fibroblasts (MEFs, see Fig. S1). Figure?2. Engagement of sphingolipids in the autophagic process. (A) Semiquantitative flow cytometry analysis of autophagy performed after human fibroblast staining with anti-LC3-II antibody. The numbers represent the mean SD of the … In order to clarify the role of GD3 during autophagic process, a small interfering RNA (siRNA) was employed to knock down ST8SIA1.