Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells

Abstract

Cellular autophagy is an evolutionarily highly conserved degradation pathway for both stochastic and selective degradation of cytoplasmic cargo within the lysosomal compartment. As such, this process of self-eating secures cellular homeostasis and survival, while malfunction contributes to the initiation and development of many agerelated human diseases including diabetes, tumorigenesis, and neurodegeneration ( 1-4 ). Autophagy is constitutively active on a low basal level resulting in constant cytoplasmic turnover and the specifi c elimination of macromolecule aggregates and damaged organelles. Following a great variety of cellular insults such as nutrient starvation, autophagy is induced above basal level and produces monomers and energy for subsequent recycling processes.Autophagy is hallmarked by the formation of doublemembrane vesicles called autophagosomes that are generated from initial preautophagosomal membrane precursors or phagophores. Elongation of the phagophore and subsequent vesicle closure complete the formation of autophagosomes that sequester the cytoplasmic cargo and communicate with the lysosomal compartment to acquire acidic hydrolases for cargo breakdown ( 5-7 ).Abstract Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1) , both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism. -Pfi sterer, S. G., D. Bakula, T. Frickey, A. Cezanne, D. Brigger, M. P. Tschan, H. Robenek, and T. ProikasCezanne. Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome

“…2C). However, the role of the WIPI members in recruiting ATG2 in human cells warrants further detailed studies, as it was also found that ATG2 functions hierarchically at the same position as WIPI1 (Itakura and Mizushima, 2010;Velikkakath et al, 2012), and if WIPI1 is not bound to PtdIns3P at omegasomes, ATG2 is not recruited to the initiation site of autophagosome formation (Pfisterer et al, 2014). Strikingly, WIPI4 was found to be mutated de novo and causative for SENDA (static encephalopathy of childhood with neurodegeneration in adulthood), a sporadic NBIA (neurodegeneration with brain iron accumulation) subtype (Haack et al, 2012;Hayflick et al, 2013;Saitsu et al, 2013).…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…77 Deletion of ATG14/Atg14 impairs autophagosome formation, 75,77,78,142 characterized by accumulation of LC3-II and ubiquitinated protein inclusions. 75,77 During autophagosome formation, ATG14 translocates to phagophore and/or omegasome membranes, 143 and it is likely to target a proportion of PIK3C3 to the ER, which is crucial for PtdIns3P production and autophagy induction. 142,144 Indeed, the Barkor/Atg14(L) autophagosome targeting sequence/BATS domain of ATG14 has been confirmed to be required for targeting the PIK3C3 complex to the phagophore and/or omegasome membrane and for the initiation of autophagosome formation.…”

WIPI β-propellers at the crossroads of autophagosome and lipid droplet dynamics

“…Hence autophagosome formation in close proximity to LDs seems to facilitate the use of LD-stored lipids for the de novo synthesis of the autophagosomal membrane [33]. Whereas DFCP1, WIPI-1, WIPI-2B and WIPI-2D specifically localized to the forming autophagosomal membrane, some Atg proteins such as LC3, Atg2A and Atg14L have also been found to localize directly to LDs [34][35][36]. Moreover, functional down-regulation of LC3 has been shown to impair LD formation [37], whereas Atg2A/Atg2B [35] and Atg2A or Atg14L [36] depletion increased LD abundance and size.…”

“…Moreover, functional down-regulation of LC3 has been shown to impair LD formation [37], whereas Atg2A/Atg2B [35] and Atg2A or Atg14L [36] depletion increased LD abundance and size. Upon starvation-induced autophagy and depending on PtdIns3P production, Atg14L and Atg2A co-localized with DFCP1 and WIPI-1, whereas the Atg2A/Atg14L colocalization at LDs was maintained in addition to, and appeared to be independent of, autophagosomal PtdIns3P production [36].…”

“…Regardless, both scenarios detail both (i) dual essential functions of Atg proteins in LD and autophagosome biogenesis, and (ii) possible roles of Atg proteins in interconnecting lipid exchange between LDs and the forming autophagosome, and thereby warrant further detailed molecular characterization. [34][35][36]. Atg2A constitutively localizes to adipocyte differentiation-related protein (ADRP)-positive LDs, and a pool of Atg2A-positive LDs is also decorated with Atg14L independent of autophagic status [36].…”