An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure.

The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.

Atg2A/B deficiency switches cytoprotective autophagy to non-canonical caspase-8 activation and apoptosis.

Autophagosomal membranes are emerging as platforms for various cell survival and death signaling networks beyond autophagy. While autophagy-dependent cell death has been reported in response to a variety of stimuli, the underlying molecular mechanisms remain far from clear. Here, we demonstrate that inhibition of autophagosome completion by Atg2A/B deletion accumulates immature autophagosomal membranes that promote non-canonical caspase-8 activation in response to nutrient starvation via an intracellular death-inducing signaling complex (iDISC). Importantly, iDISC-induced caspase-8 dimerization and activation occurs on accumulated autophagosomal membranes and requires the LC3 conjugation machinery but is independent from the extrinsic pathway of apoptosis. Moreover, we have identified NF-κB signaling and c-FLIP as negative regulators of iDISC-mediated caspase-8 activation and apoptosis. Collectively, these findings reveal autophagosomal membrane completion as a novel target to switch cytoprotective autophagy to apoptosis.

Endophilin B2 facilitates endosome maturation in response to growth factor stimulation, autophagy induction, and influenza A virus infection.

Endocytosis, and the subsequent trafficking of endosomes, requires dynamic physical alterations in membrane shape that are mediated in part by endophilin proteins. The endophilin B family of proteins contains an N-terminal Bin/amphiphysin/Rvs (N-BAR) domain that induces membrane curvature to regulate intracellular membrane dynamics. Whereas endophilin B1 (SH3GLB1/Bif-1) is known to be involved in a number of cellular processes, including apoptosis, autophagy, and endocytosis, the cellular function of endophilin B2 (SH3GLB2) is not well understood. In this study, we used genetic approaches that revealed that endophilin B2 is not required for embryonic development in vivo but that endophilin B2 deficiency impairs endosomal trafficking in vitro, as evidenced by suppressed endosome acidification, EGFR degradation, autophagic flux, and influenza A viral RNA nuclear entry and replication. Mechanistically, although the loss of endophilin B2 did not affect endocytic internalization and lysosomal function, endophilin B2 appeared to regulate the trafficking of endocytic vesicles and autophagosomes to late endosomes or lysosomes. Moreover, we also found that despite having an intracellular localization and tissue distribution similar to endophilin B1, endophilin B2 is dispensable for mitochondrial apoptosis. Taken together, our findings suggest that endophilin B2 positively regulates the endocytic pathway in response to growth factor signaling, autophagy induction, and viral entry.

The Bif-1-Dynamin 2 membrane fission machinery regulates Atg9-containing vesicle generation at the Rab11-positive reservoirs.

Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy.

Bif-1 deficiency impairs lipid homeostasis and causes obesity accompanied by insulin resistance.

Bif-1 is a membrane-curvature inducing protein that is implicated in the regulation of autophagy and tumorigenesis. Here, we report that Bif-1 plays a critical role in regulating lipid catabolism to control the size of lipid droplets and prevent the development of obesity and insulin resistance upon aging or dietary challenge. Our data show that Bif-1 deficiency promotes the expansion of adipose tissue mass without altering food intake or physical activities. While Bif-1 is dispensable for adipose tissue development, its deficiency reduces the basal rate of adipose tissue lipolysis and results in adipocyte hypertrophy upon aging. The importance of Bif-1 in lipid turnover is not limited to adipose tissue since fasting and refeeding-induced lipid droplet clearance is also attenuated by Bif-1 loss in the liver. Interestingly, obesity induced by a high fat-diet or Bif-1 deficiency downregulates the expression of proteins involved in the autophagy-lysosomal pathway, including Atg9a and Lamp1 in the adipose tissue. These findings thus identify Bif-1 as a novel regulator of lipid homeostasis to prevent the pathogenesis of obesity and its associated metabolic complications.

Sh3glb1/Bif-1 and mitophagy: acquisition of apoptosis resistance during Myc-driven lymphomagenesis.

Evasion of apoptosis, which enables cells to survive and proliferate under metabolic stress, is one of the hallmarks of cancer. We have recently reported that SH3GLB1/Bif-1 functions as a haploinsufficient tumor suppressor to prevent the acquisition of apoptosis resistance and malignant transformation during Myc-driven lymphomagenesis. SH3GLB1 is a membrane curvature-inducing protein that interacts with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the upregulation of anti-apoptotic proteins, including MCL1 and BCL2L1. Notably, we found that Sh3glb1 haploinsufficiency increases mitochondrial mass in overproliferated prelymphomatous Eµ-Myc cells. Moreover, loss of Sh3glb1 suppresses autophagy-dependent mitochondrial clearance (mitophagy) in PARK2/Parkin-expressing mouse embryonic fibroblasts (MEFs) treated with the mitochondrial uncoupler CCCP. Interestingly, PARK2-expressing Sh3glb1-deficient cells accumulate ER-associated immature autophagosome-like structures after treatment with CCCP. Taken together, we propose a model of mitophagy in which SH3GLB1 together with the class III phosphatidylinositol 3-kinase complex II (PIK3C3CII) (PIK3R4-PIK3C3-BECN1-UVRAG) regulates the trafficking of ATG9A-containing Golgi-derived membranes (A9(+)GDMs) to damaged mitochondria for autophagosome formation to counteract oncogene-driven tumorigenesis.

Bif-1 haploinsufficiency promotes chromosomal instability and accelerates Myc-driven lymphomagenesis via suppression of mitophagy.

Malignant transformation by oncogenes requires additional genetic/epigenetic changes to overcome enhanced susceptibility to apoptosis. In the present study, we report that Bif-1 (Sh3glb1), a gene encoding a membrane curvature­driving endophilin protein, is a haploinsufficient tumor suppressor that plays a key role in the prevention of chromosomal instability and suppresses the acquisition of apoptosis resistance during Myc-driven lymphomagenesis. Although a large portion of Bif-1­deficient mice harboring an Eµ-Myc transgene displayed embryonic lethality, allelic loss of Bif-1 dramatically accelerated the onset of Myc-induced lymphoma. At the premalignant stage, hemizygous deletion of Bif-1 resulted in an increase in mitochondrial mass, accumulation of DNA damage, and up-regulation of the antiapoptotic protein Mcl-1. Consistently, allelic loss of Bif-1 suppressed the activation of caspase-3 in Myc-induced lymphoma cells. Moreover, we found that Bif-1 is indispensable for the autophagy-dependent clearance of damaged mitochondria (mitophagy), because loss of Bif-1 resulted in the accumulation of endoplasmic reticulum­associated immature autophagosomes and suppressed the maturation of autophagosomes. The results of the present study indicate that Bif-1 haploinsufficiency attenuates mitophagy and results in the promotion of chromosomal instability, which enables tumor cells to efficiently bypass the oncogenic/metabolic pressures for apoptosis. .

Neuroglial expression of the MHCI pathway and PirB receptor is upregulated in the hippocampus with advanced aging.

The hippocampus undergoes changes with aging that impact neuronal function, such as synapse loss and altered neurotransmitter release. Nearly half of the aged population also develops deficits in spatial learning and memory. To identify age-related hippocampal changes that may contribute to cognitive decline, transcriptomic analysis of synaptosome preparations from adult (12 months) and aged (28 months) Fischer 344-Brown Norway rats assessed for spatial learning and memory was performed. Bioinformatic analysis identified the MHCI pathway as significantly upregulated with aging. Age-related increases in mRNAs encoding the MHCI genes RT1-A1, RT1-A2, and RT1-A3 were confirmed by qPCR in synaptosomes and in CA1 and CA3 dissections. Elevated levels of the MHCI cofactor (B2m), antigen-loading components (Tap1, Tap2, Tapbp), and two known MHCI receptors (PirB, Klra2) were also confirmed. Protein expression of MHCI was elevated with aging in synaptosomes, CA1, and DG, while PirB protein expression was induced in both CA1 and DG. MHCI expression was localized to microglia and neuronal excitatory postsynaptic densities, and PirB was localized to neuronal somata, axons, and dendrites. Induction of the MHCI antigen processing and presentation pathway in hippocampal neurons and glia may contribute to age-related hippocampal dysfunction by increasing neuroimmune signaling or altering synaptic homeostasis.