Krueger, Eugene

Chronic hepatitis B (CHB), a major global public health burden, is characterized by the presence of high circulating levels of non-infectious subviral particles (SVPs). These SVPs are mainly composed of small hepatitis B surface antigen (SHBs) and are associated with a dysfunctional HBV-specific immune response. Previous studies exploring the cellular mechanisms and molecular pathways by which SVPs are secreted from hepatocytes have utilized either non-hepatic cells or whole-genome HBV-integrated hepatoma cell lines in which SHBs can also be secreted as a component of the virion envelope. In this study, we provide a detailed examination of SHBs trafficking using biochemical approaches coupled with high-resolution confocal microscopy and live-cell imaging via a novel fluorophore-tagged SHBs probe in both HepG2 and primary human hepatocytes stably expressing SHBs. Our results provide evidence that SHBs utilize a modified ER-Golgi secretory pathway with transit from the ER to the multivesicular body and lysosomal/late endosomal compartments. Importantly, we identified the autophagy adapters ATG5 and LC3B, and the ER-phagy protein atlastin 3 (ATL3), central to mediating SHBs secretion. These findings suggest that SHBs is trafficked through a non-canonical path for secretion and provides new potential therapeutic targets for the treatment of CHB.

It is well established that many tumor types possess defective autophagic pathways. Several studies have reported that the transmembrane, autophagic lipid scramblase ATG9B is altered in multiple cancers, suggesting that this dysregulation could contribute to oncogenesis. Therefore, the goal of this study was to define the cellular distribution of ATG9B in two different tumor cell types and to provide insights into its cellular function. Surprisingly, we found that ATG9B shows a modest association with autophagic structures and exhibits a unique and prominent localization to mitochondria, in contrast to its related form ATG9A. Upon expression of tagged ATG9B forms, this mitochondrial distribution was accompanied by aberrant changes in mitochondrial morphology as well as a reduction in the mitochondrial membrane potential and the release of mtDNA. Few indicators for ATG9B-dependent mitophagy were noted. Instead, ATG9B overexpression led to pronounced apoptotic cell death as assessed by a variety of indicators. Further, we find that the N-terminal sequence of ATG9B acts as a mitochondrial targeting domain and that expression of this peptide alone can induce apoptotic cell death. These findings provide new insights into a putative cellular localization and function for ATG9B.

Abstract Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions – to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut’s neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.