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

Abstract

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 but that endophilin B2 deficiency impairs endosomal trafficking, 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.

An N-terminal conserved region in human Atg3 couples membrane curvature sensitivity to conjugase activity during autophagy

“…To determine whether the NT Cons region is essential for LC3B–PE conjugation and autophagic flux in the presence of additional autophagy machinery in mammalian cells, we reintroduced wild-type hAtg3, hAtg3 L23T , or hAtg3 P21A variant into Atg3 −/− mouse embryonic fibroblasts (MEFs) by lentiviral transduction and examined LC3B lipidation and autophagic flux by monitoring the lysosomal turnover of LC3B-II in the presence or absence of the lysosomal inhibitor bafilomycin A1 (BafA1), as described previously 23 . While the lipidation of LC3B was absent in Atg3 −/− MEFs, the expression of hAtg3 rescued not only LC3B–PE conjugation, but also starvation-induced autophagic flux (Fig.…”

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

“…Total cell lysates were prepared in radio-immunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate, 5 mM EDTA, pH 8.0) containing protease and phosphatase inhibitors and subjected to SDS-PAGE followed by IB with the indicated antibodies. The signal intensities were quantified using the Image Studio version 5 software (LI-COR Biotechnology) 45 . Uncropped scans of all the blots are shown in Supplementary Fig.…”

Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers

“…To further investigate this, we performed immunostaining with the early endosome marker EEA1 (early endosome antigen 1) following aptamer incubation ( Figure 5 A). 33 , 34 , 35 The selected aptamer showed little colocalization (yellow) with EEA1-stained endosomal structures as indicated by the white arrowheads in the overlay image. Moreover, endosomal internalization was further investigated by assessing as early as 30 min and up to 120 min with also minimal colocalization (arrowheads) observed at any magnification and time point tested ( Figure 5 B; Figure S5 ).…”

“…Upon aptamer-mediated delivery of saporin to the cytoplasm, the toxin exerted its ribosome inactivating protein effects, thus leading to cell death. 34 , 35 Another possible explanation could be that the aptamer internalizes through more than one pathway where one of these is a non-endosome-related pathway, hence the red fluorescence ( Figure 5 ). The latter is supported by the work of Van der Aa et al., 62 who demonstrated interchangeable internalization of two well-established cationic polymers through two different endocytic routes (clathrin- and caveolae-dependent endocytosis).…”