Autophagic Degradation Is Involved in Cell Protection against Ricin Toxin.

Autophagy is a complex and highly regulated degradative process, which acts as a survival pathway in response to cellular stress, starvation and pathogen infection. Ricin toxin is a plant toxin produced by the castor bean and classified as a category B biothreat agent. Ricin toxin inhibits cellular protein synthesis by catalytically inactivating ribosomes, leading to cell death. Currently, there is no licensed treatment for patients exposed to ricin. Ricin-induced apoptosis has been extensively studied; however, whether its intoxication via protein synthesis inhibition affects autophagy is not yet resolved. In this work, we demonstrated that ricin intoxication is accompanied by its own autophagic degradation in mammalian cells. Autophagy deficiency, by knocking down ATG5, attenuates ricin degradation, thus aggravating ricin-induced cytotoxicity. Additionally, the autophagy inducer SMER28 (Small Molecule Enhancer 28) partially protects cells against ricin cytotoxicity, an effect not observed in autophagy-deficient cells. These results demonstrate that autophagic degradation acts as a survival response of cells against ricin intoxication. This suggests that stimulation of autophagic degradation may be a strategy to counteract ricin intoxication.

Robust Local Thickness Estimation of Sub-Micrometer Specimen by 4D-STEM.

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.

Commercially Available Eye Drops Containing Trehalose Protect Against Dry Conditions via Autophagy Induction.

Purpose: Evaluation of marketed eye drops with or without trehalose, a nonreducing natural osmoprotector disaccharide, in autophagy modulation and its role in cell survival during desiccation. Methods: Eye drops containing either sodium hyaluronate (SH) (Hyabak®, Thea, France) or a combination of SH with trehalose (Thealose Duo®, Thea, France) were compared with control conditions to evaluate the ability to modulate autophagy in human epithelial cells in vitro. Autophagy was monitored using LC3, a marker of the autophagic machinery, by fluorescence microscopy and immunoblot analysis. Control and autophagy-deficient cells treated with eye drops were exposed to desiccation to mimic dry eyes and cell survival was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay. Trehalose, a known autophagy inducer was used as a positive control. Results: Artificial tears containing SH with and without trehalose induce a complete autophagic flux, as indicated by an increase in the number of autophagosomes and autolysosomes, and the accumulation of the lipidated form of LC3 associated with complete autophagy. In addition, there was a synergistic effect of SH for autophagy induction when combined with trehalose, compared with each of the components alone. Survival of cells treated with both eye drops and exposed to desiccation was decreased in autophagy-deficient cells, demonstrating the essential role of autophagy on eye drop protection. Conclusions: Autophagic flux is induced by SH-containing eye drops, and this phenomenon is enhanced in combination with trehalose. We also demonstrated that autophagy induction is involved in the osmoprotective effects of both trehalose and SH-containing eye drops, to maintain epithelial cell homeostasis in dry conditions.

Human Cytomegalovirus Inhibits Autophagy of Renal Tubular Epithelial Cells and Promotes Cellular Enlargement.

Human Cytomegalovirus (HCMV) is a frequent opportunistic pathogen in immunosuppressed patients, which can be involved in kidney allograft dysfunction and rejection. In order to study the pathophysiology of HCMV renal diseases, we concentrated on the impact of HCMV infection on human renal tubular epithelial HK-2 cells. Our aim was to develop a model of infection of HK-2 cells by using the viral strain TB40/E, that contains the extended cell tropism of clinical isolates and the efficient viral multiplication in cell culture of laboratory-adapted strains. We observed that HK-2 cells can be infected by HCMV and expressed viral antigens, but they do not produce extracellular viral particles. We then studied the interplay of HCMV with ciliogenesis and autophagy. Primary cilium (PC) is a stress sensor important to maintain renal tissue homeostasis that projects from the apical side into the lumen of tubule cells. PC formation and length were not modified by HCMV infection. Autophagy, another stress response process critically required for normal kidney functions, was inhibited by HCMV in HK-2 cells with a reduction in the autophagic flux. HCMV classically induces an enlargement of infected cells in vivo and in vitro, and we observed that HCMV infection led to an enlargement of the HK-2 cell volume. Our results constitute therefore an excellent starting point to further explore the role of these mechanisms in renal cells dysfunction.

Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles.

During its life cycle, Human cytomegalovirus (HCMV) tightly modulates autophagy, a vesicular pathway allowing degradation and recycling of cellular components. To study the interplay between autophagy and the viral life cycle, we established various autophagy-deficient human fibroblastic cell lines. By knocking down the expression or activity of five autophagy-related proteins, we confirmed the proviral function that the autophagic machinery exerts on HCMV production. Using 3D reconstruction from confocal microscopy and electron microscopy, we demonstrated that lipidated LC3-positive vesicles accumulated at the viral assembly compartment (vAC). The vAC is a juxtanuclear ring-shaped structure containing several organelles and membranes, where assembly and final envelopment of HCMV particles occur. Two LC3 homologs, GABARAPL1 and GATE16, also accumulated during HCMV infection and were associated with the vAC, in proximity with fragmented Golgi stacks. Additionally, we observed the formation of a pre-assembly compartment (PrAC) in infected cells, which consists of a juxtanuclear structure containing both fragmented Golgi and LC3-positive vesicles. Finally, we showed that highly purified extracellular viral particles were associated with various autophagy proteins. Our results thus suggest that autophagy machinery participates to the final cytoplasmic envelopment of HCMV viral particles into the vAC and that autophagy-related proteins can be spotted in the virions.