Showing papers by "Eeva-Liisa Eskelinen published in 2017"

Molecular definitions of autophagy and related processes

Abstract: Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

Abstract: Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

Disruption of the vacuolar-type H(+)-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

Abstract: The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.

Basal Autophagy Is Altered in Lagotto Romagnolo Dogs with an ATG4D Mutation.

Abstract: A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.

Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin

Abstract: CAPNS1 is essential for stability and function of the ubiquitous calcium-dependent proteases micro- and milli-calpain. Upon inhibition of the endoplasmic reticulum Ca2+ ATPase by 100 nM thapsigargin, both micro-calpain and autophagy are activated in human U2OS osteosarcoma cells in a CAPNS1-dependent manner. As reported for other autophagy triggers, thapsigargin treatment induces Golgi fragmentation and fusion of Atg9/Bif-1-containing vesicles with LC3 bodies in control cells. By contrast, CAPNS1 depletion is coupled with an accumulation of LC3 bodies and Rab5 early endosomes. Moreover, Atg9 and Bif-1 remain in the GM130-positive Golgi stacks and Atg9 fails to interact with the endocytic route marker transferrin receptor and with the core autophagic protein Vps34 in CAPNS1-depleted cells. Ectopic expression of a Bif-1 point mutant resistant to calpain processing is coupled to endogenous p62 and LC3-II accumulation. Altogether, these data indicate that calpain allows dynamic flux of Atg9/Bif-1 vesicles from the Golgi toward the budding autophagosome.

Correction: TRIM17 contributes to autophagy of midbodies while actively sparing other targets from degradation.

Abstract: There was an error published in J. Cell Sci. 129 , [3562-3573][1]. The affiliation was incorrect for the author Terje Johansen. The correct author affiliations are given below. Michael A. Mandell1,*, Ashish Jain2,3,4, Suresh Kumar1, Moriah J. Castleman1, Tahira Anwar5, Eeva-Liisa Eskelinen5,

Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples.

Abstract: Electron tomography has significantly contributed to recent findings regarding the biogenesis of the phagophore, an organelle which initiates autophagic sequestration. The information obtained from 1.9nm slices through the tomograms have revealed that during biogenesis the phagophore is in contact with the membranes of apposing organelles to form tubular connections and membrane contact sites (MCSs). The most reported and established tubular connections occur between the phagophore and the endoplasmic reticulum. However, as the phagophore continues to grow and expand, connections and MCSs have also been reported to occur between the phagophore and several other organelles in a possible attempt to utilize lipids for membrane expansion from alternative sources. Since the lifespan of the phagophore is only a few minutes and membrane connections and MCSs are very dynamic, capturing these two events requires precision during fixation. Up to date there is no quicker alternative for sample preservation in transmission electron microscopy than cryoimmobilization. In this report, we describe our protocol for cryoimmobilization using high-pressure freezing and freeze substitution, and report our first findings on phagophore morphology using this approach.