National University of Cuyo

About: National University of Cuyo is a education organization based out in Mendoza, Argentina. It is known for research contribution in the topics: Population & Exocytosis. The organization has 3175 authors who have published 4872 publications receiving 83221 citations. The organization is also known as: National University of Cuyo.

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.

Consensus guidelines for the detection of immunogenic cell death

Abstract: Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Rab7 is required for the normal progression of the autophagic pathway in mammalian cells

Abstract: Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy.

A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation

Abstract: Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.