Showing papers by "María Isabel Colombo published in 2012"

Guidelines for the use and interpretation of assays for monitoring autophagy

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy

Abstract: Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosom...

ATP is released from autophagic vesicles to the extracellular space in a VAMP7-dependent manner

Abstract: Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called autophagosome SNAREs proteins are key molecules of the vesicle fusion machinery Our results indicate that in a mammalian tumor cell line a subset of VAMP7 (V-SNARE)-positive vacuoles colocalize with LC3 at the cell periphery (focal adhesions) upon starvation The re-distribution of VAMP7 positive structures is a microtubule-dependent event, with the participation of the motor protein KIF5 and the RAB7 effector RILP Interestingly, most of the VAMP7-labeled vesicles were loaded with ATP Moreover, in cells subjected to starvation, these structures fuse with the plasma membrane to release the nucleotide to the extracellular medium Summarizing, our results show the molecular components involved in the release of ATP to extracellular space, which is recognized as an important autocrine/paracrine signal molecule that participates in the regulation of several cellular functions such as immunogenicity of cancer cell death or inflammation

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

Abstract: The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process Lysosomal-dependent or -independent T cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells In this context, we have previously shown that induction of autophagy in host cells before infection favors T cruzi invasion Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T cruzi entry sites and that the newly formed T cruzi parasitophorous vacuole has characteristics of an autophagolysosome This review summarizes the current knowledge of the molecular and cellular mechanisms of T cruzi invasion in nonphagocytic cells Based on our findings, we propose a new model in which T cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells

cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response

Abstract: Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus–secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus–containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.

The Coxiella burnetii Parasitophorous Vacuole

Abstract: Coxiella burnetii is a bacterial intracellular parasite of eucaryotic cells that replicates within a membrane-bound compartment, or "parasitophorous vacuole" (PV). With the exception of human macrophages/monocytes, the consensus model of PV trafficking in host cells invokes endolysosomal maturation culminating in lysosome fusion. C. burnetii resists the degradative functions of the vacuole while at the same time exploiting the acidic pH for metabolic activation. While at first glance the mature PV resembles a large phagolysosome, an increasing body of evidence indicates the vacuole is in fact a specialized compartment that is actively modified by the pathogen. Adding to the complexity of PV biogenesis is new data showing vacuole engagement with autophagic and early secretory pathways. In this chapter, we review current knowledge of PV nature and development, and discuss disparate data related to the ultimate maturation state of PV harboring virulent or avirulent C. burnetii lipopolysaccharide phase variants in human mononuclear phagocytes.

Implications of PNPLA3 polymorphism in chronic hepatitis C patients receiving peginterferon plus ribavirin

Abstract: Summary Background Homozygosity for the PNPLA3 p.I148M polymorphism influences steatosis and fibrogenesis in chronic hepatitis C (CHC). Aim To evaluate the effect of p.148M/M on sustained virological response (SVR) and viral kinetics in patients who underwent antiviral therapy with peg-interferon and ribavirin, stratified according to viral genotype and fibrosis severity, and secondarily, the interaction with interleukin-28B (IL28B) genotype on liver damage. Methods In this observational study, we considered 602 treatment-naive consecutive patients from tertiary referral centres in Milan and Vienna [61% genotype 1 (G1), 30% advanced fibrosis, 33% IL28B rs12979860 CC]. Results The p.148M/M genotype, detected in 8% of patients, did not influence SVR in the overall series (P = 0.29), but it was associated with SVR (3/17, 17% vs. 56/121, 46%; P = 0.034) and complete early viral response (4/17, 23% vs. 68/121, 56%; P = 0.018) in G1/4 patients with advanced fibrosis. After adjustment for age, viral load, IL28BCC genotype, treatment dose, and steatosis, p.148M/M remained a predictor of SVR in G1/4 patients with advanced fibrosis (OR 0.23, 95% CI 0.04–0.87). The p.148M/M genotype was associated with more advanced fibrosis in the overall series (P = 0.049), whereas the rs12979860 IL28BCC genotype only in patients negative for p.148M/M (P = 0.017), independently of age, BMI and alanine transaminase levels (OR 1.51, 95% CI 1.01–2.27). Conclusions PNPLA3 p.148M/M genotype was negatively associated with SVR and early viral kinetics independently of steatosis, albeit only in difficult-to-cure G1/4 patients with advanced fibrosis, whereas stratification for the p.148M/M PNPLA3 genotype unmasked an association between IL28BCC genotype and more severe liver fibrosis.

Staphylococcus aureus promotes autophagy by decreasing intracellular cAMP levels.

Abstract: Staphylococcus aureus is an intracellular bacterium responsible for serious infectious processes. This pathogen escapes from the phagolysosomal pathway into the cytoplasm, a strategy that allows intracellular bacterial replication and survival with the consequent killing of the eukaryotic host cell and spreading of the infection. S. aureus is able to secrete several virulence factors such as enzymes and toxins. Our recent findings indicate that the main virulence factor of S. aureus, the pore-forming toxin α-hemolysin (Hla), is the secreted factor responsible for the activation of an alternative autophagic pathway. We have demonstrated that this noncanonical autophagic response is inhibited by artificially elevating the intracellular levels of cAMP. This effect is mediated by RAPGEF3/EPAC (Rap guanine nucleotide exchange factor (GEF)3/exchange protein activated by cAMP), a cAMP downstream effector that functions as a GEF for the small GTPase Rap. We have presented evidence that RAPGEF3 and RAP2B, through ...

Cortactin Is Involved in the Entry of Coxiella burnetii into Non-Phagocytic Cells

Abstract: Background Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood.