Publications by authors named "Tonia Akoumianaki"

13 Publications

  • Page 1 of 1

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Author Correction: Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Nat Commun 2018 11 22;9(1):5015. Epub 2018 Nov 22.

Department of Medicine, University of Crete, Foundation for Research and Technology, 71300, Heraklion, Crete, Greece.

The original version of this Article contained an error in the spelling of the author Emilien Etienne, which was incorrectly given as Emilien Ettiene. These errors have now been corrected in both the PDF and HTML versions of the Article.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07301-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250689PMC
November 2018

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Nat Commun 2018 08 20;9(1):3333. Epub 2018 Aug 20.

Department of Medicine, University of Crete, Foundation for Research and Technology, 71300, Heraklion, Crete, Greece.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-05820-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102248PMC
August 2018

Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis.

Nat Microbiol 2018 07 30;3(7):791-803. Epub 2018 May 30.

Department of Medicine, University of Crete, Heraklion, Crete, Greece.

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca signalling pathway that depends on intracellular Ca sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca-CaM signalling in aspergillosis. Finally, we demonstrate that Ca sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca-CaM signalling to inhibit LAP. These findings reveal the important role of Ca-CaM signalling in antifungal immunity and identify an immunological function of Ca binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-018-0167-xDOI Listing
July 2018

DAPK1 Keeps the Peace in Antifungal Inflammation.

Cell Host Microbe 2016 Dec;20(6):695-697

Department of Clinical Microbiology and Microbial Pathogenesis, School of Medicine, University of Crete, 71300 Heraklion, Crete, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71300 Heraklion, Crete, Greece. Electronic address:

Unabated inflammation and impaired antifungal immunity underlie genetic defects in NOX-2-dependent activation of LC3-associated phagocytosis (LAP). In this issue of Cell Host & Microbe, Oikonomou et al. (2016) identify a molecular link between IFN-γ/DAPK1 signaling, the proteosomal degradation pathway, and LAP that is critical for dampening Aspergillus-triggered immunopathology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2016.11.008DOI Listing
December 2016

Melanin targets LC3-associated phagocytosis (LAP): A novel pathogenetic mechanism in fungal disease.

Autophagy 2016 05 30;12(5):888-9. Epub 2016 Mar 30.

d Unité des Aspergillus, and Plateforme de Microscopie electronique Institut Pasteur , Paris , France.

Intracellular swelling of conidia of the major human airborne fungal pathogen Aspergillus fumigatus results in surface exposure of immunostimulatory pathogen-associated molecular patterns (PAMPs) and triggers activation of a specialized autophagy pathway called LC3-associated phagocytosis (LAP) to promote fungal killing. We have recently discovered that, apart from PAMPs exposure, cell wall melanin removal during germination of A. fumigatus is a prerequisite for activation of LAP. Importantly, melanin promotes fungal pathogenicity via targeting LAP, as a melanin-deficient A. fumigatus mutant restores its virulence upon conditional inactivation of Atg5 in hematopoietic cells of mice. Mechanistically, fungal cell wall melanin selectively excludes the CYBA/p22phox subunit of NADPH oxidase from the phagosome to inhibit LAP, without interfering with signaling regulating cytokine responses. Notably, inhibition of LAP is a general property of melanin pigments, a finding with broad physiological implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2016.1157242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854543PMC
May 2016

Aspergillus Cell Wall Melanin Blocks LC3-Associated Phagocytosis to Promote Pathogenicity.

Cell Host Microbe 2016 Jan 31;19(1):79-90. Epub 2015 Dec 31.

Department of Medicine, University of Crete, Foundation for Research and Technology, 71300 Heraklion, Crete, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71300 Heraklion, Crete, Greece. Electronic address:

Concealing pathogen-associated molecular patterns (PAMPs) is a principal strategy used by fungi to avoid immune recognition. Surface exposure of PAMPs during germination can leave the pathogen vulnerable. Accordingly, β-glucan surface exposure during Aspergillus fumigatus germination activates an Atg5-dependent autophagy pathway termed LC3-associated phagocytosis (LAP), which promotes fungal killing. We found that LAP activation also requires the genetic, biochemical or biological (germination) removal of A. fumigatus cell wall melanin. The attenuated virulence of melanin-deficient A. fumigatus is restored in Atg5-deficient macrophages and in mice upon conditional inactivation of Atg5 in hematopoietic cells. Mechanistically, Aspergillus melanin inhibits NADPH oxidase-dependent activation of LAP by excluding the p22phox subunit from the phagosome. Thus, two events that occur concomitantly during germination of airborne fungi, surface exposure of PAMPs and melanin removal, are necessary for LAP activation and fungal killing. LAP blockade is a general property of melanin pigments, a finding with broad physiological implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2015.12.002DOI Listing
January 2016

Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae.

Antimicrob Agents Chemother 2015 Dec 7;60(3):1226-33. Epub 2015 Dec 7.

Department of Medicine, University of Crete, Heraklion, Crete, Greece

The modest in vitro activity of echinocandins against Aspergillus implies that host-related factors augment the action of these antifungal agents in vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against various Aspergillus species under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P = 0. 0005). Importantly, the enhanced activity of caspofungin against Aspergillus spp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinating Aspergillus hyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin with Aspergillus hyphae (P < 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery to Aspergillus hyphae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02026-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775923PMC
December 2015

Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling.

J Immunol 2013 Aug 1;191(3):1287-99. Epub 2013 Jul 1.

Department of Medicine, University of Crete, 71300 Heraklion, Crete, Greece.

Aspergillus fumigatus is the predominant airborne fungal pathogen in immunocompromised patients. Genetic defects in NADPH oxidase (chronic granulomatous disease [CGD]) and corticosteroid-induced immunosupression lead to impaired killing of A. fumigatus and unique susceptibility to invasive aspergillosis via incompletely characterized mechanisms. Recent studies link TLR activation with phagosome maturation via the engagement of autophagy proteins. In this study, we found that infection of human monocytes with A. fumigatus spores triggered selective recruitment of the autophagy protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans and was mediated by activation of the Dectin-1 receptor. LC3 II recruitment in A. fumigatus phagosomes required spleen tyrosine kinase (Syk) kinase-dependent production of reactive oxygen species and was nearly absent in monocytes of patients with CGD. This pathway was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. In vivo and ex vivo administration of corticosteroids blocked LC3 II recruitment in A. fumigatus phagosomes via rapid inhibition of phosphorylation of Src and Syk kinases and downstream production of reactive oxygen species. Our studies link Dectin-1/Syk kinase signaling with autophagy-dependent maturation of A. fumigatus phagosomes and uncover a potential mechanism for development of invasive aspergillosis in the setting of CGD and corticosteroid-induced immunosupression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1300132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883106PMC
August 2013

Phosphorylation at serine 331 is required for Aurora B activation.

J Cell Biol 2011 Oct 24;195(3):449-66. Epub 2011 Oct 24.

Department of Biology, University of Crete, Heraklion 71409, Greece.

Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201104023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206340PMC
October 2011

Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells.

J Cell Sci 2009 Apr 19;122(Pt 8):1111-8. Epub 2009 Mar 19.

Department of Biochemistry, University of Crete, School of Medicine, 71 003 Heraklion, Greece.

We have investigated the subcellular distribution and dynamics of soluble tubulin in unperturbed and transfected HeLa cells. Under normal culture conditions, endogenous alpha/beta tubulin is confined to the cytoplasm. However, when the soluble pool of subunits is elevated by combined cold-nocodazole treatment and when constitutive nuclear export is inhibited by leptomycin B, tubulin accumulates in the cell nucleus. Transfection assays and FRAP experiments reveal that GFP-tagged beta-tubulin shuttles between the cytoplasm and the cell nucleus. Nuclear import seems to occur by passive diffusion, whereas exit from the nucleus appears to rely on nuclear export signals (NESs). Several such motifs can be identified by sequence criteria along the beta-tubulin molecule and mutations in one of these (NES-1) cause a significant accumulation in the nuclear compartment. Under these conditions, the cells are arrested in the G0-G1 phase and eventually die, suggesting that soluble tubulin interferes with important nuclear functions. Consistent with this interpretation, soluble tubulin exhibits stoichiometric binding to recombinant, normally modified and hyper-phosphorylated/acetylated histone H3. Tubulin-bound H3 no longer interacts with heterochromatin protein 1 and lamin B receptor, which are known to form a ternary complex under in vitro conditions. Based on these observations, we suggest that nuclear accumulation of soluble tubulin is part of an intrinsic defense mechanism, which tends to limit cell proliferation under pathological conditions. This readily explains why nuclear tubulin has been detected so far only in cancer or in transformed cells, and why accumulation of this protein in the nucleus increases after treatment with chemotherapeutic agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.043034DOI Listing
April 2009

Synthesis and antitumor activity of peptide-paclitaxel conjugates.

J Pept Sci 2007 Oct;13(10):662-71

Department of Chemistry, University of Ioannina, GR-45110 Ioannina, Greece.

Paclitaxel (Pac) is the most important anticancer drug used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. In this work, we present the synthesis of five 2'-paclitaxel-substituted analogs in which paclitaxel was covalently bound to peptides or as multiple copies to synthetic carriers. Ac-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Arg-NH(2), Folyl-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Ser-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](2)-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](3)-NH(2) and Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) were synthesized using 2'-halogeno-acetylated paclitaxel derivatives. Paclitaxel conjugates showed greater solubility in water than paclitaxel and inhibited the proliferation of human breast, prostate, and cervical cancer cell lines. Although all synthesized compounds had an antiproliferative activity, the Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) derivative showed improved biological activity in comparison with paclitaxel in cervical and prostate human cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/psc.899DOI Listing
October 2007

Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence.

BMC Immunol 2006 Sep 6;7:20. Epub 2006 Sep 6.

Biochemistry, University of Crete, School of Medicine, P,O, Box 2208, Heraklion 71003, Greece.

Background: Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera.

Methods: We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde.

Results: We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells.

Conclusion: Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2172-7-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1574344PMC
September 2006