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      Assays to Monitor Autophagy Progression in Cell Cultures

      review-article
      Author(s): , *
      Publication date (Electronic):
      Journal: Cells
      Publisher: MDPI
      Keywords: autophagy, autophagosome, phagophore, autophagic flux, measurement, assay, method

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          Abstract

          The vast number of implications of autophagy in multiple areas of life sciences and medicine has attracted the interest of numerous scientists that aim to unveil the role of this process in specific physiological and pathological contexts. Cell cultures are one of the most frequently used experimental setup for the investigation of autophagy. As a result, it is essential to assess this highly regulated molecular pathway with efficient and reliable methods. Each method has its own advantages and disadvantages. Here, we present a review summarizing the most established assays used to monitor autophagy induction and progression in cell cultures, in order to guide researchers in the selection of the most optimal solution for their experimental setup and design.

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          Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

          Elizabeth L. Axe, Simon A. Walker, Maria Manifava … (2008)
          Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain–containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.
          Article 0 comments Cited 615 times – based on 0 reviews     Review now
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            A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

            Vladimir Kirkin, Trond Lamark, Yu-Shin Sou … (2009)
            Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.
            Article 0 comments Cited 365 times – based on 0 reviews     Review now
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              Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.

              Joungmok Kim, Young Chul Kim, Chong Fang … (2013)
              Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the nonautophagy Vps34 complex by phosphorylating T163/S165 in Vps34 and therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the proautophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy-essential gene present only in the proautophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy. Copyright © 2013 Elsevier Inc. All rights reserved.
              Article 0 comments Cited 287 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Cells
                Journal ID (iso-abbrev): Cells
                Journal ID (publisher-id): cells
                Title: Cells
                Publisher: MDPI
                ISSN (Electronic): 2073-4409
                Publication date (Electronic): 07 July 2017
                Publication date Collection: September 2017
                Volume: 6
                Issue: 3
                Electronic Location Identifier: 20
                Affiliations
                Department of Cell Biology, University of Groningen, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands; i.orhon@ 123456umcg.nl
                Author notes
                [* ]Correspondence: f.m.reggiori@ 123456umcg.nl
                Author information
                https://orcid.org/0000-0003-2652-2686
                Article
                Publisher ID: cells-06-00020
                DOI: 10.3390/cells6030020
                PMC ID: 5617966
                PubMed ID: 28686195
                SO-VID: c4757783-3735-4c6d-9bea-471131f808b6
                Copyright © © 2017 by the authors.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 05 June 2017
                Date accepted : 04 July 2017
                Categories
                Subject: Review

                Keywords: autophagy,autophagosome,phagophore,autophagic flux,measurement,assay,method
                Data availability:
                Keywords: autophagy, autophagosome, phagophore, autophagic flux, measurement, assay, method

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