live-cell-imaging-of-early-autophagy-events-omegasomes-and-beyond

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

evaluating-autophagy-levels-two-different-pancreatic-cell-models

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

The amino terminus of phospholipase D1 (PLD1) contains three potential membrane-interacting determinants: a phox homology (PX) domain, a pleckstrin homology (PH) domain and two adjacent cysteines at positions 240 and 241 within the PH domain that are fatty acylated in vivo. To understand how these determinants contribute to membrane localization, we have mutagenized critical residues of the PLD1 PH domain in the wild type or palmitate-free background in the intact protein, in a fragment that deletes the first 210 amino acids including the PX domain, and in the isolated PH domain. Mutants were expressed in COS-7 cells and examined for membrane residence, intracellular localization, palmitoylation, and catalytic activity. Our results are as follows. 1) Mutagenesis of critical residues of the PH domain results in redistribution of PLD1 from membranes to cytosol, independently of fatty acylation sites. Importantly, PH domain mutants in the wild type background showed greatly reduced fatty acylation, despite the presence of all relevant cysteines. 2) The isolated PH domain did not co-localize with PLD1 and was not palmitoylated. 3) The PX deletion mutant showed similar distribution and palmitoylation to the intact protein. Interestingly, PH domain mutants in this background showed significant palmitoylation and incomplete cytosolic redistribution. 4) PH domain mutants in the wild type or palmitate-free background maintained catalytic activity. We propose that membrane targeting of PLD1 involves a hierarchy of signals with a functional PH domain allowing fatty acylation leading to strong membrane binding. The PX domain may modulate function of the PH domain.

Hierarchy of membrane-targeting signals of phospholipase D1 involving lipid modification of a pleckstrin homology domain.

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.

Sphingosine kinase 1 is an intracellular effector of phosphatidic acid.

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG(-/-) neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG(-/-) neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.

RhoG regulates the neutrophil NADPH oxidase.

PA binding of phosphatidylinositol 4-phosphate 5-kinase.

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.

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

Autophagy presents a topological challenge for the cell because it requires delivery of cytosolic material to the lumen of a membrane-bound compartment, the lysosome. This is solved in an ingenious way by the formation of a double-membrane vesicle, the autophagosome, which captures cytosolic proteins and organelles during its transformation from a planar membrane disk into a sphere. In this way, cytosolic material first becomes lumenal and is then delivered for degradation to the lysosome. An unsolved set of questions in autophagy concerns the membrane of the autophagosome: what are the signals for its formation and what is its identity? Recently we provided some clues that may help answer these questions. By following the dynamics of several phosphatidylinositol 3-phosphate (PI3P)-binding proteins during amino acid starvation (and autophagy induction) we concluded that at least some autophagosomes are formed in a starvation-induced, PI3P-enriched membrane compartment dynamically connected to the endoplasmic reticulum (ER). We termed the membranes of this compartment omegasomes (from their omega-like shape). Our data suggest that PI3P is important for providing localization clues and perhaps for facilitating the fusion step at the final stage of autophagosome formation.

Making autophagosomes: localized synthesis of phosphatidylinositol 3-phosphate holds the clue.

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.

Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors).

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.

Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels.

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate.

The simple phosphoinositide PtdIns3P has been shown to control cell growth downstream of amino acid signalling and autophagy downstream of amino acid withdrawal. These opposing effects depend in part on the existence of distinct complexes of Vps34 (vacuolar protein sorting 34), the kinase responsible for the majority of PtdIns3P synthesis in cells: one complex is activated after amino acid withdrawal to induce autophagy and another regulates mTORC1 (mammalian target of rapamycin complex 1) activation when amino acids are present. However, lipid-dependent signalling almost always exhibits a spatial dimension, related to the site of formation of the lipid signal. In the case of PtdIns3P-regulated autophagy induction, recent data suggest that PtdIns3P accumulates in a membrane compartment dynamically connected to the endoplasmic reticulum that constitutes a platform for the formation of some autophagosomes. For PtdIns3P-regulated mTORC1 activity, a spatial context is not yet known: several possibilities can be envisaged based on the known effects of PtdIns3P on the endocytic system and on recent data suggesting that activation of mTORC1 depends on its localization on lysosomes.

How phosphoinositide 3-phosphate controls growth downstream of amino acids and autophagy downstream of amino acid withdrawal.

Basal autophagy-here defined as macroautophagic activity during cellular growth in normal medium containing amino acids and serum-appears to be highly active in many cell types and in animal tissues. Here we characterized this pathway in mammalian HEK 293 cells. First, we examined, side by side, three compounds that are widely used to reveal basal autophagy by blocking maturation of autophagosomes: bafilomycin A 1 (BafA1), chloroquine and vinblastine. Only BafA1 appeared to be without complicating side effects. Chloroquine partially inhibited mechanistic target of rapamycin (MTOR) activity, which would induce autophagy induction as well as block autophagosome maturation. Vinblastine caused the distribution of early omegasome components into punctate phagophore assembly sites, and therefore it would also induce autophagy, complicating interpretation. Basal autophagy was significantly sensitive to inhibition by wortmannin, and therefore required formation of phosphatidylinositol 3-phosphate (PtdIns3P), but it was twice as resistant to wortmannin as starvation-induced autophagy. We also determined that basal autophagy was significantly suppressed by MTOR activation brought about by overexpression of RHEB or activated RAGs. Finally we investigated the spatial relationship of nascent autophagosomes to the endoplasmic reticulum (ER) or to mitochondria by live imaging experiments under conditions that reveal basal autophagy (with BafA1 treatment), or upon MTOR inactivation (which would result in autophagy induction). Side-by-side comparison showed that under both basal and induced autophagy, 100% of autophagosomes first appeared in close proximity to ER strands. In parallel measurements, 40% were in close proximity to mitochondria under both conditions. We concluded that in HEK 293 cells, basal autophagy is mechanistically similar to that induced by MTOR inactivation in all aspects examined.

Characteristics and requirements of basal autophagy in HEK 293 cells.

Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

Dynamic association of the ULK1 complex with omegasomes during autophagy induction.

Autophagy is a membrane-trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic tagging of the POI allows its visualization in live cells, while immunostaining of the POI requires the fixation of cells and the permeabilization of cell membranes. Here we describe detailed protocols on how to visualize autophagy dynamics using fluorescence microscopy in live and fixed cells. We discuss the critical parameters of each technique, their advantages, and why the robustness is increased when they are used in tandem.

Imaging autophagy.

Autophagosomes form in eukaryotic cells in response to starvation or to other stress conditions brought about by the unwanted presence in the cytosol of pathogens, damaged organelles or aggregated protein assemblies. The uniqueness of autophagosomes is that they form de novo and that they are the only double-membraned vesicles known in cells, having arisen from flat membrane sheets which have expanded and self-closed. The various steps describing their formation as well as most of the protein and lipid components involved have been identified. Furthermore, the hierarchical relationships among the components are well documented, and the mechanistic rationale for some of these hierarchies has been revealed. In the present review, we try to provide a current view of the process of autophagosome formation in mammalian cells, emphasizing along the way gaps in our knowledge that need additional work.

Dynamics of autophagosome formation: a pulse and a sequence of waves.

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles.

During macroautophagy/autophagy, the ULK complex nucleates autophagic precursors, which give rise to autophagosomes. We analyzed, by live imaging and mathematical modeling, the translocation of ATG13 (part of the ULK complex) to the autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In nonselective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution, whereas wortmannin reduced this effect and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13 with increasing time between peaks were observed. We hypothesized that these multiple translocations arise because the engulfment of mitochondrial fragments required successive nucleation of phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behavior. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Thus, our data provide novel insights into the early dynamics of selective and nonselective autophagy. ATG: autophagy related 13; CFP: cyan fluorescent protein; dsRED:  red fluorescent protein; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IVM: ivermectin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: PtdIns-3-phosphate; ULK: unc-51 like autophagy activating kinase.

ATG13 dynamics in nonselective autophagy and mitophagy: insights from live imaging studies and mathematical modeling.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

ArticleTotal : 2	Year
Publication title Cited by 13	2013
Publication title Cited by 3	2023

Article	Year
Hierarchy of membrane-targeting signals of phospholipase D1 involving lipid modification of a pleckstrin homology domain. The Journal of biological chemistry\| PubMed ID: 12021265	2002
Sphingosine kinase 1 is an intracellular effector of phosphatidic acid. The Journal of biological chemistry\| PubMed ID: 15310762	2004
RhoG regulates the neutrophil NADPH oxidase. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16621998	2006
PA binding of phosphatidylinositol 4-phosphate 5-kinase. Advances in enzyme regulation\| PubMed ID: 18167315	2008
Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of cell biology\| PubMed ID: 18725538	2008
Making autophagosomes: localized synthesis of phosphatidylinositol 3-phosphate holds the clue. Autophagy\| PubMed ID: 18927492	2008
Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors). Biochimica et biophysica acta\| PubMed ID: 19345277	2009
Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels. The Journal of cell biology\| PubMed ID: 19349583	2009
Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy\| PubMed ID: 21799305	2011
How phosphoinositide 3-phosphate controls growth downstream of amino acids and autophagy downstream of amino acid withdrawal. Biochemical Society transactions\| PubMed ID: 22260663	2012
Characteristics and requirements of basal autophagy in HEK 293 cells. Autophagy\| PubMed ID: 23800949	2013
Dynamic association of the ULK1 complex with omegasomes during autophagy induction. Journal of cell science\| PubMed ID: 24013547	2013
Imaging autophagy. Current protocols in cytometry\| PubMed ID: 24984962	2014
Dynamics of autophagosome formation: a pulse and a sequence of waves. Biochemical Society transactions\| PubMed ID: 25233420	2014
Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nature communications\| PubMed ID: 27510922	2016
ATG13 dynamics in nonselective autophagy and mitophagy: insights from live imaging studies and mathematical modeling. Autophagy\| PubMed ID: 32320309	2021
TFG binds LC3C to regulate ULK1 localization and autophagosome formation. The EMBO journal\| PubMed ID: 33932238	2021

Maria Manifava

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